Sample records for factor-affinity-labeled species represents

  1. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    PubMed

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  2. Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beck, J.T.; Ullman, B.

    1989-08-22

    An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using activated derivatives of the ligands. These activated derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 {mu}M concentration of either activated ({sup 3}H)folate or activated ({sup 3}H)methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labelingmore » of a 46,000-dalton protein was observed when equimolar concentrations of activated radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with activated ligand. These studies provide biochemical evidence that the folate-methotrexate transporter of Leishmania donovani can be identified in crude extracts by an affinity labeling technique and serve as a prerequisite to further analysis of the transport protein by providing a vehicle for subsequent purification of this membrane carrier. Moreover, these investigations suggest that the affinity labeling technique using EDC-activated ligands may be exploitable to analyze other cell surface binding proteins in Leishmania donovani, as well as in other organisms.« less

  3. Pushing antibody-based labeling systems to higher sensitivity by linker-assisted affinity enhancement.

    PubMed

    Gorris, Hans H; Bade, Steffen; Röckendorf, Niels; Fránek, Milan; Frey, Andreas

    2011-08-17

    The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.

  4. Design and Application of Synthetic Receptors for Recognition of Methylated Lysine and Supramolecular Affinity Labeling

    NASA Astrophysics Data System (ADS)

    Gober, Isaiah Nathaniel

    This dissertation involves the design and synthesis of new synthetic receptors and their application in the molecular recognition of methylated lysine and their use as tools for chemical biology. The dissertation is divided into four parts. The first section focuses on the development of a novel labeling method that is based on ligand-directed affinity labeling principles. In this labeling method, a synthetic receptor that binds to trimethyl lysine (Kme3) is attached through a linker to an electrophilic tag group that can react with a nucleophilic amine in a histone peptide. This affinity labeling probe, which we called CX4-ONBD, is equipped with an electrophilic tag that allows for turn-on fluorescence labeling of Kme3 histone peitdes. We show that the probe gives a pronounced turn-on fluorescence response when it is incubated with a histone peptide that contains Kme3 and a nearby reactive lysine. This probe also displays >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. This represents the first time a synthetic receptor has been used for affinity labeling purposes, and it also expands on the chemical toolkit that is available for sensing PTMs like lysine methylation. In the second section, the supramolecular affinity labeling method that was optimized using CX4-ONBD was applied to the development of a real-time assay for measuring enzymatic activity. More specifically, the probe was used to create a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Most commercial kits for HDAC activity have limited substrate scope, and other common methods used for characterizing enzymatic activity often require chromatographic separation and are therefore not high-throughput. This small molecule receptor-mediated affinity labeling strategy allowed for facile readout of HDAC activity and inhibition. Overall, this application of supramolecular affinity labeling expands on the

  5. Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

    PubMed

    Roelofs, Anke J; Stewart, Charlotte A; Sun, Shuting; Błażewska, Katarzyna M; Kashemirov, Boris A; McKenna, Charles E; Russell, R Graham G; Rogers, Michael J; Lundy, Mark W; Ebetino, Frank H; Coxon, Fraser P

    2012-04-01

    Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the

  6. Peptide affinity labels for thrombin and other trypsin-like proteases

    DOEpatents

    Shaw, E.N.; Kettner, C.A.

    1982-03-09

    A peptide affinity label is disclosed of the formula (I): as given in the patent wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C[sub 1]--C[sub 4] alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C[sub 1]--C[sub 6] acyl, and Q--(A)--[sub n], wherein Q = hydrogen, aroyl, or C[sub 1]--C[sub 6] acyl, n = 1--10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereofcontaining, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH[sub 2]--, --CH[sub 2]--CH[sub 2]--, --CH[sub 2]--CH[sub 2]--CH[sub 2]--, --CH[double bond]CH-- and --CH(OH)--CH[sub 2]. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent. 2 figs.

  7. Peptide affinity labels for thrombin and other trypsin-like proteases

    DOEpatents

    Shaw, Elliott N.; Kettner, Charles A.

    1982-03-09

    A peptide affinity label of the formula (I): ##STR1## wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C.sub.1 -C.sub.4 alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C.sub.1 -C.sub.6 acyl, and Q--(A)--.sub.n, wherein Q=hydrogen, aroyl, or C.sub.1 -C.sub.6 acyl, n=1-10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereof-containing, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH.sub.2 --, --CH.sub.2 --CH.sub.2 --,--CH.sub.2 --CH.sub.2 --CH.sub.2 --, --CH.dbd.CH-- and --CH(OH)--CH.sub.2. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent.

  8. [Serological affinity of some species of nonpathogenic corynebacteria].

    PubMed

    Furtat, I M; Nohina, T M; Mikhal's'kyĭ, L O; Vedenieieva, O A

    2002-01-01

    Serological peculiarities of the species strains Corynebacterium glutamicum, C. ammoniagenes, C. vitaeruminis, C. variabilis and strain of Corynebacterium sp. (Brevibacterium stationis) UCM Ac-719 have been investigated with the help of immunoenzyme analysis ELISA with the use of mice immune serum, specific to C. ammoniagenes UCM Ac-732T, C. vitaeruminis UCM Ac-718T, C. variabilis UCM Ac-717T, C. glutamicum UCM Ac-733 and Corynebacterium sp. UCM Ac-719. It has been established that the species of nonpathogenic corynebacteria differ between themselves as to the degree of serological affinity. C. variabilis, C. ammoniagenes and C. glutamicum are the least similar as to this indication. Weak antigenic relations have been revealed in C. vitaeruminis and C. ammoniagenes. The latter displayed the higher, as compared with other strains, affinity for Corynebacterium sp. UCM Ac-719. The highest degree of serological affinity within the species was registered in strains C. glutamicum and C. variabilis. Data obtained evidence that the ELISA method permits conducting the high-reliability species diagnosis of nonpathogenic corynebacteria on the basis of their antigenic characteristics.

  9. Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

    NASA Astrophysics Data System (ADS)

    Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

    2009-02-01

    Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

  10. Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

    PubMed

    Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

    2002-02-01

    We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

  11. Thermal affinity as the dominant factor changing Mediterranean fish abundances.

    PubMed

    Givan, Or; Edelist, Dor; Sonin, Oren; Belmaker, Jonathan

    2018-01-01

    Recent decades have seen profound changes in species abundance and community composition. In the marine environment, the major anthropogenic drivers of change comprise exploitation, invasion by nonindigenous species, and climate change. However, the magnitude of these stressors has been widely debated and we lack empirical estimates of their relative importance. In this study, we focused on Eastern Mediterranean, a region exposed to an invasion of species of Red Sea origin, extreme climate change, and high fishing pressure. We estimated changes in fish abundance using two fish trawl surveys spanning a 20-year period, and correlated these changes with estimated sensitivity of species to the different stressors. We estimated sensitivity to invasion using the trait similarity between indigenous and nonindigenous species; sensitivity to fishing using a published composite index based on the species' life-history; and sensitivity to climate change using species climatic affinity based on occurrence data. Using both a meta-analytical method and random forest analysis, we found that for shallow-water species the most important driver of population size changes is sensitivity to climate change. Species with an affinity to warm climates increased in relative abundance and species with an affinity to cold climates decreased suggesting a strong response to warming local sea temperatures over recent decades. This decrease in the abundance of cold-water-associated species at the trailing "warm" end of their distribution has been rarely documented. Despite the immense biomass of nonindigenous species and the presumed high fishing pressure, these two latter factors seem to have only a minor role in explaining abundance changes. The decline in abundance of indigenous species of cold-water origin indicates a future major restructuring of fish communities in the Mediterranean in response to the ongoing warming, with unknown impacts on ecosystem function. © 2017 John Wiley & Sons

  12. Affinity Versus Label-Free Isolation of Circulating Tumor Cells: Who Wins?

    PubMed

    Murlidhar, Vasudha; Rivera-Báez, Lianette; Nagrath, Sunitha

    2016-09-01

    The study of circulating tumor cells (CTCs) has been made possible by many technological advances in their isolation. Their isolation has seen many fronts, but each technology brings forth a new set of challenges to overcome. Microfluidics has been a key player in the capture of CTCs and their downstream analysis, with the aim of shedding light into their clinical application in cancer and metastasis. Researchers have taken diverging paths to isolate such cells from blood, ranging from affinity-based isolation targeting surface antigens expressed on CTCs, to label-free isolation taking advantage of the size differences between CTCs and other blood cells. For both major groups, many microfluidic technologies have reported high sensitivity and specificity for capturing CTCs. However, the question remains as to the superiority among these two isolation techniques, specifically to identify different CTC populations. This review highlights the key aspects of affinity and label-free microfluidic CTC technologies, and discusses which of these two would be the highest benefactor for the study of CTCs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine.

    PubMed

    Köhrle, J; Rasmussen, U B; Rokos, H; Leonard, J L; Hesch, R D

    1990-04-15

    125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27

  14. Automatic face naming by learning discriminative affinity matrices from weakly labeled images.

    PubMed

    Xiao, Shijie; Xu, Dong; Wu, Jianxin

    2015-10-01

    Given a collection of images, where each image contains several faces and is associated with a few names in the corresponding caption, the goal of face naming is to infer the correct name for each face. In this paper, we propose two new methods to effectively solve this problem by learning two discriminative affinity matrices from these weakly labeled images. We first propose a new method called regularized low-rank representation by effectively utilizing weakly supervised information to learn a low-rank reconstruction coefficient matrix while exploring multiple subspace structures of the data. Specifically, by introducing a specially designed regularizer to the low-rank representation method, we penalize the corresponding reconstruction coefficients related to the situations where a face is reconstructed by using face images from other subjects or by using itself. With the inferred reconstruction coefficient matrix, a discriminative affinity matrix can be obtained. Moreover, we also develop a new distance metric learning method called ambiguously supervised structural metric learning by using weakly supervised information to seek a discriminative distance metric. Hence, another discriminative affinity matrix can be obtained using the similarity matrix (i.e., the kernel matrix) based on the Mahalanobis distances of the data. Observing that these two affinity matrices contain complementary information, we further combine them to obtain a fused affinity matrix, based on which we develop a new iterative scheme to infer the name of each face. Comprehensive experiments demonstrate the effectiveness of our approach.

  15. Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins.

    PubMed

    Konziase, Benetode

    2015-08-01

    We studied the target proteins of artemisinin in Trypanosoma brucei brucei using the affinity-labeling method. We designed and synthesized four biotinylated probes of artemisinin for use as molecular tools. Their in vitro trypanocidal activities (data not shown) proved that they mimicked the biological action of artemisinin. We assessed the chemical stability for all of the probes in the parasite culture medium and lysate using reversed-phase high-performance liquid chromatography (HPLC). After 3-h incubations, the probes remained undecomposed in a range of 40 to 65% in the parasite culture medium, whereas approximately 80% of the probes remained stable in the parasite lysate. Using liquid chromatography mass spectrometry (LC-MS), we demonstrated that, with respect to all of the probes, uptakes into the parasite ranging from 81 to 96% occurred after 30-min incubations. In a competitive binding assay between artemisinin and the four biotinylated probes, we searched for the trypanosomal target protein of artemisinin. Consequently, we observed that only the diazirine-free probe 5 could provide the desired result with high affinity-labeling efficiency. Using the horseradish peroxidase-tagged streptavidin-biotin method, we showed that artemisinin could specifically bind to candidate target proteins of approximately 60, 40, and 39 kDa. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

    PubMed

    Chang, Tsung-Che; Adak, Avijit K; Lin, Ting-Wei; Li, Pei-Jhen; Chen, Yi-Ju; Lai, Chain-Hui; Liang, Chien-Fu; Chen, Yu-Ju; Lin, Chun-Cheng

    2016-03-15

    The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

    NASA Astrophysics Data System (ADS)

    Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

    2011-10-01

    Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

  18. Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies.

    PubMed

    Ahlqvist, Josefin; Kumar, Ashok; Sundström, Heléne; Ledung, Erika; Hörnsten, E Gunnar; Enfors, Sven-Olof; Mattiasson, Bo

    2006-03-23

    A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

  19. High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies

    PubMed Central

    Anthony, Kelsey C.; You, Changjiang; Piehler, Jacob; Pomeranz Krummel, Daniel A.

    2014-01-01

    SUMMARY There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle (AuNPtris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of AuNPtris-NTA labeled proteins by electron microscopy is further ensured by the reagent’s short conformationally restricted linker. We have employed AuNPtris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. AuNPtris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our new labeling reagent should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. PMID:24560806

  20. Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

    PubMed

    Kudlow, J E; Leung, Y

    1984-06-15

    Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP

  1. Sequence2Vec: a novel embedding approach for modeling transcription factor binding affinity landscape.

    PubMed

    Dai, Hanjun; Umarov, Ramzan; Kuwahara, Hiroyuki; Li, Yu; Song, Le; Gao, Xin

    2017-11-15

    An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these hidden Markov models into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA datasets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods. Our program is freely available at https://github.com/ramzan1990/sequence2vec. xin.gao@kaust.edu.sa or lsong@cc.gatech.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  2. Mixed infection of Galleria mellonella with two entomopathogenic nematode (Nematoda: Rhabditida) species: Steinernema affine benefits from the presence of Steinernema kraussei.

    PubMed

    Půza, Vladimír; Mrácek, Zdenek

    2009-09-01

    The interactions of two sympatric entomopathogenic nematodes Steinernema affine and Steinernema. kraussei were studied in a series of laboratory experiments. Single species, simultaneous and sequential infections of Galleria mellonella were performed in Eppendorf tubes and the invasion rate, nematode progeny production and the number of hosts producing nematode progeny were observed. The invasion rate of S. affine was not affected by the mixed infection whereas the invasion of the latter species was strongly reduced. S. affine out-competed S. kraussei in all treatments and the progeny production of the latter species occurred only sporadically. In comparison to single species infections, per-host progeny production of S. affine was affected only in the treatments with a low dose of S. affine, where it was 30-50% lowered. Generally, in the presence of the latter species, S. affine was able to infect and multiply in a higher number of hosts in comparison to single species infection, especially at a low inoculation rate. S. affine invaded and multiplied also in hosts already infected and even killed by S. kraussei producing a normal amount of progeny. Generally the results suggest that the interactions between steinernematid species can be more complex, including a positive effect of one species on another.

  3. Fibrin matrices with affinity-based delivery systems and neurotrophic factors promote functional nerve regeneration.

    PubMed

    Wood, Matthew D; MacEwan, Matthew R; French, Alexander R; Moore, Amy M; Hunter, Daniel A; Mackinnon, Susan E; Moran, Daniel W; Borschel, Gregory H; Sakiyama-Elbert, Shelly E

    2010-08-15

    Glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity-based delivery of GDNF or NGF from fibrin-filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity-based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury.

  4. Species diversity and phylogeographical affinities of the Branchiopoda (Crustacea) of Churchill, Manitoba, Canada.

    PubMed

    Jeffery, Nicholas W; Elías-Gutiérrez, Manuel; Adamowicz, Sarah J

    2011-01-01

    The region of Churchill, Manitoba, contains a wide variety of habitats representative of both the boreal forest and arctic tundra and has been used as a model site for biodiversity studies for nearly seven decades within Canada. Much previous work has been done in Churchill to study the Daphnia pulex species complex in particular, but no study has completed a wide-scale survey on the crustacean species that inhabit Churchill's aquatic ecosystems using molecular markers. We have employed DNA barcoding to study the diversity of the Branchiopoda (Crustacea) in a wide variety of freshwater habitats and to determine the likely origins of the Churchill fauna following the last glaciation. The standard animal barcode marker (COI) was sequenced for 327 specimens, and a 3% divergence threshold was used to delineate potential species. We found 42 provisional and valid branchiopod species from this survey alone, including several cryptic lineages, in comparison with the 25 previously recorded from previous ecological works. Using published sequence data, we explored the phylogeographic affinities of Churchill's branchiopods, finding that the Churchill fauna apparently originated from all directions from multiple glacial refugia (including southern, Beringian, and high arctic regions). Overall, these microcrustaceans are very diverse in Churchill and contain multiple species complexes. The present study introduces among the first sequences for some understudied genera, for which further work is required to delineate species boundaries and develop a more complete understanding of branchiopod diversity over a larger spatial scale.

  5. Species Diversity and Phylogeographical Affinities of the Branchiopoda (Crustacea) of Churchill, Manitoba, Canada

    PubMed Central

    Jeffery, Nicholas W.; Elías-Gutiérrez, Manuel; Adamowicz, Sarah J.

    2011-01-01

    The region of Churchill, Manitoba, contains a wide variety of habitats representative of both the boreal forest and arctic tundra and has been used as a model site for biodiversity studies for nearly seven decades within Canada. Much previous work has been done in Churchill to study the Daphnia pulex species complex in particular, but no study has completed a wide-scale survey on the crustacean species that inhabit Churchill's aquatic ecosystems using molecular markers. We have employed DNA barcoding to study the diversity of the Branchiopoda (Crustacea) in a wide variety of freshwater habitats and to determine the likely origins of the Churchill fauna following the last glaciation. The standard animal barcode marker (COI) was sequenced for 327 specimens, and a 3% divergence threshold was used to delineate potential species. We found 42 provisional and valid branchiopod species from this survey alone, including several cryptic lineages, in comparison with the 25 previously recorded from previous ecological works. Using published sequence data, we explored the phylogeographic affinities of Churchill's branchiopods, finding that the Churchill fauna apparently originated from all directions from multiple glacial refugia (including southern, Beringian, and high arctic regions). Overall, these microcrustaceans are very diverse in Churchill and contain multiple species complexes. The present study introduces among the first sequences for some understudied genera, for which further work is required to delineate species boundaries and develop a more complete understanding of branchiopod diversity over a larger spatial scale. PMID:21610864

  6. Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

    PubMed

    Sarpong, Kwabena; Bose, Ron

    2017-03-15

    A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies.

    PubMed

    Wright, Michael; Miller, Andrew D

    2006-02-15

    Tandem synthetic-biosynthetic procedures were used to prepare two novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies. These compounds (dial-mant-Ap4A and azido-mant-Ap4A) are shown to clearly distinguish known Ap4A-binding proteins from Escherichia coli (LysU and GroEL) and a variety of other control proteins. Successful labelling of chaperonin GroEL appears to be allosteric with respect to the well-characterized adenosine 5'-triphosphate (ATP)-binding site, suggesting that GroEL possesses a distinct Ap4A-binding site.

  8. Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

    PubMed

    Lopata, Anna; Hughes, Ruth; Tiede, Christian; Heissler, Sarah M; Sellers, James R; Knight, Peter J; Tomlinson, Darren; Peckham, Michelle

    2018-04-26

    Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

  9. A sensitive and selective resonance Rayleigh scattering method for quick detection of avidin using affinity labeling Au nanoparticles

    NASA Astrophysics Data System (ADS)

    Wang, Qi; Huang, Xi; Fu, Xuan; Deng, Huan; Ma, Meihu; Cai, Zhaoxia

    2016-06-01

    Avidin is a glycoprotein with antinutritional property, which should be limited in daily food. We developed an affinity biosensor system based on resonance Rayleigh scattering (RRS) and using affinity biotin labeling Au nanoparticles (AuNPs). This method was selective and sensitive for quick avidin detection due to the avidin-biotin affinitive interaction. Under optimal conditions, RRS intensity of biotin-AuNPs increase linearly with an increasing concentration of avidin from 5 to 160 ng/mL. The lower limit of detection was 0.59 ng/mL. This rapid and selective avidin detection method was used in synthetic samples and egg products with recoveries of between 102.97 and 107.92%, thereby demonstrating the feasible and practical application of this assay.

  10. 76 FR 69585 - Testing and Labeling Pertaining to Product Certification Regarding Representative Samples for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-08

    ... 1107 Testing and Labeling Pertaining to Product Certification Regarding Representative Samples for... [CPSC Docket No. CPSC-2011-0082] Testing and Labeling Pertaining to Product Certification Regarding... to Product Certification.'' The proposed rule was intended to implement what was then known as...

  11. Adjoint affine fusion and tadpoles

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Urichuk, Andrew, E-mail: andrew.urichuk@uleth.ca; Walton, Mark A., E-mail: walton@uleth.ca; International School for Advanced Studies

    2016-06-15

    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 writtenmore » 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.« less

  12. High sensitivity and high Q-factor nanoslotted parallel quadrabeam photonic crystal cavity for real-time and label-free sensing

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Daquan; State Key Laboratory of Information Photonics and Optical Communications, School of Information and Communication Engineering, Beijing University of Posts and Telecommunications, Beijing 100876; School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138

    We experimentally demonstrate a label-free sensor based on nanoslotted parallel quadrabeam photonic crystal cavity (NPQC). The NPQC possesses both high sensitivity and high Q-factor. We achieved sensitivity (S) of 451 nm/refractive index unit and Q-factor >7000 in water at telecom wavelength range, featuring a sensor figure of merit >2000, an order of magnitude improvement over the previous photonic crystal sensors. In addition, we measured the streptavidin-biotin binding affinity and detected 10 ag/mL concentrated streptavidin in the phosphate buffered saline solution.

  13. Distribution, habitat affinities and phenology of the Micrargus herbigradus-species group (Araneae: Linyphiidae) in Poland.

    PubMed

    Wiśniewski, Konrad; Rozwałka, Robert; Wesołowska, Wanda

    2018-01-01

    We review the known information on the distribution and habitat affinities of the Micrargus herbigradus -species group in Poland. The analysis is based on a thorough literature survey, our own materials, and verification of some older collections. We give new diagnostic drawings and review the characters that are useful in identification of species within the group. Three species are present in Poland: M. herbigradus (Blackwall, 1854), M. apertus (O.-P. Cambridge, 1870) and M. georgescuae Millidge, 1976. The latter is recorded for the first time in the country, and we add numerous new localities for the two former species. Micrargus herbigradus is common and widespread in Poland, living in various habitats, with only a slight preference to forests. In contrast, M. apertus is widely distributed but rarely found, while its affinity to forests is the highest within the group. The records of this species are most numerous in lowland forests (up to c. 300 m a.s.l), but it can also be found at higher altitudes. M. georgescuae is found only in montane habitats, both in the Sudetes and the Carpathian Mountains, from above 650 m a.s.l. The adults of all three species occur the whole year round, but seem to be most abundant in May and June.

  14. Synthesis and characterization of (18)F-labeled active site inhibited factor VII (ASIS).

    PubMed

    Erlandsson, Maria; Nielsen, Carsten H; Jeppesen, Troels E; Kristensen, Jesper B; Petersen, Lars C; Madsen, Jacob; Kjaer, Andreas

    2015-05-15

    Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Label-Free Bioanalyte Detection from Nanometer to Micrometer Dimensions-Molecular Imprinting and QCMs †.

    PubMed

    Mujahid, Adnan; Mustafa, Ghulam; Dickert, Franz L

    2018-06-01

    Modern diagnostic tools and immunoassay protocols urges direct analyte recognition based on its intrinsic behavior without using any labeling indicator. This not only improves the detection reliability, but also reduces sample preparation time and complexity involved during labeling step. Label-free biosensor devices are capable of monitoring analyte physiochemical properties such as binding sensitivity and selectivity, affinity constants and other dynamics of molecular recognition. The interface of a typical biosensor could range from natural antibodies to synthetic receptors for example molecular imprinted polymers (MIPs). The foremost advantages of using MIPs are their high binding selectivity comparable to natural antibodies, straightforward synthesis in short time, high thermal/chemical stability and compatibility with different transducers. Quartz crystal microbalance (QCM) resonators are leading acoustic devices that are extensively used for mass-sensitive measurements. Highlight features of QCM devices include low cost fabrication, room temperature operation, and most importantly ability to monitor extremely low mass shifts, thus potentially a universal transducer. The combination of MIPs with quartz QCM has turned out as a prominent sensing system for label-free recognition of diverse bioanalytes. In this article, we shall encompass the potential applications of MIP-QCM sensors exclusively label-free recognition of bacteria and virus species as representative micro and nanosized bioanalytes.

  16. Biomarker Candidates of Chlamydophila pneumoniae Proteins and Protein Fragments Identified by Affinity-Proteomics Using FTICR-MS and LC-MS/MS

    NASA Astrophysics Data System (ADS)

    Susnea, Iuliana; Bunk, Sebastian; Wendel, Albrecht; Hermann, Corinna; Przybylski, Michael

    2011-04-01

    We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.

  17. Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

    PubMed Central

    Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

    2016-01-01

    Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

  18. Scaffold design of trivalent chelator heads dictates high-affinity and stable His-tagged protein labeling in vitro and in cellulo.

    PubMed

    Gatterdam, Karl; Joest, Eike F; Gatterdam, Volker; Tampé, Robert

    2018-05-29

    Small chemical/biological interaction pairs are at the forefront in tracing proteins' function and interaction at high signal-to-background ratio in cellular pathways. Pharma ventures have eager plans to develop trisNTA probes for in vitro and in vivo screening of His-tagged protein targets. However, the optimal design of scaffold, linker, and chelator head yet deserves systematic investigations to achieve highest affinity and kinetic stability for in vitro and especially cell applications. In this study, we report on a library of N-nitrilotriacetic acid (NTA) based multivalent chelator heads (MCHs) built up on linear, cyclic, and dendritic scaffolds and contrast these with regard to their binding affinity and stability for labeling of cellular His-tagged proteins. Furthermore, we assign a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we describe fundamental differences between the MCH scaffold and define a cyclic trisNTA chelator, which displays the highest affinity and kinetic stability of all reversible, low-molecular weight interaction pairs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

    PubMed Central

    Tulloch, Lindsay B.; Menzies, Stefanie K.; Fraser, Andrew L.; Gould, Eoin R.; King, Elizabeth F.; Zacharova, Marija K.; Florence, Gordon J.

    2017-01-01

    Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or

  20. Preliminary Crystallographic Study of Hemoglobin from Buffalo (Bubalus bubalis): A Low Oxygen Affinity Species.

    PubMed

    Balasubramanian, Moovarkumudalvan; Moorthy, Ponnuraj Sathya; Neelagandan, Kamariah; Ponnuswamy, Mondikalipudur Nanjappa Gounder

    2009-01-01

    Hemoglobin is a tetrameric, iron-containing metalloprotein, which plays a vital role in the transportation of oxygen from lungs to tissues and carbon dioxide back to lungs. Though good amount of work has already been done on hemoglobins, the scarcity of data on three dimensional structures pertaining to low oxygen affinity hemoglobins from mammalian species, motivated our group to work on this problem specifically. Herein, we report the preliminary crystallographic analysis of buffalo hemoglobin, which belongs to low oxygen affinity species. The buffalo blood was collected, purified by anion exchange chromatography and crystallized with PEG 3350 using 50mM phosphate buffer at pH 6.7 as a precipitant by hanging drop vapor diffusion method. Data collection was carried out using mar345dtb image plate detector system. Buffalo hemoglobin crystallizes in orthorhombic space group P2(1)2(1)2(1) with one whole biological molecule (alpha2beta2) in the asymmetric unit with cell dimensions a=63.064A, b=74.677A, c=110.224A.

  1. Label-free proteomics assisted by affinity enrichment for elucidating the chemical reactivity of the liver mitochondrial proteome toward adduction by the lipid electrophile 4-hydroxy-2-nonenal (HNE)

    NASA Astrophysics Data System (ADS)

    Maier, Claudia

    2016-03-01

    The analysis of oxidative stress-induced post-translational modifications remains challenging due to the chemical diversity of these modifications, the possibility of the presence of positional isomers and the low stoichiometry of the modified proteins present in a cell or tissue proteome. Alcoholic liver disease (ALD) is a multifactorial disease in which mitochondrial dysfunction and oxidative stress have been identified as being critically involved in the progression of the disease from steatosis to cirrhosis. Ethanol metabolism leads to increased levels of reactive oxygen species (ROS), glutathione depletion and lipid peroxidation. Posttranslational modification of proteins by electrophilic products of lipid peroxidation has been associated with governing redox-associated signaling mechanisms, but also as contributing to protein dysfunction leading to organelle and liver injury. In particular the prototypical α,β-unsaturated aldehyde, 4-hydroxy-2-nonenal (HNE), has been extensively studied as marker of increased oxidative stress in hepatocytes. In this study, we combined a LC-MS label-free quantification method and affinity enrichment to assess the dose-dependent insult by HNE on the proteome of rat liver mitochondria. We used a carbonyl-selective probe, the ARP probe, to label HNE-protein adducts and to perform affinity capture at the protein level. Using LC-MS to obtain protein abundance estimates, a list of protein targets was obtained with increasing concentration of HNE used in the exposure studies. In parallel, we performed affinity capture at the peptide level to acquire site-specific information. Examining the concentration-dependence of the protein modifications, we observed distinct reactivity profiles for HNE-protein adduction. Pathway analysis indicated that proteins associated with metabolic processes, including amino acid, fatty acid and glyoxylate and dicarboxylate metabolism, bile acid synthesis and TCA cycle, showed enhanced reactivity to HNE

  2. Label-free quantitative 1H NMR spectroscopy to study low-affinity ligand–protein interactions in solution: A contribution to the mechanism of polyphenol-mediated astringency

    PubMed Central

    Delius, Judith; Frank, Oliver

    2017-01-01

    Nuclear magnetic resonance (NMR) spectroscopy is well-established in assessing the binding affinity between low molecular weight ligands and proteins. However, conventional NMR-based binding assays are often limited to small proteins of high purity and may require elaborate isotopic labeling of one of the potential binding partners. As protein–polyphenol complexation is assumed to be a key event in polyphenol-mediated oral astringency, here we introduce a label-free, ligand-focused 1H NMR titration assay to estimate binding affinities and characterize soluble complex formation between proteins and low molecular weight polyphenols. The method makes use of the effects of NMR line broadening due to protein–ligand interactions and quantitation of the non-bound ligand at varying protein concentrations by quantitative 1H NMR spectroscopy (qHNMR) using electronic reference to access in vivo concentration (ERETIC 2). This technique is applied to assess the interaction kinetics of selected astringent tasting polyphenols and purified mucin, a major lubricating glycoprotein of human saliva, as well as human whole saliva. The protein affinity values (BC50) obtained are subsequently correlated with the intrinsic mouth-puckering, astringent oral sensation imparted by these compounds. The quantitative NMR method is further exploited to study the effect of carboxymethyl cellulose, a candidate “anti-astringent” protein binding antagonist, on the polyphenol–protein interaction. Consequently, the NMR approach presented here proves to be a versatile tool to study the interactions between proteins and low-affinity ligands in solution and may find promising applications in the discovery of bioactives. PMID:28886151

  3. Affimer proteins are versatile and renewable affinity reagents

    PubMed Central

    Tiede, Christian; Bedford, Robert; Heseltine, Sophie J; Smith, Gina; Wijetunga, Imeshi; Ross, Rebecca; AlQallaf, Danah; Roberts, Ashley PE; Balls, Alexander; Curd, Alistair; Hughes, Ruth E; Martin, Heather; Needham, Sarah R; Zanetti-Domingues, Laura C; Sadigh, Yashar; Peacock, Thomas P; Tang, Anna A; Gibson, Naomi; Kyle, Hannah; Platt, Geoffrey W; Ingram, Nicola; Taylor, Thomas; Coletta, Louise P; Manfield, Iain; Knowles, Margaret; Bell, Sandra; Esteves, Filomena; Maqbool, Azhar; Prasad, Raj K; Drinkhill, Mark; Bon, Robin S; Patel, Vikesh; Goodchild, Sarah A; Martin-Fernandez, Marisa; Owens, Ray J; Nettleship, Joanne E; Webb, Michael E; Harrison, Michael; Lippiat, Jonathan D; Ponnambalam, Sreenivasan; Peckham, Michelle; Smith, Alastair; Ferrigno, Paul Ko; Johnson, Matt; McPherson, Michael J; Tomlinson, Darren Charles

    2017-01-01

    Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001 PMID:28654419

  4. Recognition of genetically modified product based on affinity propagation clustering and terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, Jianjun; Kan, Jianquan

    2018-04-01

    In this paper, based on the terahertz spectrum, a new identification method of genetically modified material by support vector machine (SVM) based on affinity propagation clustering is proposed. This algorithm mainly uses affinity propagation clustering algorithm to make cluster analysis and labeling on unlabeled training samples, and in the iterative process, the existing SVM training data are continuously updated, when establishing the identification model, it does not need to manually label the training samples, thus, the error caused by the human labeled samples is reduced, and the identification accuracy of the model is greatly improved.

  5. Category labels versus feature labels: category labels polarize inferential predictions.

    PubMed

    Yamauchi, Takashi; Yu, Na-Yung

    2008-04-01

    What makes category labels different from feature labels in predictive inference? This study suggests that category labels tend to make inductive reasoning polarized and homogeneous. In two experiments, participants were shown two schematic pictures of insects side by side and predicted the value of a hidden feature of one insect on the basis of the other insect. Arbitrary verbal labels were shown above the two pictures, and the meanings of the labels were manipulated in the instructions. In one condition, the labels represented the category membership of the insects, and in the other conditions, the same labels represented attributes of the insects. When the labels represented category membership, participants' responses became substantially polarized and homogeneous, indicating that the mere reference to category membership can modify reasoning processes.

  6. Differentiation of some species of Neisseriaceae and other bacterial groups by DNA-DNA hybridization.

    PubMed

    Tønjum, T; Bukholm, G; Bøvre, K

    1989-05-01

    DNA-DNA hybridization using total genomic DNA probes may represent a way of differentiating between miscellaneous bacterial species. This was studied with type and reference strains of 20 species in Moraxella, Kingella, and other selected Gram-negative groups. Both radioactive and biotin labelling were employed. Most of the species examined were easily distinguished, such as Moraxella (Branhamella) catarrhalis, M.(B.) ovis, M. atlantae, M. phenylpyruvica, M. osloensis, Neisseria elongata, N. meningitidis, Kingella kingae, K. indologenes, K. dentrificans, Oligella urethralis, Eikenella corrodens, Cardiobacterium hominis, Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Gardnerella vaginalis, and DF-2. This reflected the extent of the genetic distances between them as a basis for identification by hybridization. There was some clustering in the Moraxella group. Especially the closely related Moraxella nonliquefaciens, M. lacunata and M. bovis showed strong hybridization affinities. This leads to potential problems in distinguishing these three species from each other by DNA-DNA hybridization with total genomic probes alone.

  7. Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

    PubMed Central

    Zhao, Wenjing; McCallum, Scott A.; Xiao, Zhongping; Zhang, Fuming; Linhardt, Robert J.

    2011-01-01

    Heparin and heparan sulphate (HS) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS-binding to vascular endothelial growth factor (VEGF) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and that a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that VEGF binding affinity likely depends on the specific structural features of these oligosaccharides including their degree of sulphation and sugar ring stereochemistry and conformation. Notably, the unique 3-O-sulpho group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs. PMID:21658003

  8. The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

    PubMed

    Yandell, C A; Dunbar, A J; Wheldrake, J F; Upton, Z

    1999-09-17

    The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

  9. Exploring Strategies for Labeling Viruses with Gold Nanoclusters through Non-equilibrium Molecular Dynamics Simulations.

    PubMed

    Pohjolainen, Emmi; Malola, Sami; Groenhof, Gerrit; Häkkinen, Hannu

    2017-09-20

    Biocompatible gold nanoclusters can be utilized as contrast agents in virus imaging. The labeling of viruses can be achieved noncovalently but site-specifically by linking the cluster to the hydrophobic pocket of a virus via a lipid-like pocket factor. We have estimated the binding affinities of three different pocket factors of echovirus 1 (EV1) in molecular dynamics simulations combined with non-equilibrium free-energy calculations. We have also studied the effects on binding affinities with a pocket factor linked to the Au 102 pMBA 44 nanocluster in different protonation states. Although the absolute binding affinities are over-estimated for all the systems, the trend is in agreement with recent experiments.3 Our results suggest that the natural pocket factor (palmitic acid) can be replaced by molecules pleconaril (drug) and its derivative Kirtan1 that have higher estimated binding affinities. Our results also suggest that including the gold nanocluster does not decrease the affinity of the pocket factor to the virus, but the affinity is sensitive to the protonation state of the nanocluster, i.e., to pH conditions. The methodology introduced in this work helps in the design of optimal strategies for gold-virus bioconjugation for virus detection and manipulation.

  10. The identification of high-affinity G protein-coupled receptor ligands from large combinatorial libraries using multicolor quantum dot-labeled cell-based screening

    PubMed Central

    Fu, Junjie; Lee, Timothy; Qi, Xin

    2014-01-01

    G protein-coupled receptors (GPCRs), which are involved in virtually every biological process, constitute the largest family of transmembrane receptors. Many top-selling and newly approved drugs target GPCRs. In this review, we aim to recapitulate efforts and progress in combinatorial library-assisted GPCR ligand discovery, particularly focusing on one-bead-one-compound library synthesis and quantum dot-labeled cell-based assays, which both effectively enhance the rapid identification of GPCR ligands with higher affinity and specificity. PMID:24941874

  11. [Interaction of human factor X with thromboplastin].

    PubMed

    Kiselev, S V; Zubairov, D M; Timarbaev, V N

    2003-01-01

    The binding of 125I-labeled human factor X to native and papaine-treated tissue tromboplastin in the presence of CaCl2 or EDTA was studied. The Scatchard analysis suggests the existence of high (Kd=l,8 x10(-9) M) and low affinity binding sites on the thromboplastin surface. The removal of Ca2+ reduced affinity of factor X to the high affinity sites. This was accompanied by some increase of their number. Proteolysis by papaine decreased affinity of high affinity sites and caused the increase of their number in the presence of Ca2+. In the absence of Ca2+ the affinity remained unchanged, but the number of sites decreased. At low concentrations of factor X positive cooperativity for high affinity binding sites was observed. It did not depend on the presence of Ca2+. The results indirectly confirm the role of hydrophobic interactons in Ca2+ dependent binding of factor X to thromboplastin and the fact that heterogeneity of this binding is determined by mesophase structure of the thromboplastin phospholipids.

  12. Acoustic classification of multiple simultaneous bird species: a multi-instance multi-label approach

    Treesearch

    F. Briggs; B. Lakshminarayanan; L. Neal; X.Z. Fern; R. Raich; S.F. Hadley; A.S. Hadley; M.G. Betts

    2012-01-01

    Although field-collected recordings typically contain multiple simultaneously vocalizing birds of different species, acoustic species classification in this setting has received little study so far. This work formulates the problem of classifying the set of species present in an audio recording using the multi-instance multi-label (MIML) framework for machine learning...

  13. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

    PubMed Central

    Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

    2016-01-01

    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

  14. Leaf trait variations associated with habitat affinity of tropical karst tree species.

    PubMed

    Geekiyanage, Nalaka; Goodale, Uromi Manage; Cao, Kunfang; Kitajima, Kaoru

    2018-01-01

    Karst hills, that is, jagged topography created by dissolution of limestone and other soluble rocks, are distributed extensively in tropical forest regions, including southern parts of China. They are characterized by a sharp mosaic of water and nutrient availability, from exposed hilltops with poor soil development to valleys with occasional flooding, to which trees show species-specific distributions. Here we report the relationship of leaf functional traits to habitat preference of tropical karst trees. We described leaf traits of 19 tropical tree species in a seasonal karst rainforest in Guangxi Province, China, 12 species in situ and 13 ex situ in a non-karst arboretum, which served as a common garden, with six species sampled in both. We examined how the measured leaf traits differed in relation to species' habitat affinity and evaluated trait consistency between natural habitats vs . the arboretum. Leaf mass per area (LMA) and optical traits (light absorption and reflectance characteristics between 400 and 1,050 nm) showed significant associations with each other and habitats, with hilltop species showing high values of LMA and low values of photochemical reflectance index (PRI). For the six species sampled in both the karst forest and the arboretum, LMA, leaf dry matter content, stomatal density, and vein length per area showed inconsistent within-species variations, whereas some traits (stomatal pore index and lamina thickness) were similar between the two sites. In conclusion, trees specialized in exposed karst hilltops with little soils are characterized by thick leaves with high tissue density indicative of conservative resources use, and this trait syndrome could potentially be sensed remotely with PRI.

  15. Synthesis, characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels.

    PubMed

    Pragl, Bernt; Koschak, Alexandra; Trieb, Maria; Obermair, Gerald; Kaufmann, Walter A; Gerster, Uli; Blanc, Eric; Hahn, Christoph; Prinz, Heino; Schütz, Gerhard; Darbon, Herve; Gruber, Hermann J; Knaus, Hans-Günther

    2002-01-01

    Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.

  16. Purification, crystallization and preliminary X-ray diffraction studies on goat (Capra hircus) hemoglobin - a low oxygen affinity species.

    PubMed

    Moorthy, Ponnuraj Sathya; Neelagandan, Kamariah; Balasubramanian, Moovarkumudalvan; Ponnuswamy, Mondikalipudur Nanjappa Gounder

    2009-01-01

    Hemoglobin is a vital protein present in almost all higher species. It is a transport protein involved in carrying oxygen from lungs to tissues and carbon dioxide back to lungs by an intrinsically coordinated manner. Even though a good amount of work has been carried out in this direction there exists scarcity of structural insight on low oxygen affinity species. Attempts are being made to unravel the structural insight of this low oxygen affinity species. Goat blood plasma was collected, treated with EDTA to avoid blood clotting and purification was accomplished using DEAE-anion chromatographic column. The goat hemoglobin was crystallized using 50mM of phosphate buffer at pH 6.7 with 1M NaCl and PEG 3350 as precipitant by hanging drop vapor diffusion method. Crystals obtained are screened and suitable crystals are taken for data collection using mar345dtb as image plate detector system. Goat hemoglobin crystal diffracted up to 2.61 A resolution. Goat hemoglobin crystallizes in orthorhombic space group P212(1)2(1) as a whole biological molecule in the asymmetric unit with cell dimensions a=53.568A, b=67.365A, c=154.183A.

  17. Proteomic validation of protease drug targets: pharmacoproteomics of matrix metalloproteinase inhibitor drugs using isotope-coded affinity tag labelling and tandem mass spectrometry.

    PubMed

    Butler, G S; Overall, C M

    2007-01-01

    We illustrate the use of quantitative proteomics, namely isotope-coded affinity tag labelling and tandem mass spectrometry, to assess the targets and effects of the blockade of matrix metalloproteinases by an inhibitor drug in a breast cancer cell culture system. Treatment of MT1-MMP-transfected MDA-MB-231 cells with AG3340 (Prinomastat) directly affected the processing a multitude of matrix metalloproteinase substrates, and indirectly altered the expression of an array of other proteins with diverse functions. Therefore, broad spectrum blockade of MMPs has wide-ranging biological consequences. In this human breast cancer cell line, secreted substrates accumulated uncleaved in the conditioned medium and plasma membrane protein substrates were retained on the cell surface, due to reduced processing and shedding of these proteins (cell surface receptors, growth factors and bioactive molecules) to the medium in the presence of the matrix metalloproteinase inhibitor. Hence, proteomic investigation of drug-perturbed cellular proteomes can identify new protease substrates and at the same time provides valuable information for target validation, drug efficacy and potential side effects prior to commitment to clinical trials.

  18. Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).

    PubMed

    Meek, Megan E; Van Dolah, Frances M

    2016-05-01

    Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165)

    NASA Astrophysics Data System (ADS)

    Koide, Hiroyuki; Yoshimatsu, Keiichi; Hoshino, Yu; Lee, Shih-Hui; Okajima, Ai; Ariizumi, Saki; Narita, Yudai; Yonamine, Yusuke; Weisman, Adam C.; Nishimura, Yuri; Oku, Naoto; Miura, Yoshiko; Shea, Kenneth J.

    2017-07-01

    Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.

  20. Affinity labeling of a cysteine at or near the catalytic center of Escherichia coli B DNA-dependent RNA polymerase.

    PubMed

    Miller, J A; Serio, G F; Bear, J L; Howard, R A; Kimball, A P

    1980-03-14

    9-beta-D-Arabinofuranosyl-6-thiopurine was used to affinity label DNA-dependent RNA polymerase isolated from Escherichia coli B. This substrate analogue displayed competitive type inhibition which could be reversed by addition of a thiol reagent, such as dithiothreitol, while exposure to hydrogen peroxide, a mild oxidizing agent, caused an increase in both the inhibitory and enzyme binding capability of arabinofuranosyl thiopurine. Chromatographic analysis of the products obtained by pronase digestion of the 9-beta-D-arabinofuranosyl-6-[35S]thiopurine-enzyme complex suggests that disulfide bond formation occurs between the inhibitor and a cysteine residue located in or near the active center of the enzyme. In addition, polyacrylamide gel electrophoresis indicated that the arabinofuranosyl thiopurine moeity was bound to the beta' subunit of the enzyme.

  1. Factors associated with self-reported menu labeling use among US adults

    PubMed Central

    Lee-Kwan, Seung Hee; Pan, Liping; Maynard, Leah M.; McGuire, Lisa C.; Park, Sohyun

    2016-01-01

    Background Menu labeling may help people select foods and beverages with lower calories and is a potential population-based strategy to reduce obesity and diet-related chronic diseases in the United States. Objectives The aim of this cross-sectional study was to examine the prevalence of menu labeling use among adults and its association with sociodemographic, behavioral, and policy factors. Methods 2012 Behavioral Risk Factor Surveillance System data from 17 states that included 100,141 adults who noticed menu labeling at fast food/chain restaurants (“When calorie information is available in the restaurant, how often does this information help you decide what to order?”) were used. Menu labeling use was categorized: frequent (always/most of the time), moderate (half the time/sometimes), and never. Multinomial logistic regression was used to examine associations of sociodemographic, behavioral, and policy factors with menu labeling use. Results Overall, of adults who noticed menu labeling, 25.6% reported frequent use of menu labeling, 31.6% reported moderate use, and 42.7% reported that they never use menu labeling. Compared to never users, frequent users were significantly more likely to be younger, female, non-white, more educated, high-income, overweight or obese, physically active, former- or never-smokers, with no or lower (<1 time/day) sugar-sweetened beverage intake, and living in states where menu labeling legislation was enacted or proposed. Conclusions Menu labeling is one method that consumers can use to help reduce their calorie consumption from restaurants. These findings can be used to develop targeted interventions to increase menu labeling use among subpopulations with lower use. PMID:26875022

  2. Factors Associated with Self-Reported Menu-Labeling Usage among US Adults.

    PubMed

    Lee-Kwan, Seung Hee; Pan, Liping; Maynard, Leah M; McGuire, Lisa C; Park, Sohyun

    2016-07-01

    Menu labeling can help people select foods and beverages with fewer calories and is a potential population-based strategy to reduce obesity and diet-related chronic diseases in the United States. The aim of this cross-sectional study was to examine the prevalence of menu-labeling use among adults and its association with sociodemographic, behavioral, and policy factors. The 2012 Behavioral Risk Factor Surveillance System data from 17 states, which included 100,141 adults who noticed menu labeling at fast-food or chain restaurants ("When calorie information is available in the restaurant, how often does this information help you decide what to order?") were used. Menu-labeling use was categorized as frequent (always/most of the time), moderate (half the time/sometimes), and never. Multinomial logistic regression was used to examine associations among sociodemographic, behavioral, and policy factors with menu-labeling use. Overall, of adults who noticed menu labeling, 25.6% reported frequent use of menu labeling, 31.6% reported moderate use, and 42.7% reported that they never use menu labeling. Compared with never users, frequent users were significantly more likely to be younger, female, nonwhite, more educated, high-income, adults who were overweight or obese, physically active, former- or never-smokers, less than daily (<1 time/day) consumers of sugar-sweetened beverage, and living in states where menu-labeling legislation was enacted or proposed. Menu labeling is one method that consumers can use to help reduce their calorie consumption from restaurants. These findings can be used to develop targeted interventions to increase menu-labeling use among subpopulations with lower use. Published by Elsevier Inc.

  3. Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.

    PubMed

    Ferry, D R; Goll, A; Glossmann, H

    1987-04-01

    The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

  4. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  5. Biogeographical affinities of fish associated to the shrimp trawl fishery in the Gulf of Tehuantepec, Mexico.

    PubMed

    Martínez-Muñoz, Marco A; Lloris, Domènec; Gracia, Adolfo; Ramírez-Murillo, Ricardo; Sarmiento-Nafáte, Saul; Ramos-Cruz, Sebastián; Fernández, Felipe

    2016-06-01

    Fish by-catch of shrimp fishery from the Gulf of Tehuantepec is composed of several species that are mainly discarded. In this study, fish by-catch species composition, distribution and biogeographical affinities were analyzed. For this, a total of 15 cruises were carried out on the continental shelf, at depths from 15 to 64 m, during 2003, 2004, 2005 and 2013. Results showed that fish by-catch was represented by 58 families, 129 genera and 242 species. The families Haemulidae, Sciaenidae, Paralichthyidae, Gerreidae and Carangidae accounted for > 70 % of the catch. Haemulopsis axillaris, Syacium ovale, Selene peruviana, Diapterus peruvianus, Larimus acclivins and Stellifer erycimba were the most frequent species at < 40 m depth (inner shelf), and Prionotus stephanophrys, Scorpaena russula, Porichthys analis and Synodus scituliceps were dominant at 40-60 m depth (outer shelf). Analysis of biogeographical affinities showed that 36.1 % of species had a wide distribution, from San Diego Province to the Panamic Province, while 13.2 % had a restricted distribution in the Mexican and Panamic Provinces. The ichthyofaunal composition was markedly influenced by the local environment and seasonal conditions.

  6. Methods for determining the genetic affinity of microorganisms and viruses

    NASA Technical Reports Server (NTRS)

    Fox, George E. (Inventor); Willson, III, Richard C. (Inventor); Zhang, Zhengdong (Inventor)

    2012-01-01

    Selecting which sub-sequences in a database of nucleic acid such as 16S rRNA are highly characteristic of particular groupings of bacteria, microorganisms, fungi, etc. on a substantially phylogenetic tree. Also applicable to viruses comprising viral genomic RNA or DNA. A catalogue of highly characteristic sequences identified by this method is assembled to establish the genetic identity of an unknown organism. The characteristic sequences are used to design nucleic acid hybridization probes that include the characteristic sequence or its complement, or are derived from one or more characteristic sequences. A plurality of these characteristic sequences is used in hybridization to determine the phylogenetic tree position of the organism(s) in a sample. Those target organisms represented in the original sequence database and sufficient characteristic sequences can identify to the species or subspecies level. Oligonucleotide arrays of many probes are especially preferred. A hybridization signal can comprise fluorescence, chemiluminescence, or isotopic labeling, etc.; or sequences in a sample can be detected by direct means, e.g. mass spectrometry. The method's characteristic sequences can also be used to design specific PCR primers. The method uniquely identifies the phylogenetic affinity of an unknown organism without requiring prior knowledge of what is present in the sample. Even if the organism has not been previously encountered, the method still provides useful information about which phylogenetic tree bifurcation nodes encompass the organism.

  7. Novel Peptide Sequence (“IQ-tag”) with High Affinity for NIR Fluorochromes Allows Protein and Cell Specific Labeling for In Vivo Imaging

    PubMed Central

    McCarthy, Jason R.; Weissleder, Ralph

    2007-01-01

    Background Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes. Methods Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes. The developed peptide sequence (“IQ-tag”) allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging. Significance The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development. PMID:17653285

  8. Temporal Hierarchy of Gene Expression Mediated by Transcription Factor Binding Affinity and Activation Dynamics

    PubMed Central

    Gao, Rong

    2015-01-01

    ABSTRACT Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. PMID:26015501

  9. Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

    PubMed

    Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

    2013-02-01

    We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

  10. Pediatric off-label drug use in China: risk factors and management strategies.

    PubMed

    Zhang, Lingli; Li, Youping; Liu, Yi; Zeng, Linan; Hu, Die; Huang, Liang; Chen, Min; Lv, Juan; Yang, Chunsong

    2013-02-01

    To analyze the risk factors of pediatric off-label drug use, and propose management strategies for policy making of the pediatric off-label drug use in China. (i) We applied stratified random sampling to select recipes of children aged 0 to 18 years in pediatric clinics and wards of the West China Second University Hospital in 2010. (ii) All included prescriptions were categorized as off-label use or on-label use, according to the latest package insert licensed by the State Food and Drug Administration. (iii) Risk factors and the weights were calculated using logistic regression. (iv) The correlation between risk factors and the different kinds of off-label prescriptions was presented using adjusted odds ratio, and the impact of the risk factors was measured using standardized partial regression coefficient. (v) SPSS 16.0 was used for statistic analysis. (vi) From the perspective of the medical institutions, pharmaceutical enterprises, professional institutions, and the public, we combined the results of the Evidence-based research on the policy of the off-label drug use in 15 countries and the results of risk factor analysis, in order to propose management strategies for the policy making of pediatric off-label drug use in China. (i) Using the method of sampling, we received 2640 recipes from outpatients and 14,374 prescriptions from 749 inpatients. (ii) The neonates (0 to 27 days) had higher risk in off-label drug use than the other three children age groups. (iii) The dermatological medicines (D), nervous system medicines (N), traditional Chinese medicines, and respiratory drugs (R) were high-risk off-label medicines whose labels should be updated more frequently. (iv) The great factors of off-label drug use are those influence health status and relate to health services (ICD-10:Z00-Z99) (mainly in the clinic of child care and growth development, and in the ward of chemotherapy). (v) Off-label drug use in the ward was 4.4 times than that in clinic service (P < 0

  11. Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

    PubMed Central

    Owczarek, C M; Layton, M J; Metcalf, D; Lock, P; Willson, T A; Gough, N M; Nicola, N A

    1993-01-01

    Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins. Images PMID:8253075

  12. Floristic affinities of the lowland savannahs of Belize and southern Mexico.

    PubMed

    Canché-Estrada, Idalia Arely; Ortiz-Díaz, Juan Javier; Tun-Garrido, Juan

    2018-01-01

    Environmental heterogeneity of Belize and southern Mexico savannahs as well as their geographical location suggest that these plant communities share floristic elements, making them conducive to a phytogeographical analysis. The aim of this study was to analyse the floristic affinities of nine savannahs of Belize and southern Mexico and to explain the similarities and differences amongst them. A binary data matrix containing 915 species was built based on the authors' own collections and on nine floristic lists already published. A second data matrix, consisting of 113 species representing trees, was also used since most literature on neotropical savannahs has focused on this life form. In addition, the ten most species-rich families as well as the characteristic species present in more than five savannahs were analysed. Floristic similarities were calculated using the Jaccard index. Dendrograms obtained in both types of analysis showed clusters with low similarity values, corresponding to geographic locations formed by the savannahs of Belize-Tabasco and the Yucatan Peninsula. The floristic affinities of the savannahs may be explained in terms of heterogeneity in climate and physiography. The Yucatan Peninsula and Belize-Tabasco groups have differences in climate type and the amount of rainfall. In addition, the Yucatan Peninsula savannahs are established at the bottom of karstic valleys, while the Belize and Tabasco savannahs develop on extensive flatlands. The savannahs of Oaxaca have the same climate type and amount of rainfall as those of the Yucatan Peninsula but they are distributed along peaks and the slopes of shale hills. Fabaceae and Poaceae mainly dominated the local floras with 121 and 116 species each; remarkably, Melastomataceae was absent in the Yucatan Peninsula and Oaxaca. Nine species occurred in five to seven savannahs, confirming that they are widespread in both Belize and southern Mexico, and the Neotropics. Geographic location and floristic

  13. Quantitative monitoring of two simultaneously binding species using Label-Enhanced surface plasmon resonance.

    PubMed

    Eng, Lars; Garcia, Brandon L; Geisbrecht, Brian V; Hanning, Anders

    2018-02-26

    Surface plasmon resonance (SPR) is a well-established method for biomolecular interaction studies. SPR monitors the binding of molecules to a solid surface, embodied as refractive index changes close to the surface. One limitation of conventional SPR is the universal nature of the detection that results in an inability to qualitatively discriminate between different binding species. Furthermore, it is impossible to directly discriminate two species simultaneously binding to different sites on a protein, which limits the utility of SPR, for example, in the study of allosteric binders or bi-specific molecules. It is also impossible in principle to discriminate protein conformation changes from actual binding events. Here we demonstrate how Label-Enhanced SPR can be utilized to discriminate and quantitatively monitor the simultaneous binding of two different species - one dye-labeled and one unlabeled - on a standard, single-wavelength SPR instrument. This new technique increases the versatility of SPR technology by opening up application areas where the usefulness of the approach has previously been limited. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. On the structure of self-affine convex bodies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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.

  15. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the rangemore » of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.« less

  16. Tumour necrosis factors modulate the affinity state of the leukotriene B4 receptor on human neutrophils.

    PubMed Central

    Brom, J; Knöller, J; Köller, M; König, W

    1988-01-01

    Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TNF) revealed a time- and dose-dependent reduction of the expression of leukotriene B4-receptor sites. Analysis of the binding data by Scatchard plots showed a shift from a heterologous receptor population (indicating high- and low-affinity subsets) to a homologous population. From the results it is considered that TNF can influence host defence through the modulation of leukotriene B4 receptor affinity. PMID:2851543

  17. Fundamentals of affinity cell separations.

    PubMed

    Zhang, Ye; Lyons, Veronica; Pappas, Dimitri

    2018-03-01

    Cell separations using affinity methods continue to be an enabling science for a wide variety of applications. In this review, we discuss the fundamental aspects of affinity separation, including the competing forces for cell capture and elution, cell-surface interactions, and models for cell adhesion. Factors affecting separation performance such as bond affinity, contact area, and temperature are presented. We also discuss and demonstrate the effects of nonspecific binding on separation performance. Metrics for evaluating cell separations are presented, along with methods of comparing separation techniques for cell isolation using affinity capture. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Myoglobin oxygen affinity in aquatic and terrestrial birds and mammals.

    PubMed

    Wright, Traver J; Davis, Randall W

    2015-07-01

    Myoglobin (Mb) is an oxygen binding protein found in vertebrate skeletal muscle, where it facilitates intracellular transport and storage of oxygen. This protein has evolved to suit unique physiological needs in the muscle of diving vertebrates that express Mb at much greater concentrations than their terrestrial counterparts. In this study, we characterized Mb oxygen affinity (P50) from 25 species of aquatic and terrestrial birds and mammals. Among diving species, we tested for correlations between Mb P50 and routine dive duration. Across all species examined, Mb P50 ranged from 2.40 to 4.85 mmHg. The mean P50 of Mb from terrestrial ungulates was 3.72±0.15 mmHg (range 3.70-3.74 mmHg). The P50 of cetaceans was similar to terrestrial ungulates ranging from 3.54 to 3.82 mmHg, with the exception of the melon-headed whale, which had a significantly higher P50 of 4.85 mmHg. Among pinnipeds, the P50 ranged from 3.23 to 3.81 mmHg and showed a trend for higher oxygen affinity in species with longer dive durations. Among diving birds, the P50 ranged from 2.40 to 3.36 mmHg and also showed a trend of higher affinities in species with longer dive durations. In pinnipeds and birds, low Mb P50 was associated with species whose muscles are metabolically active under hypoxic conditions associated with aerobic dives. Given the broad range of potential globin oxygen affinities, Mb P50 from diverse vertebrate species appears constrained within a relatively narrow range. High Mb oxygen affinity within this range may be adaptive for some vertebrates that make prolonged dives. © 2015. Published by The Company of Biologists Ltd.

  19. Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

    PubMed Central

    Azimi, Mohammad; Mofrad, Mohammad R. K.

    2013-01-01

    Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized. PMID:24282617

  20. Human factors in labeling and training for home healthcare technology.

    PubMed

    Patterson, Patricia A

    2010-01-01

    In this article, Patricia A. Patterson, a contributor to the recently-released standard ANSI/AAMI HE75:2009 Human factors engineering-Design of medical devices, highlights information from the standard important to developing labeling and training for homecare devices. She also describes one approach to developing labeling and training materials.

  1. Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII.

    PubMed

    Kelley, Brian D; Tannatt, Molly; Magnusson, Robert; Hagelberg, Sigrid; Booth, James

    2004-08-05

    An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.

  2. Species composition, co-occurrence, association and affinity indices of mosquito larvae (Diptera: Culicidae) in Mazandaran Province, northern Iran.

    PubMed

    Nikookar, Seyed Hassan; Azari-Hamidian, Shahyad; Fazeli-Dinan, Mahmoud; Nasab, Seyed Nouraddin Mousavi; Aarabi, Mohsen; Ziapour, Seyyed Payman; Enayati, Ahmadali

    2016-05-01

    Although considerable progress has been made in the past years in management of mosquito borne diseases such as malaria, dengue, yellow fever and West Nile fever through research in biology and ecology of the vectors, these diseases are still major threats to human health. Therefore, more research is required for better management of the diseases. This investigation provides information on the composition, co-occurrence, association and affinity indices of mosquito larvae in Mazandaran Province, northern Iran. In a large scale field study, mosquito larvae were collected from 120 sentinel sites in 16 counties in Mazandaran Province, using standard 350 ml dipper. Sampling took place monthly from May to December 2014. Collected larvae were mounted on glass slides using de Faure's medium and were diagnosed using morphological characters. Totally, 19,840 larvae were collected including three genera and 16 species from 120 larval habitats, as follows: Anopheles claviger, Anopheles hyrcanus, Anopheles maculipennis s.l., Anopheles marteri, Anopheles plumbeus, Anopheles pseudopictus, Culex pipiens, Culex tritaeniorhynchus, Culex torrentium, Culex perexiguus, Culex territans, Culex mimeticus, Culex hortensis, Culiseta annulata, Culiseta longiareolata, and Culiseta morsitans. Predominant species were Cx. pipiens and An. maculipennis s.l. which show the highest co-occurrence. The pair of species An. hyrcanus/An. pseudopictus showed significant affinity and association. High co-occurrence of the predominant species Cx. pipiens and An. maculipennis s.l. in the study area is of considerable importance in terms of vector ecology. It was also revealed that An. pseudopictus/An. hyrcanus often occur sympatrically indicating their common habitat requirements. The information may be equally important when vector control measures are considered. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity

    PubMed Central

    Zandvakili, Arya; Campbell, Ian; Weirauch, Matthew T.

    2018-01-01

    Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. Here, we show that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, we developed a method that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, we found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs. PMID:29617378

  4. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

    PubMed

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

    2016-01-01

    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Relative binding affinity of carboxylate-, phosphonate-, and bisphosphonate-functionalized gold nanoparticles targeted to damaged bone tissue

    NASA Astrophysics Data System (ADS)

    Ross, Ryan D.; Cole, Lisa E.; Roeder, Ryan K.

    2012-10-01

    Functionalized Au NPs have received considerable recent interest for targeting and labeling cells and tissues. Damaged bone tissue can be targeted by functionalizing Au NPs with molecules exhibiting affinity for calcium. Therefore, the relative binding affinity of Au NPs surface functionalized with either carboxylate ( l-glutamic acid), phosphonate (2-aminoethylphosphonic acid), or bisphosphonate (alendronate) was investigated for targeted labeling of damaged bone tissue in vitro. Targeted labeling of damaged bone tissue was qualitatively verified by visual observation and backscattered electron microscopy, and quantitatively measured by the surface density of Au NPs using field-emission scanning electron microscopy. The surface density of functionalized Au NPs was significantly greater within damaged tissue compared to undamaged tissue for each functional group. Bisphosphonate-functionalized Au NPs exhibited a greater surface density labeling damaged tissue compared to glutamic acid- and phosphonic acid-functionalized Au NPs, which was consistent with the results of previous work comparing the binding affinity of the same functionalized Au NPs to synthetic hydroxyapatite crystals. Targeted labeling was enabled not only by the functional groups but also by the colloidal stability in solution. Functionalized Au NPs were stabilized by the presence of the functional groups, and were shown to remain well dispersed in ionic (phosphate buffered saline) and serum (fetal bovine serum) solutions for up to 1 week. Therefore, the results of this study suggest that bisphosphonate-functionalized Au NPs have potential for targeted delivery to damaged bone tissue in vitro and provide motivation for in vivo investigation.

  6. Two distinct DNA sequences recognized by transcription factors represent enthalpy and entropy optima

    PubMed Central

    Yin, Yimeng; Das, Pratyush K; Jolma, Arttu; Zhu, Fangjie; Popov, Alexander; Xu, You; Nilsson, Lennart

    2018-01-01

    Most transcription factors (TFs) can bind to a population of sequences closely related to a single optimal site. However, some TFs can bind to two distinct sequences that represent two local optima in the Gibbs free energy of binding (ΔG). To determine the molecular mechanism behind this effect, we solved the structures of human HOXB13 and CDX2 bound to their two optimal DNA sequences, CAATAAA and TCGTAAA. Thermodynamic analyses by isothermal titration calorimetry revealed that both sites were bound with similar ΔG. However, the interaction with the CAA sequence was driven by change in enthalpy (ΔH), whereas the TCG site was bound with similar affinity due to smaller loss of entropy (ΔS). This thermodynamic mechanism that leads to at least two local optima likely affects many macromolecular interactions, as ΔG depends on two partially independent variables ΔH and ΔS according to the central equation of thermodynamics, ΔG = ΔH - TΔS. PMID:29638214

  7. ( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Branchek, T.; Adham, N.; Macchi, M.

    1990-11-01

    The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

  8. Alternative Affinity Ligands for Immunoglobulins.

    PubMed

    Kruljec, Nika; Bratkovič, Tomaž

    2017-08-16

    The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

  9. Cadmium accumulation characteristics of the winter farmland weeds Cardamine hirsuta Linn. and Gnaphalium affine D. Don.

    PubMed

    Lin, Lijin; Shi, Jun; Liu, Qihua; Liao, Ming'an; Mei, Luoyin

    2014-07-01

    In a preliminary study, we found that the cadmium (Cd) concentrations in shoots of the winter farmland weeds Cardamine hirsuta Linn. and Gnaphalium affine D. Don exceeded the critical value of a Cd-hyperaccumulator (100 mg kg(-1)), indicating that these two farmland weeds might be Cd-hyperaccumulators. In this study, we grew these species in soil containing various concentrations of Cd to further evaluate their Cd accumulation characteristics. The biomasses of C. hirsuta and G. affine decreased with increasing Cd concentrations in the soil, while the root/shoot ratio and the Cd concentrations in shoot tissues increased. The Cd concentrations in shoots of C. hirsuta and G. affine reached 121.96 and 143.91 mg kg(-1), respectively, at the soil Cd concentration of 50 mg kg(-1). Both of these concentrations exceeded the critical value of a Cd-hyperaccumulator (100 mg kg(-1)). The shoot bioconcentration factors of C. hirsuta and G. affine were greater than 1. The translocation factor of C. hirsuta was less than 1 and that of G. affine was greater than 1. These findings indicated that C. hirsuta is a Cd-accumulator and G. affine is Cd-hyperaccumulator. Both plants are distributed widely in the field, and they could be used to remediate Cd-contaminated farmland soil in winter.

  10. In vivo gene transfer to dopamine neurons of rat substantia nigra via the high-affinity neurotensin receptor.

    PubMed Central

    Alvarez-Maya, I.; Navarro-Quiroga, I.; Meraz-Ríos, M. A.; Aceves, J.; Martinez-Fong, D.

    2001-01-01

    BACKGROUND: Recently, we synthesized a nonviral gene vector capable of transfecting cell lines taking advantage of neurotensin (NT) internalization. The vector is NT cross-linked with poly-L-lysine, to which a plasmid DNA was bound to form a complex (NT-polyplex). Nigral dopamine neurons are able to internalize NT, thus representing a target for gene transfer via NT-polyplex. This hypothesis was tested here using reporter genes encoding green fluorescent protein or chloramphenicol acetyl transferase. MATERIALS AND METHODS: NT-polyplex was injected into the substantia nigra. Double immunofluorescence labeling was used to reveal the cell type involved in the propidium iodide-labeled polyplex internalization and reporter gene expression. RESULTS: Polyplex internalization was observed within dopamine neurons but not within glial cells, and was prevented by both hypertonic sucrose solution and SR-48692, a selective nonpeptide antagonist of NT receptors. Reporter gene expression was observed in dopamine neurons from 48 hr up to 15 days after NT-polyplex injection, and was prevented by SR-48692. However, no expression was seen when the NT-polyplex was injected into the ansiform lobule of the cerebellum, which contains low- but not high-affinity NT receptors. Neither internalization nor expression was observed in cultured glial cells, despite the NT-polyplex binding to those cells that was prevented by levocabastine, a low-affinity NT receptor antagonist. CONCLUSIONS: These results suggest that high-affinity NT receptors mediate the uptake of NT-polyplex with the subsequent reporter gene expression in vivo. NT polyfection may be used to transfer genes of physiologic interest to nigrostriatal dopamine neurons, and to produce transgenic animal models of dopamine-related diseases. PMID:11471555

  11. Selection of multiple umbrella species for functional and taxonomic diversity to represent urban biodiversity.

    PubMed

    Sattler, T; Pezzatti, G B; Nobis, M P; Obrist, M K; Roth, T; Moretti, M

    2014-04-01

    Surrogates, such as umbrella species, are commonly used to reduce the complexity of quantifying biodiversity for conservation purposes. The presence of umbrella species is often indicative of high taxonomic diversity; however, functional diversity is now recognized as an important metric for biodiversity and thus should be considered when choosing umbrella species. We identified umbrella species associated with high taxonomic and functional biodiversity in urban areas in Switzerland. We analyzed 39,752 individuals of 574 animal species from 96 study plots and 1397 presences of 262 plant species from 58 plots. Thirty-one biodiversity measures of 7 taxonomic groups (plants, spiders, bees, ground beetles, lady bugs, weevils and birds) were included in within- and across-taxa analyses. Sixteen measures were taxonomical (species richness and species diversity), whereas 15 were functional (species traits including mobility, resource use, and reproduction). We used indicator value analysis to identify umbrella species associated with single or multiple biodiversity measures. Many umbrella species were indicators of high biodiversity within their own taxonomic group (from 33.3% in weevils to 93.8% in birds), to a lesser extent they were indicators across taxa. Principal component analysis revealed that umbrella species for multiple measures of biodiversity represented different aspects of biodiversity, especially with respect to measures of taxonomic and functional diversity. Thus, even umbrella species for multiple measures of biodiversity were complementary in the biodiversity aspects they represented. Thus, the choice of umbrella species based solely on taxonomic diversity is questionable and may not represent biodiversity comprehensively. Our results suggest that, depending on conservation priorities, managers should choose multiple and complementary umbrella species to assess the state of biodiversity. © 2013 Society for Conservation Biology.

  12. Rapid method for controlling the correct labeling of products containing common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas) by fast real-time PCR.

    PubMed

    Espiñeira, Montserrat; Vieites, Juan M

    2012-12-15

    The TaqMan real-time PCR has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of species. This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas). This technique is notable for the combination of simplicity, speed, sensitivity and specificity in an homogeneous assay. The method can be applied to all kinds of products; fresh, frozen and processed, including those undergoing intensive processes of transformation. This methodology was validated to check how the degree of food processing affects the method and the detection of each species. Moreover, it was applied to 34 commercial samples to evaluate the labeling of products made from them. The methodology herein developed is useful to check the fulfillment of labeling regulations for seafood products and to verify traceability in commercial trade and for fisheries control. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. 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

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

    2007-12-01

    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.

  14. 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

  15. Multi-Affinity for Growing Rough Interfaces of Bacterial Colonies

    NASA Astrophysics Data System (ADS)

    Kobayashi, N.; Ozawa, T.; Saito, K.; Yamazaki, Y.; Matsuyama, T.; Matsushita, M.

    We have examined whether rough interfaces of bacterial colonies are multi-affine. We have used the bacterial species called textit{Bacillus subtilis}, which has been found to exhibit a variety of colony patterns when varying both the concentration of nutrient and solidity of agar medium. Consequently, we have found that the colony interface on a nutrient-rich, solid agar medium is multi-affine. On the other hand, the colony interface on a nutrient-rich, semi-solid agar medium is self-affine.

  16. Positron Emission Tomography and Near-Infrared Fluorescence Imaging of Vascular Endothelial Growth Factor with Dual-Labeled Bevacizumab.

    PubMed

    Zhang, Yin; Hong, Hao; Engle, Jonathan W; Yang, Yunan; Barnhart, Todd E; Cai, Weibo

    2012-01-01

    The pivotal role of vascular endothelial growth factor (VEGF) in cancer is underscored by the approval of bevacizumab (Bev, a humanized anti-VEGF monoclonal antibody) for first line treatment of cancer patients. The aim of this study was to develop a dual-labeled Bev for both positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of VEGF. Bev was conjugated to a NIRF dye (i.e. 800CW) and 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) before (64)Cu-labeling. Flow cytometry analysis of U87MG human glioblastoma cells revealed no difference in VEGF binding affinity/specificity between Bev and NOTA-Bev-800CW. (64)Cu-labeling of NOTA-Bev-800CW was achieved with high yield. Serial PET imaging of U87MG tumor-bearing female nude mice revealed that tumor uptake of (64)Cu-NOTA-Bev-800CW was 4.6 ± 0.7, 16.3 ± 1.6, 18.1 ± 1.4 and 20.7 ± 3.7 %ID/g at 4, 24, 48 and 72 h post-injection respectively (n = 4), corroborated by in vivo/ex vivo NIRF imaging and biodistribution studies. Tumor uptake as measured by ex vivo NIRF imaging had a good linear correlation with the %ID/g values obtained from PET (R(2) = 0.93). Blocking experiments and histology both confirmed the VEGF specificity of (64)Cu-NOTA-Bev-800CW. The persistent, prominent, and VEGF-specific uptake of (64)Cu-NOTA-Bev-800CW in the tumor, observed by both PET and NIRF imaging, warrants further investigation and future clinical translation of such Bev-based imaging agents.

  17. High Specific Activity Tritium-Labeled N-(2-methoxybenzyl)-2,5-dimethoxy-4-iodophenethylamine (INBMeO): A High Affinity 5-HT2A Receptor-Selective Agonist Radioligand

    PubMed Central

    Nichols, David E.; Frescas, Stewart P.; Chemel, Benjamin R.; Rehder, Kenneth S.; Zhong, Desong; Lewin, Anita H.

    2009-01-01

    The title compound ([3H]INBMeO) was prepared by an O,O-dimethylation reaction of a t-BOC protected diphenolic precursor using no carrier added tritiated iodomethane in DMF with K2CO3. Removal of the t-BOC protecting group and purification by HPLC afforded an overall yield of 43%, with a radiochemical purity of 99% and specific activity of 164 Ci/mmol. The new radioligand was suitable for labeling human 5-HT2A receptors in two heterologous cell lines and had about 20-fold higher affinity than [3H]ketanserin. PMID:18468904

  18. Comparison of receptor affinity of natSc-DOTA-TATE versus natGa-DOTA-TATE.

    PubMed

    Koumarianou, Eftychia; Pawlak, Dariusz; Korsak, Agnieszka; Mikolajczak, Renata

    2011-01-01

    44Sc as a positron emitter can be an interesting alternative to 68Ga (T½=67.71 min) due to its longer half-life (T½=3.97 h). Moreover, the b-emitter 47Sc can be used for therapy when attached to the same biomolecule vectors. DOTA as a chelating agent has been proven suitable for the radiolabelling of peptides recognising tumour cell receptors in vivo with M3+ radiometals. DOTA-derivatized peptides have been successfully labelled with 90Y and 177Lu for therapy, and with 68Ga for PET imaging. However, published data on 44Sc-labelled DOTA-biomolecules as potential PET radiotracers are still very limited. The aim of this study was to compare the affinity of natGa- and natSc-labelled DOTA-TATE to somatostatin receptors subtype 2 expressed in rat pancreatic cancer cell line AR42J. The cold complexes of DOTA-TATE with natGa and natSc were synthesized and identified by HPLC and MS analysis and evaluated in vitro for competitive binding to cancer cell line AR42J expressing somatostatin receptors subtype 2 (sstr2). The IC50 values calculated from the displacement curve of {125I-Tyr11}-SST-14 were: 0.20±0.18, 0.70±0.20, 0.64±0.22 and 0.67±0.12 for natGa-DOTA-TATE, natSc-DOTA-TATE, DOTA-TATE, and {Tyr11}-SST-14 complexes, respectively, with the affinity lowering in the decreasing order: natGa-DOTA-TATE>DOTA-TATE>Tyr11-SST-14>natSc-DOTA-TATE. The binding affinity of natGa-DOTA-TATE appeared higher than that of natSc-DOTA-TATE. Further in vitro and in vivo studies are needed to verify the influence of the chelated metal on the affinity and uptake of the respective radiolabelled compounds. This information might be crucial when the in vivo applications of peptides labelled with 68Ga and 44Sc for PET, as well as the use of 47Sc for radiotherapy are considered.

  19. Carbon isotope labeling in boreal forests to assess roles of fungal species in decomposition

    NASA Astrophysics Data System (ADS)

    Treseder, K. K.; Czimczik, C. I.; Trumbore, S. E.; Allison, S. D.

    2006-12-01

    We used 14C and 13C labeling to assess the in situ respiration of alanine-, starch-, and lignocellulose-derived carbon from the sporocarps of particular fungal species fruiting in a boreal forest in Alaska. By measuring isotopically-labeled respiration of sporocarps, which can be identified to species, we were able to attribute turnover of carbon compounds to specific fungal groups. Moreover, collection of sporocarp respiration is non-destructive, so we could return to the same sporocarps to collect a time series of measurements that spanned hours to days. We tested the hypotheses that alanine and starch turn over more quickly than lignocellulose, and that saprotrophic fungi would use starch-C and lignocellulose-C but ectomycorrhizal fungi would not. Small amounts of 14C-labeled alanine (about 100,000 permil) were dispensed into the soil within three meters of sporocarps of the ectomycorrhizal fungus Lactarius alnicola. Δ14CO2 values of sporocarp respiration climbed from 75.8 +/- 6.3 permil to 7855 +/- 3940 permil within one hour of additions, indicating that the fungus quickly acquired, transported, and transformed the alanine-C. In a separate approach, a mixture of 13C-labeled starch (about 15,000 permil) and 14C-labeled lignocellulose (about 36,000 permil) was applied in 9 m2 plots containing sporocarps of the ectomycorrhizal genera Phellodon and Sarcodon and the saprotrophic genera Lycoperdon and Polyporus. An unlabeled control plot was also established. We observed no detectable increase in 14CO2 or 13CO2 over a 144 hour period, suggesting that neither ectomycorrhizal nor saprotrophic fungi significantly broke down starch or lignocellulose during this time. The alanine experiment is one of the first to indicate that ectomycorrhizal fungi can influence the spatial distribution and storage of soil carbon over short time scales. This influence may be restricted to carbon of organic compounds like amino acids. In contrast, starch was not transformed quickly even

  20. Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns.

    PubMed

    Bi, Cong; Zheng, Xiwei; Hage, David S

    2016-02-05

    In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Determination of trace glucose and forecast of human diseases by affinity adsorption solid substrate room temperature phosphorimetry based on Triticum valgaris lectin labeled with 4.0-generation dendrimers

    NASA Astrophysics Data System (ADS)

    Li, Zhiming; Zhu, Guohui; Liu, Jiaming; Lu, Qiaomei; Yang, Minlan; Wu, Hong; Shi, Xiumei; Chen, Xinhua

    2007-08-01

    A new phosphorescence labeling reagent Triton-100X-4.0G-D (4.0G-D refers to 4.0-generation dendrimers) was found. Quantitative specific affinity adsorption (AA) reaction between Triton-100X-4.0G-D-WGA and glucose (G) was carried out on the surface of nitrocellulose membrane (NCM), and the Δ Ip of the product of AA reaction was linear correlation to the content of G. Based on the facts above, a new method for the determination of trace G was established by WGA labeled with Triton-100X-4.0G-D affinity adsorption solid substrate room temperature phosphorimetry (Triton-100X-4.0G-D-WGA-AA-SS-RTP). This research showed that AA-SS-RTP for either direct method or sandwich method could combine very well the characteristics of both the high sensitivity of SS-RTP and the specificity of the AA reaction. Detection limits (LD) were 0.24 fg spot -1 for direct method and 0.18 fg spot -1 for sandwich method, indicating both of them were of high sensitivity. The method has been applied to the determination of the content of G in human serum, and the results were coincided with those obtained by glucose oxidize enzyme method. It can also be applied to forecast accurately some human diseases, such as primary hepatic carcinoma, cirrhosis, acute and chronic hepatitis, transfer hepatocellular, etc. Meanwhile, the mechanism for the determination of G with AA-SS-RTP was discussed.

  2. Attitude and Behavior Factors Associated with Front-of-Package Label Use with Label Users Making Accurate Product Nutrition Assessments.

    PubMed

    Roseman, Mary G; Joung, Hyun-Woo; Littlejohn, Emily I

    2018-05-01

    attitude and behavior factors were associated with label use. Copyright © 2018 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.

  3. A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

    PubMed Central

    Schotte, Lise; Rombaut, Bart; Thys, Bert

    2012-01-01

    In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to

  4. Angiosperm flora on the páramos of northwestern Colombia: diversity and affinities

    PubMed Central

    Alzate-Guarín, Fernando; Murillo-Serna, Jhon Steven

    2016-01-01

    Abstract Páramos are high-elevation isolated ecosystems in the Andes characterized by specific flora. This flora includes a number of endemic species and some taxa phylogenetically related to temperate lineages (van der Hammen and Cleef 1986). There are six páramo units or complexes in the Department of Antioquia, located in northwestern Colombia. For five years, we conducted botanic explorations in order to quantify the richness of angiosperm flora in these units. We estimate the richness of angiosperms in these páramos at 693 species, 277 genera, and 86 families, which represent almost 10% of the floral diversity in Antioquia, but contained in only 0.7% of its area. We found that Frontino-Urrao is the most species-rich páramo with 465 species from 225 genera. Our results show that the most diverse angiosperm families of the páramos of Antioquia are Asteraceae, Orchidaceae, Melastomataceae, and Poaceae, which together represent 245 species. Groupings between páramos by Sørensen’s similarity index show that the complexes of the Central Andes Cordillera form a cluster of greater affinity than Páramos from other regions. Of the species found, 80 have a CITES or IUCN diagnosis. The expeditions allowed the identification of 21 species not previously registered in Antioquia and a considerable number of endemisms (35 species), further proof of the high plant diversity in these ecosystems. PMID:27829798

  5. Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components

    PubMed Central

    Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, UA

    2014-01-01

    Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and Impact of the Study Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. PMID:24935714

  6. Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters.

    PubMed

    Basu, Sankha S; Mesaros, Clementina; Gelhaus, Stacy L; Blair, Ian A

    2011-02-15

    Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

  7. Biogeographic affinity helps explain productivity-richness relationships at regional and local scales

    USGS Publications Warehouse

    Harrison, S.; Grace, J.B.

    2007-01-01

    The unresolved question of what causes the observed positive relationship between large-scale productivity and species richness has long interested ecologists and evolutionists. Here we examine a potential explanation that we call the biogeographic affinity hypothesis, which proposes that the productivity-richness relationship is a function of species' climatic tolerances that in turn are shaped by the earth's climatic history combined with evolutionary niche conservatism. Using botanical data from regions and sites across California, we find support for a key prediction of this hypothesis, namely, that the productivity-species richness relationship differs strongly and predictably among groups of higher taxa on the basis of their biogeographic affinities (i.e., between families or genera primarily associated with north-temperate, semiarid, or desert zones). We also show that a consideration of biogeographic affinity can yield new insights on how productivity-richness patterns at large geographic scales filter down to affect patterns of species richness and composition within local communities. ?? 2007 by The University of Chicago. All rights reserved.

  8. Projections of climate-driven changes in tuna vertical habitat based on species-specific differences in blood oxygen affinity.

    PubMed

    Mislan, K A S; Deutsch, Curtis A; Brill, Richard W; Dunne, John P; Sarmiento, Jorge L

    2017-10-01

    Oxygen concentrations are hypothesized to decrease in many areas of the ocean as a result of anthropogenically driven climate change, resulting in habitat compression for pelagic animals. The oxygen partial pressure, pO 2 , at which blood is 50% saturated (P 50 ) is a measure of blood oxygen affinity and a gauge of the tolerance of animals for low ambient oxygen. Tuna species display a wide range of blood oxygen affinities (i.e., P 50 values) and therefore may be differentially impacted by habitat compression as they make extensive vertical movements to forage on subdaily time scales. To project the effects of end-of-the-century climate change on tuna habitat, we calculate tuna P 50 depths (i.e., the vertical position in the water column at which ambient pO 2 is equal to species-specific blood P 50 values) from 21st century Earth System Model (ESM) projections included in the fifth phase of the Climate Model Intercomparison Project (CMIP5). Overall, we project P 50 depths to shoal, indicating likely habitat compression for tuna species due to climate change. Tunas that will be most impacted by shoaling are Pacific and southern bluefin tunas-habitat compression is projected for the entire geographic range of Pacific bluefin tuna and for the spawning region of southern bluefin tuna. Vertical shifts in P 50 depths will potentially influence resource partitioning among Pacific bluefin, bigeye, yellowfin, and skipjack tunas in the northern subtropical and eastern tropical Pacific Ocean, the Arabian Sea, and the Bay of Bengal. By establishing linkages between tuna physiology and environmental conditions, we provide a mechanistic basis to project the effects of anthropogenic climate change on tuna habitats. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  9. Measurement of free glucocorticoids: quantifying corticosteroid-binding globulin binding affinity and its variation within and among mammalian species.

    PubMed

    Delehanty, Brendan; Hossain, Sabrina; Jen, Chao Ching; Crawshaw, Graham J; Boonstra, Rudy

    2015-01-01

    Plasma glucocorticoids (GCs) are commonly used as measures of stress in wildlife. A great deal of evidence indicates that only free GC (GC not bound by the specific binding protein, corticosteroid-binding globulin, CBG) leaves the circulation and exerts biological effects on GC-sensitive tissues. Free hormone concentrations are difficult to measure directly, so researchers estimate free GC using two measures: the binding affinity and the binding capacity in plasma. We provide an inexpensive saturation binding method for calculating the binding affinity (equilibrium dissociation constant, K d) of CBG that can be run without specialized laboratory equipment. Given that other plasma proteins, such as albumin, also bind GCs, the method compensates for this non-specific binding. Separation of bound GC from free GC was achieved with dextran-coated charcoal. The method provides repeatable estimates (12% coefficient of variation in the red squirrel, Tamiasciurus hudsonicus), and there is little evidence of inter-individual variation in K d (range 2.0-7.3 nM for 16 Richardson's ground squirrels, Urocitellus richardsonii). The K d values of 28 mammalian species we assessed were mostly clustered around a median of 4 nM, but five species had values between 13 and 61 nM. This pattern may be distinct from birds, for which published values are more tightly distributed (1.5-5.1 nM). The charcoal separation method provides a reliable and robust method for measuring the K d in a wide range of species. It uses basic laboratory equipment to provide rapid results at very low cost. Given the importance of CBG in regulating the biological activity of GCs, this method is a useful tool for physiological ecologists.

  10. A HIGH-LEVEL CALCULATION OF THE PROTON AFFINITY OF DIBORANE

    EPA Science Inventory

    The experimental proton affinity of diborane (B2H6) is based on an unstable species, B2H,+, 4 which has been observed only at low temperatures. The present work calculates the proton 5 affinity of diborane using the Gaussian-3 method and other high-level compound ab initio 6 met...

  11. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Semicircular canals in Anolis lizards: ecomorphological convergence and ecomorph affinities of fossil species.

    PubMed

    Dickson, Blake V; Sherratt, Emma; Losos, Jonathan B; Pierce, Stephanie E

    2017-10-01

    Anoli s lizards are a model system for the study of adaptive radiation and convergent evolution. Greater Antillean anoles have repeatedly evolved six similar forms or ecomorphs: crown-giant, grass-bush, twig, trunk, trunk-crown and trunk-ground. Members of each ecomorph category possess a specific set of morphological, ecological and behavioural characteristics which have been acquired convergently. Here we test whether the semicircular canal system-the organ of balance during movement-is also convergent among ecomorphs, reflecting the shared sensory requirements of their ecological niches. As semicircular canal shape has been shown to reflect different locomotor strategies, we hypothesized that each Anolis ecomorph would have a unique canal morphology. Using three-dimensional semilandmarks and geometric morphometrics, semicircular canal shape was characterized in 41 Anolis species from the Greater Antilles and the relationship between canal shape and ecomorph grouping, phylogenetic history, size, head dimensions, and perch characteristics was assessed. Further, canal morphology of modern species was used to predict the ecomorph affinity of five fossil anoles from the Miocene of the Dominican Republic. Of the covariates tested, our study recovered ecomorph as the single-most important covariate of canal morphology in modern taxa; although phylogenetic history, size, and head dimensions also showed a small, yet significant correlation with shape. Surprisingly, perch characteristics were not found to be significant covariates of canal shape, even though they are important habitat variables. Using posterior probabilities, we found that the fossil anoles have different semicircular canals shapes to modern ecomorph groupings implying extinct anoles may have been interacting with their Miocene environment in different ways to modern Anolis species.

  13. Semicircular canals in Anolis lizards: ecomorphological convergence and ecomorph affinities of fossil species

    NASA Astrophysics Data System (ADS)

    Dickson, Blake V.; Sherratt, Emma; Losos, Jonathan B.; Pierce, Stephanie E.

    2017-10-01

    Anolis lizards are a model system for the study of adaptive radiation and convergent evolution. Greater Antillean anoles have repeatedly evolved six similar forms or ecomorphs: crown-giant, grass-bush, twig, trunk, trunk-crown and trunk-ground. Members of each ecomorph category possess a specific set of morphological, ecological and behavioural characteristics which have been acquired convergently. Here we test whether the semicircular canal system-the organ of balance during movement-is also convergent among ecomorphs, reflecting the shared sensory requirements of their ecological niches. As semicircular canal shape has been shown to reflect different locomotor strategies, we hypothesized that each Anolis ecomorph would have a unique canal morphology. Using three-dimensional semilandmarks and geometric morphometrics, semicircular canal shape was characterized in 41 Anolis species from the Greater Antilles and the relationship between canal shape and ecomorph grouping, phylogenetic history, size, head dimensions, and perch characteristics was assessed. Further, canal morphology of modern species was used to predict the ecomorph affinity of five fossil anoles from the Miocene of the Dominican Republic. Of the covariates tested, our study recovered ecomorph as the single-most important covariate of canal morphology in modern taxa; although phylogenetic history, size, and head dimensions also showed a small, yet significant correlation with shape. Surprisingly, perch characteristics were not found to be significant covariates of canal shape, even though they are important habitat variables. Using posterior probabilities, we found that the fossil anoles have different semicircular canals shapes to modern ecomorph groupings implying extinct anoles may have been interacting with their Miocene environment in different ways to modern Anolis species.

  14. Pushing the limits for amplifying BrdU-labeled DNA encoding 16S rRNA: DNA polymerase as the determining factor.

    PubMed

    Roux-Michollet, Dad D; Schimel, Joshua P; Holden, Patricia A

    2010-12-01

    Identifying microorganisms that are active under specific conditions in ecosystems is a challenge in microbial ecology. Recently, the bromodeoxyuridine (BrdU) technique was developed to label actively growing cells. BrdU, a thymidine analog, is incorporated into newly synthesized DNA, and the BrdU-labeled DNA is then isolated from total extractable DNA by immunocapture using a BrdU-specific antibody. Analyzing the BrdU-labeled DNA allows for assessing the actively growing community, which can then be compared to the unlabeled DNA that represents the total community. However, applying the BrdU approach to study soils has been problematic due to low DNA amounts and soil contaminants. To address these challenges, we developed a protocol, optimizing specificity and reproducibility, to amplify BrdU-labeled gene fragments encoding 16S rRNA. We found that the determining factor was the DNA polymerase: among the 13 different polymerases we tested, only 3 provided adequate yields with minimal contamination, and only two of those three produced similar amplification patterns of community DNA. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schwarz, Toni M.; Edwards, Megan R.; Diederichs, Audrey

    ABSTRACT Zaire ebolavirus(EBOV),Bundibugyo ebolavirus(BDBV), andReston ebolavirus(RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA bindingmore » affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. IMPORTANCEThe interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of theEbolavirusgenus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA

  16. VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability.

    PubMed

    Schwarz, Toni M; Edwards, Megan R; Diederichs, Audrey; Alinger, Joshua B; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

    2017-02-15

    Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition

  17. Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Blanton, M.P.; Wang, H.H.

    1990-02-06

    A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchRmore » {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.« less

  18. Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

    PubMed

    Tome, Jacob M; Ozer, Abdullah; Pagano, John M; Gheba, Dan; Schroth, Gary P; Lis, John T

    2014-06-01

    RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

  19. Novel interactions of mitochondria and reactive oxygen/nitrogen species in alcohol mediated liver disease

    PubMed Central

    Mantena, Sudheer K; King, Adrienne L; Andringa, Kelly K; Landar, Aimee; Darley-Usmar, Victor; Bailey, Shannon M

    2007-01-01

    Mitochondrial dysfunction is known to be a contributing factor to a number of diseases including chronic alcohol induced liver injury. While there is a detailed understanding of the metabolic pathways and proteins of the liver mitochondrion, little is known regarding how changes in the mitochondrial proteome may contribute to the development of hepatic pathologies. Emerging evidence indicates that reactive oxygen and nitrogen species disrupt mitochondrial function through post-translational modifications to the mitochondrial proteome. Indeed, various new affinity labeling reagents are available to test the hypothesis that post-translational modification of proteins by reactive species contributes to mitochondrial dysfunction and alcoholic fatty liver disease. Specialized proteomic techniques are also now available, which allow for identification of defects in the assembly of multi-protein complexes in mitochondria and the resolution of the highly hydrophobic proteins of the inner membrane. In this review knowledge gained from the study of changes to the mitochondrial proteome in alcoholic hepatotoxicity will be described and placed into a mechanistic framework to increase understanding of the role of mitochondrial dysfunction in liver disease. PMID:17854139

  20. Habitat affinity of resident natural enemies of the invasive Aphis glycines (Hemiptera: Aphididae), on soybean, with comments on biological control.

    PubMed

    Brewer, Michael J; Noma, Takuji

    2010-06-01

    We integrated a natural enemy survey of the broader landscape into a more traditional survey for Aphis glycines Matsumura (Hemiptera: Aphididae), parasitoids and predatory flies on soybean using A. glycines-infested soybean, Glycine max (L.) Merr., placed in cropped and noncropped plant systems to complement visual field observations. Across three sites and 5 yr, 18 parasitoids and predatory flies in total (Hymenoptera: Aphelinidae [two species] and Bracondae [seven species], Diptera: Cecidomyiidae [one species], Syrphidae [seven species], Chamaemyiidae [one species]) were detected, with significant variability in recoveries detected across plant system treatments and strong contrasts in habitat affinity detected among species. Lysiphlebus testaceipes Cresson was the most frequently detected parasitoid, and no differences in abundance were detected in cropped (soybean, wheat [Triticum aestivum L.], corn [Zea mays L.], and alfalfa [Medicago sativa L.]) and noncropped (poplar [Populus euramericana (Dode) Guinier] and early successional vegetation) areas. In contrast, Binodoxys kelloggensis Pike, Starý & Brewer had strong habitat affinity for poplar and early successional vegetation. The low recoveries seasonally and across habitats of Aphelinus asychis Walker, Aphelinus sp., and Aphidius colemoni Viereck make their suitability to A. glycines on soybean highly suspect. The widespread occurrence of many of the flies reflects their broad habitat affinity and host aphid ranges. The consistent low field observations of parasitism and predation suggest that resident parasitoids and predatory flies are unlikely to contribute substantially to A. glycines suppression, at least during the conventional time period early in the pest invasion when classical biological control activities are considered. For selected species that were relatively well represented across plant systems (i.e., L. testaceipes and Aphidoletes aphidimyza Rondani), conservation biological control efforts

  1. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Deng-Liang; Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou; Song, Yan-Ling

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the idealmore » antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.« less

  2. The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: I. Statistically annotated datasets for peptide sequences and proteins identified via the application of ICAT and tandem mass spectrometry to proteins copurifying with T cell lipid rafts.

    PubMed

    von Haller, Priska D; Yi, Eugene; Donohoe, Samuel; Vaughn, Kelly; Keller, Andrew; Nesvizhskii, Alexey I; Eng, Jimmy; Li, Xiao-jun; Goodlett, David R; Aebersold, Ruedi; Watts, Julian D

    2003-07-01

    Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same isotope-coded affinity tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin tag component of the ICAT reagents by avidin-affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.

  3. Protein sorption on polymer surfaces measured by fluorescence labels.

    PubMed

    Brynda, E; Drobník, J; Vacík, J; Kálal, J

    1978-01-01

    Fluorescence labeling can be used in studying protein sorption on various surfaces with a sensitivity of about 10(-8) g/cm2, commensurate with radioactive labeling. Fluorescamine proved to be the most suitable compound for studying protein sorption on hydrophilic gels, because, unlike fluoresceine isothiocyanate and dansylchloride, free fluorochrome does not interfere with measurements. Sorption properties of labeled serum albumin were tested on poly(2-hydroxyethyl methacrylate), on the copolymer of 2-hydroxyethyl methacrylate with methyl methacrylate, and on polyethylene. Labeling does not cause aggregation of the protein, but, as expected, it shifts and somewhat broadens its electrophoretic band while at the same time slightly raising its affinity toward hydrophobic surfaces.

  4. Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

    PubMed

    de Vega-Bartol, José J; Santos, Raquen Raissa; Simões, Marta; Miguel, Célia M

    2013-05-01

    Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

  5. Optimized molecular design of ADAPT-based HER2-imaging probes labelled with 111In and 68Ga.

    PubMed

    Lindbo, Sarah; Garousi, Javad; Mitran, Bogdan; Vorobyeva, Anzhelika; Oroujeni, Maryam; Orlova, Anna; Hober, Sophia; Tolmachev, Vladimir

    2018-06-04

    Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of 111 In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE) 3 DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C 59 - DEAVDANS-ADAPT6-GSSC and DOTA-C 61 -(HE) 3 DANS-ADAPT6-GSSC) were stably labeled with 111 In for SPECT and 68 Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. 111 In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to 68 Ga-labeled counterparts. The best performing variant was DOTA-C 61 -(HE) 3 DANS-ADAPT6-GSSC, providing tumor-to-blood ratios of 208±36 and 109±17 at 3 h for 111 In and 68 Ga labels, respectively.

  6. Active Learning by Querying Informative and Representative Examples.

    PubMed

    Huang, Sheng-Jun; Jin, Rong; Zhou, Zhi-Hua

    2014-10-01

    Active learning reduces the labeling cost by iteratively selecting the most valuable data to query their labels. It has attracted a lot of interests given the abundance of unlabeled data and the high cost of labeling. Most active learning approaches select either informative or representative unlabeled instances to query their labels, which could significantly limit their performance. Although several active learning algorithms were proposed to combine the two query selection criteria, they are usually ad hoc in finding unlabeled instances that are both informative and representative. We address this limitation by developing a principled approach, termed QUIRE, based on the min-max view of active learning. The proposed approach provides a systematic way for measuring and combining the informativeness and representativeness of an unlabeled instance. Further, by incorporating the correlation among labels, we extend the QUIRE approach to multi-label learning by actively querying instance-label pairs. Extensive experimental results show that the proposed QUIRE approach outperforms several state-of-the-art active learning approaches in both single-label and multi-label learning.

  7. Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

    PubMed

    Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

    2014-10-01

    Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

  8. Biological factors contributing to bark and ambrosia beetle species diversification.

    PubMed

    Gohli, Jostein; Kirkendall, Lawrence R; Smith, Sarah M; Cognato, Anthony I; Hulcr, Jiri; Jordal, Bjarte H

    2017-05-01

    The study of species diversification can identify the processes that shape patterns of species richness across the tree of life. Here, we perform comparative analyses of species diversification using a large dataset of bark beetles. Three examined covariates-permanent inbreeding (sibling mating), fungus farming, and major host type-represent a range of factors that may be important for speciation. We studied the association of these covariates with species diversification while controlling for evolutionary lag on adaptation. All three covariates were significantly associated with diversification, but fungus farming showed conflicting patterns between different analyses. Genera that exhibited interspecific variation in host type had higher rates of species diversification, which may suggest that host switching is a driver of species diversification or that certain host types or forest compositions facilitate colonization and thus allopatric speciation. Because permanent inbreeding is thought to facilitate dispersal, the positive association between permanent inbreeding and diversification rates suggests that dispersal ability may contribute to species richness. Bark beetles are ecologically unique; however, our results indicate that their impressive species diversity is largely driven by mechanisms shown to be important for many organism groups. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

  9. Notch and affinity boundaries in Drosophila.

    PubMed

    Herranz, Héctor; Milán, Marco

    2006-02-01

    Cells in multicellular organisms often do not intermingle freely with each other. Differential cell affinities can contribute to organizing cells into different tissues. Drosophila limbs and the vertebrate central nervous system are subdivided into compartments. Cells in adjacent compartments do not mix. Cell interactions mediated by Notch-family receptors have been implicated in the specification of these compartment boundaries. Two recent reports analyze the role of the Notch signaling pathway in the generation of an affinity boundary in the Drosophila wing. The first report analyzes the connection between Notch and the actin cytoskeleton. The second report analyzes the differential requirements of Notch and the transcription factor Suppressor of Hairless in generating the affinity boundary.

  10. Increasing the affinity of selective bZIP-binding peptides through surface residue redesign.

    PubMed

    Kaplan, Jenifer B; Reinke, Aaron W; Keating, Amy E

    2014-07-01

    The coiled-coil dimer is a prevalent protein interaction motif that is important for many cellular processes. The basic leucine-zipper (bZIP) transcription factors are one family of proteins for which coiled-coil mediated dimerization is essential for function, and misregulation of bZIPs can lead to disease states including cancer. This makes coiled coils attractive protein-protein interaction targets to disrupt using engineered molecules. Previous work designing peptides to compete with native coiled-coil interactions focused primarily on designing the core residues of the interface to achieve affinity and specificity. However, folding studies on the model bZIP GCN4 show that coiled-coil surface residues also contribute to binding affinity. Here we extend a prior study in which peptides were designed to bind tightly and specifically to representative members of each of 20 human bZIP families. These "anti-bZIP" peptides were designed with an emphasis on target-binding specificity, with contributions to design-target specificity and affinity engineered considering only the coiled-coil core residues. High-throughput testing using peptide arrays indicated many successes. We have now measured the binding affinities and specificities of anti-bZIPs that bind to FOS, XBP1, ATF6, and CREBZF in solution and tested whether redesigning the surface residues can increase design-target affinity. Incorporating residues that favor helix formation into the designs increased binding affinities in all cases, providing low-nanomolar binders of each target. However, changes in surface electrostatic interactions sometimes changed the binding specificity of the designed peptides. © 2014 The Protein Society.

  11. Let's get specific: the relationship between specificity and affinity.

    PubMed

    Eaton, B E; Gold, L; Zichi, D A

    1995-10-01

    The factors that lead to high-affinity binding are a good fit between the surfaces of the two molecules in their ground state and charge complementarity. Exactly the same factors give high specificity for a target. We argue that selection for high-affinity binding automatically leads to highly specific binding. This principle can be used to simplify screening approaches aimed at generating useful drugs.

  12. Electron affinities, molecular structures, and thermochemistry of the fluorine, chlorine and bromine substituted methyl radicals

    NASA Astrophysics Data System (ADS)

    Li, Qian-Shu; Zhao, Jun-Fang; Xie, Yaoming; Schaefer, Henry F., III

    Four independent density functional theory (DFT) methods have been employed to study the structures and electron affinities of the methyl and F-, Cl- and Br-substituted methyl radicals and their anions. The methods used have been carefully calibrated against a comprehensive tabulation of experimental electron affinities (Chemical Reviews, 2002, 102, 231). The first dissociation energies together with the vibrational frequencies of these species are also reported. The basis sets used in this work are of double- ζ plus polarization quality with additional s- and p-type diffuse functions, labelled as DZP++. Previously observed trends in the prediction of bond lengths by the DFT methods are also demonstrated for the F-, Cl- and Br-substituted methyl radicals and their anions. Generally, the Hartree-Fock/DFT hybrid methods predict shorter and more reliable bond lengths than the pure DFT methods. Neutral-anion energy differences reported in this work are the adiabatic electron affinity (EAad), the vertical electron affinity (EAvert), and the vertical detachment energy (VDE). Compared with the available experimental electron affinities, the BHLYP method predicts much lower values, while the other methods predict values (EAad, EAvert, VDE) close to each other and almost within the experimental range. For those systems without reliable experimental measurements, our best adiabatic EAs predicted by BLYP are 0.78 (CHF2), 1.23 (CHFCl), 1.44 (CHFBr), 1.61 (CHClBr), 2.24 (CF2Cl), 2.42 (CF2Br), 2.56 (CFBr2), 2.36 (CCl2Br), 2.46 (CClBr2), and 2.44 eV (CFClBr). The most striking feature of these predictions is that they display an inverse relationship between halogen electronegativity and EA. The DZP++ B3LYP method determines the vibrational frequencies in best agreement with available experimental results for this series, with an average relative error of ~2%. The value of using a variety of DFT methods is observed in that BHLYP does best for geometries, BLYP for electron

  13. The labelling index: a prognostic factor in head and neck carcinoma.

    PubMed

    Chauvel, P; Courdi, A; Gioanni, J; Vallicioni, J; Santini, J; Demard, F

    1989-03-01

    The thymidine labelling index (LI), representing the percentage of cells in the DNA-synthesis phase, was measured in vitro prior to therapy in 87 patients with squamous cell carcinoma of the head and neck, who were treated between 1977 and 1982. The LI was not related to patient age, site of the tumour, clinical stage or histological grade. Overall survival was 44.5%. Univariate analysis demonstrated that survival was affected by the following factors: (1) age: patients older than 55 had a better outcome (p = 0.03); (2) site of the tumour (p = 0.005): laryngeal tumours had the best survival; (3) clinical stage (p = 0.05). Histological grade did not influence the survival (p = 0.41). Patients having a tumour LI higher than 15.5% (mean + 1 S.D.) had a significantly lower survival than patients with lower tumour LI (p = 0.008). A multivariate analysis using the Cox model showed that clinical stage and LI kept their prognostic impact with regard to survival. Finally, survival after relapse was lower in patients with a high tumour LI. These results demonstrate that a high tumour proliferation rate is an additional factor influencing the disease outcome in head and neck carcinoma. Patients with bad prognosis defined by this parameter could be offered a more energetic treatment.

  14. 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.

  15. An improved affine projection algorithm for active noise cancellation

    NASA Astrophysics Data System (ADS)

    Zhang, Congyan; Wang, Mingjiang; Han, Yufei; Sun, Yunzhuo

    2017-08-01

    Affine projection algorithm is a signal reuse algorithm, and it has a good convergence rate compared to other traditional adaptive filtering algorithm. There are two factors that affect the performance of the algorithm, which are step factor and the projection length. In the paper, we propose a new variable step size affine projection algorithm (VSS-APA). It dynamically changes the step size according to certain rules, so that it can get smaller steady-state error and faster convergence speed. Simulation results can prove that its performance is superior to the traditional affine projection algorithm and in the active noise control (ANC) applications, the new algorithm can get very good results.

  16. Optimality of affine control system of several species in competition on a sequential batch reactor

    NASA Astrophysics Data System (ADS)

    Rodríguez, J. C.; Ramírez, H.; Gajardo, P.; Rapaport, A.

    2014-09-01

    In this paper, we analyse the optimality of affine control system of several species in competition for a single substrate on a sequential batch reactor, with the objective being to reach a given (low) level of the substrate. We allow controls to be bounded measurable functions of time plus possible impulses. A suitable modification of the dynamics leads to a slightly different optimal control problem, without impulsive controls, for which we apply different optimality conditions derived from Pontryagin principle and the Hamilton-Jacobi-Bellman equation. We thus characterise the singular trajectories of our problem as the extremal trajectories keeping the substrate at a constant level. We also establish conditions for which an immediate one impulse (IOI) strategy is optimal. Some numerical experiences are then included in order to illustrate our study and show that those conditions are also necessary to ensure the optimality of the IOI strategy.

  17. Effectiveness of protected areas for representing species and populations of terrestrial mammals in Costa Rica.

    PubMed

    González-Maya, José F; Víquez-R, Luis R; Belant, Jerrold L; Ceballos, Gerardo

    2015-01-01

    Costa Rica has one of the greatest percentages (26%) of protected land in the world. The National Protected Areas System (NPAS) of Costa Rica was established in 1976 and currently includes >190 protected areas within seven different protection categories. The effectiveness of the NPAS to represent species, populations, and areas with high species richness has not been properly evaluated. Such evaluations are fundamental to understand what is necessary to strengthen the NPAS and better protect biodiversity. We present a novel assessment of NPAS effectiveness in protecting mammal species. We compiled the geographical ranges of all terrestrial Costa Rican mammals then determined species lists for all protected areas and the estimated proportion of each species' geographic range protected. We also classified mammal species according to their conservation status using the IUCN Red List of Threatened Species. We found almost complete representation of mammal species (98.5%) in protected areas, but low relative coverage (28.3% on average) of their geographic ranges in Costa Rica and 25% of the species were classified as underprotected according to a priori representation targets. Interestingly, many species-rich areas are not protected, and at least 43% of cells covering the entire country are not included in protected areas. Though protected areas in Costa Rica represent species richness well, strategic planning for future protected areas to improve species complementarity and range protection is necessary. Our results can help to define sites where new protected areas can have a greater impact on mammal conservation, both in terms of species richness and range protection.

  18. The Cutting Edge of Affinity Electrophoresis Technology

    PubMed Central

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  19. Using digital soil maps to infer edaphic affinities of plant species in Amazonia: Problems and prospects.

    PubMed

    Moulatlet, Gabriel Massaine; Zuquim, Gabriela; Figueiredo, Fernando Oliveira Gouvêa; Lehtonen, Samuli; Emilio, Thaise; Ruokolainen, Kalle; Tuomisto, Hanna

    2017-10-01

    Amazonia combines semi-continental size with difficult access, so both current ranges of species and their ability to cope with environmental change have to be inferred from sparse field data. Although efficient techniques for modeling species distributions on the basis of a small number of species occurrences exist, their success depends on the availability of relevant environmental data layers. Soil data are important in this context, because soil properties have been found to determine plant occurrence patterns in Amazonian lowlands at all spatial scales. Here we evaluate the potential for this purpose of three digital soil maps that are freely available online: SOTERLAC, HWSD, and SoilGrids. We first tested how well they reflect local soil cation concentration as documented with 1,500 widely distributed soil samples. We found that measured soil cation concentration differed by up to two orders of magnitude between sites mapped into the same soil class. The best map-based predictor of local soil cation concentration was obtained with a regression model combining soil classes from HWSD with cation exchange capacity (CEC) from SoilGrids. Next, we evaluated to what degree the known edaphic affinities of thirteen plant species (as documented with field data from 1,200 of the soil sample sites) can be inferred from the soil maps. The species segregated clearly along the soil cation concentration gradient in the field, but only partially along the model-estimated cation concentration gradient, and hardly at all along the mapped CEC gradient. The main problems reducing the predictive ability of the soil maps were insufficient spatial resolution and/or georeferencing errors combined with thematic inaccuracy and absence of the most relevant edaphic variables. Addressing these problems would provide better models of the edaphic environment for ecological studies in Amazonia.

  20. Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy.

    PubMed

    Congdon, Erin E; Lin, Yan; Rajamohamedsait, Hameetha B; Shamir, Dov B; Krishnaswamy, Senthilkumar; Rajamohamedsait, Wajitha J; Rasool, Suhail; Gonzalez, Veronica; Levenga, Josien; Gu, Jiaping; Hoeffer, Charles; Sigurdsson, Einar M

    2016-08-30

    A few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures. Surprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer's paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6's efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly. Overall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.

  1. Prostate Cancer and Bone: The Elective Affinities

    PubMed Central

    2014-01-01

    The onset of metastases dramatically changes the prognosis of prostate cancer patients, determining increased morbidity and a drastic fall in survival expectancy. Bone is a common site of metastases in few types of cancer, and it represents the most frequent metastatic site in prostate cancer. Of note, the prevalence of tumor relapse to the bone appears to be increasing over the years, likely due to a longer overall survival of prostate cancer patients. Bone tropism represents an intriguing challenge for researchers also because the preference of prostate cancer cells for the bone is the result of a sequential series of targetable molecular events. Many factors have been associated with the peculiar ability of prostate cancer cells to migrate in bone marrow and to determine mixed osteoblastic/osteolytic lesions. As anticipated by the success of current targeted therapy aimed to block bone resorption, a better understanding of molecular affinity between prostate cancer and bone microenvironment will permit us to cure bone metastasis and to improve prognosis of prostate cancer patients. PMID:24971315

  2. Effectiveness of Protected Areas for Representing Species and Populations of Terrestrial Mammals in Costa Rica

    PubMed Central

    González-Maya, José F.; Víquez-R, Luis R.; Belant, Jerrold L.; Ceballos, Gerardo

    2015-01-01

    Costa Rica has one of the greatest percentages (26%) of protected land in the world. The National Protected Areas System (NPAS) of Costa Rica was established in 1976 and currently includes >190 protected areas within seven different protection categories. The effectiveness of the NPAS to represent species, populations, and areas with high species richness has not been properly evaluated. Such evaluations are fundamental to understand what is necessary to strengthen the NPAS and better protect biodiversity. We present a novel assessment of NPAS effectiveness in protecting mammal species. We compiled the geographical ranges of all terrestrial Costa Rican mammals then determined species lists for all protected areas and the estimated proportion of each species’ geographic range protected. We also classified mammal species according to their conservation status using the IUCN Red List of Threatened Species. We found almost complete representation of mammal species (98.5%) in protected areas, but low relative coverage (28.3% on average) of their geographic ranges in Costa Rica and 25% of the species were classified as underprotected according to a priori representation targets. Interestingly, many species-rich areas are not protected, and at least 43% of cells covering the entire country are not included in protected areas. Though protected areas in Costa Rica represent species richness well, strategic planning for future protected areas to improve species complementarity and range protection is necessary. Our results can help to define sites where new protected areas can have a greater impact on mammal conservation, both in terms of species richness and range protection. PMID:25970293

  3. Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

    PubMed Central

    Soh, Nobuaki

    2008-01-01

    Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology. PMID:27879749

  4. Intact stable isotope labeled plasma proteins from the SILAC-labeled HepG2 secretome.

    PubMed

    Mangrum, John B; Martin, Erika J; Brophy, Donald F; Hawkridge, Adam M

    2015-09-01

    The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    PubMed

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-05

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

  6. Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose

    PubMed Central

    DiScipio, Richard G.; Liddington, Robert C.; Schraufstatter, Ingrid U.

    2016-01-01

    A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

  7. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  8. Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.

    PubMed

    Fonseca, L P; Cabral, J M

    1996-01-01

    Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.

  9. High-level fluorescence labeling of gram-positive pathogens.

    PubMed

    Aymanns, Simone; Mauerer, Stefanie; van Zandbergen, Ger; Wolz, Christiane; Spellerberg, Barbara

    2011-01-01

    Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10-50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

  10. High-Level Fluorescence Labeling of Gram-Positive Pathogens

    PubMed Central

    Aymanns, Simone; Mauerer, Stefanie; van Zandbergen, Ger; Wolz, Christiane; Spellerberg, Barbara

    2011-01-01

    Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10–50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration. PMID:21731607

  11. Advances toward DNA-based identification and phylogeny of North American Armillaria species using elongation factor-1 alpha gene

    Treesearch

    Amy L. Ross-Davis; John W. Hanna; Mee-Sook Kim; Ned B. Klopfenstein

    2012-01-01

    The translation elongation factor-1 alpha gene was used to examine the phylogenetic relationships among 30 previously characterized isolates representing ten North American Armillaria species: A. solidipes (=A. ostoyae), A. gemina, A. calvescens, A. sinapina, A. mellea, A. gallica, A. nabsnona, North American biological species X, A. cepistipes, and A. tabescens. The...

  12. [Diversity and biogeographic affinities of sharks, rays and chimaeras (Chondrichthyes: Elasmobranchii, Holocephali) of Mexico].

    PubMed

    Del Moral-Flores, Luis Fernando; Morrone, Juan J; Alcocer, Javier; Pérez-Ponce de León, Gerardo

    2016-12-01

    The diversity of chondrychthyans in Mexico is described. The fauna is composed by 214 species (111 sharks, 95 rays and 8 chimaeras) and represents 17.3 % of the total number of species recorded worldwide. The families with the highest diversity comprise: Rajidae (14.5 %), Carcharhinidae (12.1 %), Pentanchidae, Triakidae, and Urotrygonidae (5.1 %). In terms of geographical distribution, the diversity on the Mexican Pacific slope reaches up to 56.1 % of those species inhabiting Mexican marine and brackish waters (120 species, 62 genera, 37 families and 14 orders); the diversity in the Atlantic slope resulted similar to that on the Mexican Pacific with 55.1 % of the species (118 species, 59 genera, 35 families and 13 orders). The biogeographical affinities of the Mexican chondrychthyan fauna are complex with 19.7 % of the species being circumglobal, 9.9 % transatlantic, 1.9 % transpacific, and 9.4 % endemic to the exclusive economic zone. Additionally, 36.6 % of the species recorded so far are endemic to the Eastern Pacific coast where the species are similar to those found in the Cortez biogeographic province (27.7 %), followed by the Californian (20.7 %), Panamanian (19.3 %), Galapagos (5.6 %) and Peruvian-Chilean (8.9 %). Likewise, 33.3 % are endemic of the Atlantic coast, where species are similar to those found in the Caribbean province (31.9 %), followed by the Carolinean (24.4 %) and the Brazilian (6.6 %).

  13. Identification of preoptic sleep neurons using retrograde labelling and gene profiling.

    PubMed

    Chung, Shinjae; Weber, Franz; Zhong, Peng; Tan, Chan Lek; Nguyen, Thuc Nghi; Beier, Kevin T; Hörmann, Nikolai; Chang, Wei-Cheng; Zhang, Zhe; Do, Johnny Phong; Yao, Shenqin; Krashes, Michael J; Tasic, Bosiljka; Cetin, Ali; Zeng, Hongkui; Knight, Zachary A; Luo, Liqun; Dan, Yang

    2017-05-25

    In humans and other mammalian species, lesions in the preoptic area of the hypothalamus cause profound sleep impairment, indicating a crucial role of the preoptic area in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry have shown the existence of sleep-active neurons in the preoptic area, especially in the ventrolateral preoptic area and median preoptic nucleus. Pharmacogenetic activation of c-Fos-labelled sleep-active neurons has been shown to induce sleep. However, the sleep-active neurons are spatially intermingled with wake-active neurons, making it difficult to target the sleep neurons specifically for circuit analysis. Here we identify a population of preoptic area sleep neurons on the basis of their projection target and discover their molecular markers. Using a lentivirus expressing channelrhodopsin-2 or a light-activated chloride channel for retrograde labelling, bidirectional optogenetic manipulation, and optrode recording, we show that the preoptic area GABAergic neurons projecting to the tuberomammillary nucleus are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification and single-cell RNA sequencing identify candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrate that several peptide markers (cholecystokinin, corticotropin-releasing hormone, and tachykinin 1) label sleep-promoting neurons. Together, these findings provide easy genetic access to sleep-promoting preoptic area neurons and a valuable entry point for dissecting the sleep control circuit.

  14. Factors affecting the efficient transformation of Colletotrichum species

    USGS Publications Warehouse

    Redman, Regina S.; Rodriguez, Rusty J.

    1994-01-01

    Factors affecting the efficient transformation of Colletotrichum species. Experimental Mycology, 18, 230-246. Twelve isolates representing four species of Colletotrichum were transformed either by enhanced protoplast, restriction enzyme-mediated integration (REMI), or electroporation-mediated protocols. The enhanced protoplast transformation protocol resulted in 100- and 50-fold increases in the transformation efficiencies of Colletotrichum lindemuthianum and C. magna , respectively. REMI transformation involved the use of Hin dIII and vector DNA linearized with HindIII to increase the number of integration events and potential gene disruptions in the fungal genome. Combining the enhanced protoplast and the REMI protocols resulted in a 22-fold increase in the number of hygromycin/nystatin-resistant mutants in C. lindemuthianum . Electroporation-mediated transformation was performed on mycelial fragments and spores of four Colletotrichum species, resulting in efficiencies of up to 1000 transformants/μg DNA. The pHA1.3 vector which confers hygromycin resistance contains telomeric sequences from Fusarium oxysporum , transforms by autonomous replication and genomic integration, and was essential for elevated transformation efficiencies of 100 to 10,000 transformants/μg DNA. Modifications of pHA1.3 occurred during bacterial amplification and post fungal transformation resulting in plasmids capable of significantly elevated transformation efficiencies in C. lindemuthianum.

  15. Fluorescein-labeled stable neurotensin derivatives.

    PubMed

    Maes, Veronique; Hultsch, Christina; Kohl, Suzann; Bergmann, Ralf; Hanke, Thomas; Tourwé, Dirk

    2006-08-01

    Neurotensin(8-13) analogs containing a glycine or 5-aminovaleroyl spacer were labeled with fluorescein through formation of an N-terminal thiourea function. The receptor binding was measured in HT-29 cell cultures and showed a substantial decrease in affinity, especially for the metabolically stabilized [MeArg(9), Tle(11)] analog. Using fluorescence microscopy, the internalization of the fluorescent neurotensin analogs into HT-29 cells was observed. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

  16. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was appliedmore » to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.« less

  17. N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging.

    PubMed

    Gourni, Eleni; Mansi, Rosalba; Jamous, Mazen; Waser, Beatrice; Smerling, Christiane; Burian, Antje; Buchegger, Franz; Reubi, Jean Claude; Maecke, Helmut R

    2014-10-01

    Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated

  18. Environmental and Individual Factors Affecting Menu Labeling Utilization: A Qualitative Research Study

    PubMed Central

    Schindler, Jennifer; Kiszko, Kamila; Abrams, Courtney; Islam, Nadia; Elbel, Brian

    2013-01-01

    Obesity is a significant public health concern that disproportionally affects low-income and minority populations. Recent policies mandating the posting of calories on menus in fast food chain restaurants have not proven to uniformly influence food choice. This qualitative research study uses focus groups to study individual and environmental factors affecting the usage of these menu labels among low-income, minority populations. Ten focus groups targeting low-income residents (n=105) were conducted at various community organizations throughout NYC in Spanish, English, or a combination of both languages, over a nine-month period in 2011. In late 2011 and early 2012, transcripts were coded through the process of thematic analysis using Atlas.ti for naturally emerging themes, influences, and determinants of food choice. Few used menu labels, despite awareness. Among the themes pertaining to menu label usage, price and time constraints, confusion and lack of understanding of caloric values, as well as the priority of preference, hunger, and habitual ordering habits were most frequently cited as barriers to menu label usage. Based on the individual and external influences on food choice that often take priority over calorie consideration, a modified approach may be necessary to make menu labels more effective and user-friendly. PMID:23402695

  19. Identification of a receptor for ADP on blood platelets by photoaffinity labelling.

    PubMed Central

    Cristalli, G; Mills, D C

    1993-01-01

    The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase. Images Figure 5 PMID:8387782

  20. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Middleton, R.E.; Cohen, J.B.

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with highmore » affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.« less

  1. Nutrition Label Viewing during a Food-Selection Task: Front-of-Package Labels vs Nutrition Facts Labels.

    PubMed

    Graham, Dan J; Heidrick, Charles; Hodgin, Katie

    2015-10-01

    Earlier research has identified consumer characteristics associated with viewing Nutrition Facts labels; however, little is known about those who view front-of-package nutrition labels. Front-of-package nutrition labels might appeal to more consumers than do Nutrition Facts labels, but it might be necessary to provide consumers with information about how to locate and use these labels. This study quantifies Nutrition Facts and front-of-package nutrition label viewing among American adult consumers. Attention to nutrition information was measured during a food-selection task. One hundred and twenty-three parents (mean age=38 years, mean body mass index [calculated as kg/m(2)]=28) and one of their children (aged 6 to 9 years) selected six foods from a university laboratory-turned-grocery aisle. Participants were randomized to conditions in which front-of-package nutrition labels were present or absent, and signage explaining front-of-package nutrition labels was present or absent. Adults' visual attention to Nutrition Facts labels and front-of-package nutrition labels was objectively measured via eye-tracking glasses. To examine whether there were significant differences in the percentages of participants who viewed Nutrition Facts labels vs front-of-package nutrition labels, McNemar's tests were conducted across all participants, as well as within various sociodemographic categories. To determine whether hypothesized factors, such as health literacy and education, had stronger relationships with front-of-package nutrition label vs Nutrition Facts label viewing, linear regression assessed the magnitude of relationships between theoretically and empirically derived factors and each type of label viewing. Overall, front-of-package nutrition labels were more likely to be viewed than Nutrition Facts labels; however, for all subgroups, higher rates of front-of-package nutrition label viewership occurred only when signage was present drawing attention to the presence and

  2. Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

    PubMed

    Hua, Weiwei; Lou, Yimin; Xu, Weiyuan; Cheng, Zhixian; Gong, Xingwen; Huang, Jianying

    2016-01-01

    Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

  3. Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sine, Steven M.; Huang, Sun; Li, Shu-Xing

    2013-09-01

    The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

  4. 2D Affine and Projective Shape Analysis.

    PubMed

    Bryner, Darshan; Klassen, Eric; Huiling Le; Srivastava, Anuj

    2014-05-01

    Current techniques for shape analysis tend to seek invariance to similarity transformations (rotation, translation, and scale), but certain imaging situations require invariance to larger groups, such as affine or projective groups. Here we present a general Riemannian framework for shape analysis of planar objects where metrics and related quantities are invariant to affine and projective groups. Highlighting two possibilities for representing object boundaries-ordered points (or landmarks) and parameterized curves-we study different combinations of these representations (points and curves) and transformations (affine and projective). Specifically, we provide solutions to three out of four situations and develop algorithms for computing geodesics and intrinsic sample statistics, leading up to Gaussian-type statistical models, and classifying test shapes using such models learned from training data. In the case of parameterized curves, we also achieve the desired goal of invariance to re-parameterizations. The geodesics are constructed by particularizing the path-straightening algorithm to geometries of current manifolds and are used, in turn, to compute shape statistics and Gaussian-type shape models. We demonstrate these ideas using a number of examples from shape and activity recognition.

  5. Factors that Predict How Women Label Their Own Childhood Sexual Abuse

    ERIC Educational Resources Information Center

    Katerndahl, David; Burge, Sandra; Kellogg, Nancy

    2006-01-01

    Despite the psychological impact of child sexual abuse, many victims do not acknowledge that their experiences were "abuse." This study sought to identify factors that predict how women label their own experiences of childhood sexual abuse. This cross-sectional study was conducted in a family medicine clinic with adult female patients. Subjects…

  6. Species delimitation using Bayes factors: simulations and application to the Sceloporus scalaris species group (Squamata: Phrynosomatidae).

    PubMed

    Grummer, Jared A; Bryson, Robert W; Reeder, Tod W

    2014-03-01

    Current molecular methods of species delimitation are limited by the types of species delimitation models and scenarios that can be tested. Bayes factors allow for more flexibility in testing non-nested species delimitation models and hypotheses of individual assignment to alternative lineages. Here, we examined the efficacy of Bayes factors in delimiting species through simulations and empirical data from the Sceloporus scalaris species group. Marginal-likelihood scores of competing species delimitation models, from which Bayes factor values were compared, were estimated with four different methods: harmonic mean estimation (HME), smoothed harmonic mean estimation (sHME), path-sampling/thermodynamic integration (PS), and stepping-stone (SS) analysis. We also performed model selection using a posterior simulation-based analog of the Akaike information criterion through Markov chain Monte Carlo analysis (AICM). Bayes factor species delimitation results from the empirical data were then compared with results from the reversible-jump MCMC (rjMCMC) coalescent-based species delimitation method Bayesian Phylogenetics and Phylogeography (BP&P). Simulation results show that HME and sHME perform poorly compared with PS and SS marginal-likelihood estimators when identifying the true species delimitation model. Furthermore, Bayes factor delimitation (BFD) of species showed improved performance when species limits are tested by reassigning individuals between species, as opposed to either lumping or splitting lineages. In the empirical data, BFD through PS and SS analyses, as well as the rjMCMC method, each provide support for the recognition of all scalaris group taxa as independent evolutionary lineages. Bayes factor species delimitation and BP&P also support the recognition of three previously undescribed lineages. In both simulated and empirical data sets, harmonic and smoothed harmonic mean marginal-likelihood estimators provided much higher marginal-likelihood estimates

  7. Nanozeolite bioconjugates labeled with 223Ra for targeted alpha therapy.

    PubMed

    Piotrowska, Agata; Męczyńska-Wielgosz, Sylwia; Majkowska-Pilip, Agnieszka; Koźmiński, Przemysław; Wójciuk, Grzegorz; Cędrowska, Edyta; Bruchertseifer, Frank; Morgenstern, Alfred; Kruszewski, Marcin; Bilewicz, Aleksander

    2017-04-01

    Alpha particle emitting isotopes are of considerable interest for radionuclide therapy because of their high cytotoxicity and short path length. Among the many α emitters, 223 Ra exhibits very attractive nuclear properties for application in radionuclide therapy. The decay of this radioisotope and its daughters is accompanied by the emission of four α-particles, releasing 27.9MeV of cumulative energy. Unfortunately the lack of an appropriate bifunctional ligand for radium has so far been a main obstacle for the application of 223 Ra in receptor targeted therapy. In our studies we investigated the use of nanozeolite-Substance P bioconjugates as vehicles for 223 Ra radionuclides for targeted α therapy. The sodium form of an A-type of nanozeolite (NaA) was synthesized using the template method. Next, the nanozeolite particles were conjugated to the Substance P (5-11) peptide fragment, which targets NK-1 receptors on glioma cells. The obtained bioconjugate was characterized by transmission emission spectroscopy, thermogravimetric analysis and dynamic light scattering analysis. The NaA-silane-PEG-SP(5-11) bioconjugates were labeled with 223 Ra by exchange of the Na + cation and the stability, receptor affinity and cytotoxicity of the obtained radiobioconjugates were tested. The 223 Ra-labeled nanozeolite bioconjugate almost quantitatively retains 223 Ra in vitro after 6days, while the retention of decay products varies from 90 to 95%. The synthesized 223 RaA-silane-PEG-SP(5-11) showed high receptor affinity toward NK-1 receptor expressing glioma cells and exhibited a high cytotoxic effect in vitro. Substance P functionalized nanozeolite-A represents a viable solution for the use of the 223 Ra in vivo generator as a therapeutic construct for targeting glioma cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe

    NASA Astrophysics Data System (ADS)

    Koh, Eunjung; Song, Ha Jeong; Kwon, Na Young; Kim, Gi Won; Lee, Kwang Ho; Jo, Soyeon; Park, Sujin; Park, Jihyun; Park, Eun Kyeong; Hwang, Seung Yong

    2017-06-01

    Real time PCR is a standard method for identification of species. One of limitations of the qPCR is that there would be false-positive result due to mismatched hybridization between target sequence and probe depending on the annealing temperature in the PCR condition. As an alternative, fluorescence melting curve analysis (FMCA) could be applied for species identification. FMCA is based on a dual-labeled probe. Even with subtle difference of target sequence, there are visible melting temperature (Tm) shift. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by species or country of origin. However, counterfeiting or distributing them without any verification procedure are becoming social problems and threatening food safety. Besides distinguishing them in naked eye is very difficult and almost impossible in any processed form. Therefore, it is necessary to identify species in molecular level. In this research three species of squids which have 1-2 base pair differences each are selected as samples since they have the same issue. We designed a probe which perfectly matches with one species and the others mismatches 2 and 1 base pair respectively and labeled with fluorophore and quencher. In an experiment with a single probe, we successfully distinguished them by Tm shift depending on the difference of base pair. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, andc furthermore, enabling results analysis with smart phone would realize point-of-care testing (POCT).

  9. Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Morris, D.I.; Robbins, J.D.; Ruoho, A.E.

    1991-07-15

    Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of (3H)forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited (3H)forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP {gamma} S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolinmore » but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP {gamma} S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.« less

  10. Modulators of haemocyanin oxygen affinity in the hypoxia- and sulphide-tolerant Baltic isopod Saduria entomon (L.).

    PubMed

    Hagerman, L; Vismann, B

    2001-11-01

    Dialysed haemocyanin from the isopod Saduria entomon had a considerably increased oxygen affinity (lower P50) and Bohr factor (-1.71) compared to native haemocyanin (Bohr factor -1.36) indicating that dialysis removes a small molecule size modulating factor decreasing the affinity of native haemolymph. Dialysed haemocyanin had a slightly lower co-operativity (2.42 +/- 0.3) than native haemocyanin (2.9 +/- 0.2). L-Lactate (10 mmol l(-1)) improved oxygen affinity by 1-1.5 torr while urate had no effect. Mg2+ affected affinity in a pH-dependent manner (Bohr-factor increased to -1.67) while Ca2+ had no effect on the Bohr factor but increased affinity with ca 1 torr. Thiosulphate changed the Bohr factor to -1.75 to -1.82, similar to dialysed blood. Co-operativity was in neither case affected. The haemocyanin characteristics of S. entomon are similar to those of crustaceans from hydrothermal vents. These characteristics are probably general for crustaceans that are more or less permanently exposed to sulphide.

  11. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    PubMed Central

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  12. Development of QSAR models for predicting the binding affinity of endocrine disrupting chemicals to eight fish estrogen receptor.

    PubMed

    He, Junyi; Peng, Tao; Yang, Xianhai; Liu, Huihui

    2018-02-01

    Endocrine disrupting effect has become a central point of concern, and various biological mechanisms involve in the disruption of endocrine system. Recently, we have explored the mechanism of disrupting hormonal transport protein, through the binding affinity of sex hormone-binding globulin in different fish species. This study, serving as a companion article, focused on the mechanism of activating/inhibiting hormone receptor, by investigating the binding interaction of chemicals with the estrogen receptor (ER) of different fish species. We collected the relative binding affinity (RBA) of chemicals with 17β-estradiol binding to the ER of eight fish species. With this parameter as the endpoints, quantitative structure-activity relationship (QSAR) models were established using DRAGON descriptors. Statistical results indicated that the developed models had satisfactory goodness of fit, robustness and predictive ability. The Euclidean distance and Williams plot verified that these models had wide application domains, which covered a large number of structurally diverse chemicals. Based on the screened descriptors, we proposed an appropriate mechanism interpretation for the binding potency. Additionally, even though the same chemical had different affinities for ER from different fish species, the affinity of ER exhibited a high correlation for fish species within the same Order (i.e., Salmoniformes, Cypriniformes, Perciformes), which consistent with that in our previous study. Hence, when performing the endocrine disrupting effect assessment, the species diversity should be taken into account, but maybe the fish species in the same Order can be grouped together. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Selective retention of basic compounds by metal aquo-ion affinity chromatography.

    PubMed

    Asakawa, Yoshiki; Yamamoto, Eiichi; Asakawa, Naoki

    2014-10-01

    A novel metal aquo-ion affinity chromatography has been developed for the analysis of basic compounds using heat-treated silica gel containing hydrated metal cations (metal aquo-ions) as the packing material. The packing materials of the metal aquo-ion affinity chromatography were prepared by the immobilization of a single metal component such as Fe(III), Al(III), Ag(I), and Ni(II) on silica gel followed by extensive heat treatment. The immobilized metals form aquo-ions to present cation-exchange ability for basic analytes and the cation-exchange ability for basic analytes depends on pKa of the immobilized metal species. In the present study, to evaluate the retention characteristics of metal aquo-ion affinity chromatography, the on-line solid-phase extraction of drugs was investigated. Obtained data clearly evidence the selective retention capability of metal aquo-ion affinity chromatography for basic analytes with sufficient capacity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Demura, T.; Driscoll, W.J.; Lee, Y.C.

    1991-01-01

    Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinctmore » from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.« less

  15. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-05-01

    Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no furthermore » increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.« less

  16. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction

    PubMed Central

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K.; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G.; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H.

    2017-01-01

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively. PMID:27899623

  17. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

    PubMed

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Factors associated with nutrition label use among female college students applying the theory of planned behavior.

    PubMed

    Lim, Hyun Jeong; Kim, Min Ju; Kim, Kyung Won

    2015-02-01

    Use of nutrition labels in food selection is recommended for consumers. The aim of this study is to examine factors, mainly beliefs explaining nutrition label use in female college students based on the Theory of Planned Behavior (TPB). The subjects were female college students from a university in Seoul, Korea. The survey questionnaire was composed of items examining general characteristics, nutrition label use, behavioral beliefs, normative beliefs, corresponding motivation to comply, and control beliefs. The subjects (n = 300) responded to the questionnaire by self-report, and data from 275 students were analyzed using t-test or χ(2)-test. The results showed that 37.8% of subjects were nutrition label users. Three out of 15 behavioral beliefs differed significantly by nutrition label use. Nutrition label users agreed more strongly on the benefits of using nutrition labels including 'comparing and selecting better foods' (P < 0.001), 'selecting healthy foods' (P < 0.05). The negative belief of 'annoying' was stronger in non-users than in users (P < 0.001). Three out of 7 sources (parents, siblings, best friend) were important in nutrition label use. Twelve out of 15 control beliefs differed significantly by nutrition label use. These included beliefs regarding constraints of using nutrition labels (e.g., time, spending money for healthy foods) and lack of nutrition knowledge (P < 0.001). Perceived confidence in understanding and applying the specifics of nutrition labels in food selection was also significantly related to nutrition label use (P < 0.001). This study found that the beliefs, especially control beliefs, suggested in the TPB were important in explaining nutrition label use. To promote nutrition label use, nutrition education might focus on increasing perceived control over constraints of using nutrition labels, acquiring skills for checking nutrition labels, as well as the benefits of using nutrition labels and receiving support from significant

  19. High affinity binding of 125I-neurotensin to dispersed cells from chicken liver and brain.

    PubMed

    Mitra, S P; Carraway, R E

    1997-01-01

    Dispersed cells from chicken brain and liver were found to possess cell surface binding sites for 125I-neurotensin (125I-NT). Scatchard analyses indicated the presence of high affinity (K4, 25-80 pM) and low affinity (Kd, 250-450 pM) components in adult tissues. Binding capacity was reduced 25-40% by incubation with pertussis toxin. Ontogenetic studies indicated that NT receptor capacity increased approximately 20-fold from the embryonic stage to adult. Cross-linking of 125I-NT to intact cells labeled one major band (52 kDa, > or = 90%) and two minor bands (40 and 90 kDa, < or = 10%) which could represent distinct NT-receptors or one receptor partly degraded or cross-linked to G-protein(s). The binding of 125I-NT to dispersed cells was enhanced by reduction with dithoithreitol and suppressed by alkylation with N-ethyl-maleimide (NEM), maleimidocaproic acid (MCA) and p-chloromercuribenzenesulfonate (PCMBS). Since MCA and PCMBS do not permeate cells, this suggests that the sulfhydryl group(s) critical to binding are located within the NT receptor itself. Preincubation of cells with NT prior to treatment with NEM diminished its inhibitory effect, suggesting that the critical SH-group(s) were within the NT binding pocket or were protected by an allosteric effect. These results suggest that one or more of the nine cysteine residues in the NT receptor is involved in the NT binding reaction.

  20. Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ooi, G.T.; Herington, A.C.

    1986-05-29

    Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When /sup 125/I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, while the remaining three (MW 37 K, 34 K and 18 K) appeared as minor bands only. Inhibitor bioactivity, followingmore » further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands.« less

  1. Effect of single point mutations of the human tachykinin NK1 receptor on antagonist affinity.

    PubMed

    Lundstrom, K; Hawcock, A B; Vargas, A; Ward, P; Thomas, P; Naylor, A

    1997-10-15

    Molecular modelling and site-directed mutagenesis were used to identify eleven amino acid residues which may be involved in antagonist binding of the human tachykinin NK1 receptor. Recombinant receptors were expressed in mammalian cells using the Semliki Forest virus system. Wild type and mutant receptors showed similar expression levels in BHK and CHO cells, verified by metabolic labelling. Binding affinities were determined for a variety of tachykinin NK1 receptor antagonists in SFV-infected CHO cells. The binding affinity for GR203040, CP 99,994 and CP 96,345 was significantly reduced by mutant Q165A. The mutant F268A significantly reduced the affinity for GR203040 and CP 99,994 and the mutant H197A had reduced affinity for CP 96,345. All antagonists seemed to bind in a similar region of the receptor, but do not all rely on the same binding site interactions. Functional coupling to G-proteins was assayed by intracellular Ca2+ release in SFV-infected CHO cells. The wild type receptor and all mutants except A162L and F268A responded to substance P stimulation.

  2. Species decline: Contaminants and other contributing factors

    USGS Publications Warehouse

    Pattee, O.H.; Rattner, B.A.; Eisler, R.

    1998-01-01

    Members of over 1,200 taxa have been listed as Threatened or Endangered, and over 4,000 additional organisms have been identified as Candidate Species or Species of Concern. Identification of critical limiting factors may result in management actions that stabilize vulnerable populations and insure their perpetuation. Both naturally-occurring and anthropogenic activities (e.g., environmental contaminants and pollution) have been demonstrated to be a significant factor in depressing populations or catalyzing the final crash of some species. The objective of this project is to develop a synthesis document and database that lists and ranks the presumed causes of decline, with special emphasis on contaminants and pollutant-related situations. This will be accomplished by synoptic review of all recovery plans (n=479) with listing packages (n=1134) serving as a secondary source of information, followed by itemization, cross-referencing, enumeration, and ranking of contributing and limiting factors. To date we have analyzed all of the recovery plans for reptiles (n=26) and amphibians (n=6). 188 causes are defined, falling into 6 major categories: habitat alteration/availability (47.8%); exploitation/harvest (19.7%); introduction of exotic species (10.1%); contaminants (9.0%); miscellaneous others (6.9%); pollution (6.4%). The applicability of these data are extensive, including facilitating reviews of Section 7 consultations and Environmental Impact Statements, reviewing permit applications, conducting environmental contaminant risk assessments, identifying specific data gaps and research needs, selecting potential management actions, and establishing priorities for broad-based research on limiting factors applicable to groups of species rather than the current species-by-species approach. However. caution must be exercised in the use of this data because of the speculative nature of the causes; most of the causes (69.7%) are based on poorly documented expert opinion and

  3. Co-Labeling for Multi-View Weakly Labeled Learning.

    PubMed

    Xu, Xinxing; Li, Wen; Xu, Dong; Tsang, Ivor W

    2016-06-01

    It is often expensive and time consuming to collect labeled training samples in many real-world applications. To reduce human effort on annotating training samples, many machine learning techniques (e.g., semi-supervised learning (SSL), multi-instance learning (MIL), etc.) have been studied to exploit weakly labeled training samples. Meanwhile, when the training data is represented with multiple types of features, many multi-view learning methods have shown that classifiers trained on different views can help each other to better utilize the unlabeled training samples for the SSL task. In this paper, we study a new learning problem called multi-view weakly labeled learning, in which we aim to develop a unified approach to learn robust classifiers by effectively utilizing different types of weakly labeled multi-view data from a broad range of tasks including SSL, MIL and relative outlier detection (ROD). We propose an effective approach called co-labeling to solve the multi-view weakly labeled learning problem. Specifically, we model the learning problem on each view as a weakly labeled learning problem, which aims to learn an optimal classifier from a set of pseudo-label vectors generated by using the classifiers trained from other views. Unlike traditional co-training approaches using a single pseudo-label vector for training each classifier, our co-labeling approach explores different strategies to utilize the predictions from different views, biases and iterations for generating the pseudo-label vectors, making our approach more robust for real-world applications. Moreover, to further improve the weakly labeled learning on each view, we also exploit the inherent group structure in the pseudo-label vectors generated from different strategies, which leads to a new multi-layer multiple kernel learning problem. Promising results for text-based image retrieval on the NUS-WIDE dataset as well as news classification and text categorization on several real-world multi

  4. Lp-dual affine surface area

    NASA Astrophysics Data System (ADS)

    Wei, Wang; Binwu, He

    2008-12-01

    According to the notion of Lp-affine surface area by Lutwak, in this paper, we introduce the concept of Lp-dual affine surface area. Further, we establish the affine isoperimetric inequality and the Blaschke-Santaló inequality for Lp-dual affine surface area. Besides, the dual Brunn-Minkowski inequality for Lp-dual affine surface area is presented.

  5. Brain tumor detection and segmentation in a CRF (conditional random fields) framework with pixel-pairwise affinity and superpixel-level features.

    PubMed

    Wu, Wei; Chen, Albert Y C; Zhao, Liang; Corso, Jason J

    2014-03-01

    Detection and segmentation of a brain tumor such as glioblastoma multiforme (GBM) in magnetic resonance (MR) images are often challenging due to its intrinsically heterogeneous signal characteristics. A robust segmentation method for brain tumor MRI scans was developed and tested. Simple thresholds and statistical methods are unable to adequately segment the various elements of the GBM, such as local contrast enhancement, necrosis, and edema. Most voxel-based methods cannot achieve satisfactory results in larger data sets, and the methods based on generative or discriminative models have intrinsic limitations during application, such as small sample set learning and transfer. A new method was developed to overcome these challenges. Multimodal MR images are segmented into superpixels using algorithms to alleviate the sampling issue and to improve the sample representativeness. Next, features were extracted from the superpixels using multi-level Gabor wavelet filters. Based on the features, a support vector machine (SVM) model and an affinity metric model for tumors were trained to overcome the limitations of previous generative models. Based on the output of the SVM and spatial affinity models, conditional random fields theory was applied to segment the tumor in a maximum a posteriori fashion given the smoothness prior defined by our affinity model. Finally, labeling noise was removed using "structural knowledge" such as the symmetrical and continuous characteristics of the tumor in spatial domain. The system was evaluated with 20 GBM cases and the BraTS challenge data set. Dice coefficients were computed, and the results were highly consistent with those reported by Zikic et al. (MICCAI 2012, Lecture notes in computer science. vol 7512, pp 369-376, 2012). A brain tumor segmentation method using model-aware affinity demonstrates comparable performance with other state-of-the art algorithms.

  6. Differential identification of Candida species and other yeasts by analysis of (/sup 35/S)methionine-labeled polypeptide profiles

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shen, H.D.; Choo, K.B.; Tsai, W.C.

    1988-12-01

    This paper describes a scheme for differential identification of Candida species and other yeasts based on autoradiographic analysis of protein profiles of (/sup 35/S)methionine-labeled cellular proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using ATCC strains as references, protein profile analysis showed that different Candida and other yeast species produced distinctively different patterns. Good agreement in results obtained with this approach and with other conventional systems was observed. Being accurate and reproducible, this approach provides a basis for the development of an alternative method for the identification of yeasts isolated from clinical specimens.

  7. Molybdenum disulfide (MoS2) nanoflakes as inherently electroactive labels for DNA hybridization detection

    NASA Astrophysics Data System (ADS)

    Loo, Adeline Huiling; Bonanni, Alessandra; Ambrosi, Adriano; Pumera, Martin

    2014-09-01

    The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide nanomaterials for sensing and biosensing purposes represents an upcoming research area which holds great promise. Hence, our findings are anticipated to have significant contributions towards the fabrication of future DNA biosensors.The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide

  8. Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

    PubMed

    Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

    2015-09-04

    The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Factors associated with physicians' reliance on pharmaceutical sales representatives.

    PubMed

    Anderson, Britta L; Silverman, Gabriel K; Loewenstein, George F; Zinberg, Stanley; Schulkin, Jay

    2009-08-01

    To examine relationships between pharmaceutical representatives and obstetrician-gynecologists and identify factors associated with self-reported reliance on representatives when making prescribing decisions. In 2006-2007, questionnaires were mailed to 515 randomly selected physicians in the American College of Obstetricians and Gynecologists' Collaborative Ambulatory Research Network. Participants were asked about the information sources used when deciding to prescribe a new drug, interactions with sales representatives, views of representatives' value, and guidelines they had read on appropriate industry interactions. Two hundred fifty-one completed questionnaires (49%) were returned. Seventy-six percent of participants see sales representatives' information as at least somewhat valuable. Twenty-nine percent use representatives often or almost always when deciding whether to prescribe a new drug; 44% use them sometimes. Physicians in private practice are more likely than those in university hospitals to interact with, value, and rely on representatives; community hospital physicians tend to fall in the middle. Gender and age are not associated with industry interaction. Dispensing samples is associated with increased reliance on representatives when making prescribing decisions, beyond what is predicted by a physician's own beliefs about the value of representatives' information. Reading guidelines on physician-industry interaction is not associated with less reliance on representatives after controlling for practice setting. Physicians' interactions with industry and their familiarity with guidelines vary by practice setting, perhaps because of more restrictive policies in university settings, professional isolation of private practice, or differences in social norms. Prescribing samples may be associated with physicians' use of information from sales representatives more than is merited by the physicians' own beliefs about the value of pharmaceutical

  10. Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration* ♦

    PubMed Central

    Koenig, Patrick; Lee, Chingwei V.; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F.; Fuh, Germaine

    2015-01-01

    The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137

  11. Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

    DOE PAGES

    Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick; ...

    2017-11-24

    Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

  12. Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick

    Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

  13. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

    PubMed

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H

    2017-01-09

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Factors associated with nutrition label use among female college students applying the theory of planned behavior

    PubMed Central

    Lim, Hyun Jeong; Kim, Min Ju

    2015-01-01

    BACKGROUND/OBJECTIVES Use of nutrition labels in food selection is recommended for consumers. The aim of this study is to examine factors, mainly beliefs explaining nutrition label use in female college students based on the Theory of Planned Behavior (TPB). SUBJECTS/METHODS The subjects were female college students from a university in Seoul, Korea. The survey questionnaire was composed of items examining general characteristics, nutrition label use, behavioral beliefs, normative beliefs, corresponding motivation to comply, and control beliefs. The subjects (n = 300) responded to the questionnaire by self-report, and data from 275 students were analyzed using t-test or χ2-test. RESULTS The results showed that 37.8% of subjects were nutrition label users. Three out of 15 behavioral beliefs differed significantly by nutrition label use. Nutrition label users agreed more strongly on the benefits of using nutrition labels including 'comparing and selecting better foods' (P < 0.001), 'selecting healthy foods' (P < 0.05). The negative belief of 'annoying' was stronger in non-users than in users (P < 0.001). Three out of 7 sources (parents, siblings, best friend) were important in nutrition label use. Twelve out of 15 control beliefs differed significantly by nutrition label use. These included beliefs regarding constraints of using nutrition labels (e.g., time, spending money for healthy foods) and lack of nutrition knowledge (P < 0.001). Perceived confidence in understanding and applying the specifics of nutrition labels in food selection was also significantly related to nutrition label use (P < 0.001). CONCLUSIONS This study found that the beliefs, especially control beliefs, suggested in the TPB were important in explaining nutrition label use. To promote nutrition label use, nutrition education might focus on increasing perceived control over constraints of using nutrition labels, acquiring skills for checking nutrition labels, as well as the benefits of using

  15. Robust multi-atlas label propagation by deep sparse representation

    PubMed Central

    Zu, Chen; Wang, Zhengxia; Zhang, Daoqiang; Liang, Peipeng; Shi, Yonghong; Shen, Dinggang; Wu, Guorong

    2016-01-01

    Recently, multi-atlas patch-based label fusion has achieved many successes in medical imaging area. The basic assumption in the current state-of-the-art approaches is that the image patch at the target image point can be represented by a patch dictionary consisting of atlas patches from registered atlas images. Therefore, the label at the target image point can be determined by fusing labels of atlas image patches with similar anatomical structures. However, such assumption on image patch representation does not always hold in label fusion since (1) the image content within the patch may be corrupted due to noise and artifact; and (2) the distribution of morphometric patterns among atlas patches might be unbalanced such that the majority patterns can dominate label fusion result over other minority patterns. The violation of the above basic assumptions could significantly undermine the label fusion accuracy. To overcome these issues, we first consider forming label-specific group for the atlas patches with the same label. Then, we alter the conventional flat and shallow dictionary to a deep multi-layer structure, where the top layer (label-specific dictionaries) consists of groups of representative atlas patches and the subsequent layers (residual dictionaries) hierarchically encode the patchwise residual information in different scales. Thus, the label fusion follows the representation consensus across representative dictionaries. However, the representation of target patch in each group is iteratively optimized by using the representative atlas patches in each label-specific dictionary exclusively to match the principal patterns and also using all residual patterns across groups collaboratively to overcome the issue that some groups might be absent of certain variation patterns presented in the target image patch. Promising segmentation results have been achieved in labeling hippocampus on ADNI dataset, as well as basal ganglia and brainstem structures, compared

  16. Robust multi-atlas label propagation by deep sparse representation.

    PubMed

    Zu, Chen; Wang, Zhengxia; Zhang, Daoqiang; Liang, Peipeng; Shi, Yonghong; Shen, Dinggang; Wu, Guorong

    2017-03-01

    Recently, multi-atlas patch-based label fusion has achieved many successes in medical imaging area. The basic assumption in the current state-of-the-art approaches is that the image patch at the target image point can be represented by a patch dictionary consisting of atlas patches from registered atlas images. Therefore, the label at the target image point can be determined by fusing labels of atlas image patches with similar anatomical structures. However, such assumption on image patch representation does not always hold in label fusion since (1) the image content within the patch may be corrupted due to noise and artifact; and (2) the distribution of morphometric patterns among atlas patches might be unbalanced such that the majority patterns can dominate label fusion result over other minority patterns. The violation of the above basic assumptions could significantly undermine the label fusion accuracy. To overcome these issues, we first consider forming label-specific group for the atlas patches with the same label. Then, we alter the conventional flat and shallow dictionary to a deep multi-layer structure, where the top layer ( label-specific dictionaries ) consists of groups of representative atlas patches and the subsequent layers ( residual dictionaries ) hierarchically encode the patchwise residual information in different scales. Thus, the label fusion follows the representation consensus across representative dictionaries. However, the representation of target patch in each group is iteratively optimized by using the representative atlas patches in each label-specific dictionary exclusively to match the principal patterns and also using all residual patterns across groups collaboratively to overcome the issue that some groups might be absent of certain variation patterns presented in the target image patch. Promising segmentation results have been achieved in labeling hippocampus on ADNI dataset, as well as basal ganglia and brainstem structures

  17. A chirality change in XPC- and Sfi1-derived peptides affects their affinity for centrin.

    PubMed

    Grecu, Dora; Irudayaraj, Victor Paul Raj; Martinez-Sanz, Juan; Mallet, Jean-Maurice; Assairi, Liliane

    2016-04-01

    The Ca(2+)-binding protein centrin binds to a hydrophobic motif (W(1)xxL(4)xxxL(8)) included in the sequence of several cellular targets: XPC (xeroderma pigmentosum group C protein), Sfi1 (suppressor of fermentation-induced loss of stress resistance protein1), and Sac3 [the central component of the transcription and mRNA export (TREX-2) complex]. However, centrin binding occurs in a reversed orientation (L(8)xxxL(4)xxW(1)) for Sfi1 and Sac3 compared with XPC. Because D-peptides have been investigated for future therapeutic use, we analyzed their centrin-binding properties. Their affinity for centrin was measured using isothermal titration calorimetry. The chirality change in the target-derived peptides affected their ability to bind centrin in a specific manner depending on the sequence orientation of the centrin-binding motif. In contrast to L-XPC-P10, D-XPC-P10 bound C-HsCen1 in a Ca(2+)-dependent manner and to a lesser extent. D-XPC-P10 exhibited a reduced affinity for C-HsCen1 (Ka=0.064 × 10(6) M(-1)) by a factor of 2000 compared with L-XPC-P10 (Ka=132 × 10(6) M(-1)). D-peptides have a lower affinity than L-peptides for centrin, and the strength of this affinity depends on the sequence orientation of the target-derived peptides. The residual affinity observed for D-XPC suggests that the use of d-peptides represents a promising strategy for inhibiting centrin binding to its targets. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

    PubMed

    Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

    2016-12-02

    Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

  19. Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

    PubMed

    Dagnino, Sonia; Bellet, Virginie; Grimaldi, Marina; Riu, Anne; Aït-Aïssa, Sélim; Cavaillès, Vincent; Fenet, Hélène; Balaguer, Patrick

    2014-02-01

    Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

  20. Important biological factors for utilizing native plant species

    Treesearch

    Loren E. Wiesner

    1999-01-01

    Native plant species are valuable resources for revegetation of disturbed ecosystems. The success of these plantings is dependent on the native species selected, quality of seed used, condition of the soil, environmental conditions before and after planting, planting equipment used, time of planting, and other factors. Most native species contain dormant seed. Dormancy...

  1. Quantification of transcription factor-DNA binding affinity in a living cell

    PubMed Central

    Belikov, Sergey; Berg, Otto G.; Wrange, Örjan

    2016-01-01

    The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

  2. Erikson Psychosocial Stage Inventory: A Factor Analysis

    ERIC Educational Resources Information Center

    Gray, Mary McPhail; And Others

    1986-01-01

    The 72-item Erikson Psychosocial Stage Inventory (EPSI) was factor analyzed for a group of 534 university freshmen and sophomore students. Seven factors emerged, which were labeled Initiative, Industry, Identity, Friendship, Dating, Goal Clarity, and Self-Confidence. Item's representing Erikson's factors, Trust and Autonomy, were dispersed across…

  3. Paper-based immune-affinity arrays for detection of multiple mycotoxins in cereals.

    PubMed

    Li, Li; Chen, Hongpu; Lv, Xiaolan; Wang, Min; Jiang, Xizhi; Jiang, Yifei; Wang, Heye; Zhao, Yongfu; Xia, Liru

    2018-03-01

    Mycotoxins produced by different species of fungi may coexist in cereals and feedstuffs, and could be highly toxic for humans and animals. For quantification of multiple mycotoxins in cereals, we developed a paper-based mycotoxin immune-affinity array. First, paper-based microzone arrays were fabricated by photolithography. Then, monoclonal mycotoxin antibodies were added in a copolymerization reaction with a cross-linker to form an immune-affinity monolith on the paper-based microzone array. With use of a competitive immune-response format, paper-based mycotoxin immune-affinity arrays were successfully applied to detect mycotoxins in samples. The detection limits for deoxynivalenol, zearalenone, T-2 toxin, and HT-2 toxin were 62.7, 10.8, 0.36, and 0.23 μg·kg -1 , respectively, which meet relevant requirements for these compounds in food. The recovery rates were 81-86% for deoxynivalenol, 89-117% for zearalenone, 79-86% for T-2 toxin, and 78-83% for HT-2 toxin, and showed the paper-based immune-affinity arrays had good reproducibility. In summary, the paper-based mycotoxin immune-affinity array provides a sensitive, rapid, accurate, stable, and convenient platform for detection of multiple mycotoxins in agro-foods. Graphical abstract Paper-based immune-affinity monolithic array. DON deoxynivalenol, HT-2 HT-2 toxin, T-2 T-2 toxin, PEGDA polyethylene glycol diacrylate, ZEN zearalenone.

  4. Label-Free Aptasensors for the Detection of Mycotoxins

    PubMed Central

    Rhouati, Amina; Catanante, Gaelle; Nunes, Gilvanda; Hayat, Akhtar; Marty, Jean-Louis

    2016-01-01

    Various methodologies have been reported in the literature for the qualitative and quantitative monitoring of mycotoxins in food and feed samples. Based on their enhanced specificity, selectivity and versatility, bio-affinity assays have inspired many researchers to develop sensors by exploring bio-recognition phenomena. However, a significant problem in the fabrication of these devices is that most of the biomolecules do not generate an easily measurable signal upon binding to the target analytes, and signal-generating labels are required to perform the measurements. In this context, aptamers have been emerged as a potential and attractive bio-recognition element to design label-free aptasensors for various target analytes. Contrary to other bioreceptor-based approaches, the aptamer-based assays rely on antigen binding-induced conformational changes or oligomerization states rather than binding-assisted changes in adsorbed mass or charge. This review will focus on current designs in label-free conformational switchable design strategies, with a particular focus on applications in the detection of mycotoxins. PMID:27999353

  5. How Much Binding Affinity Can be Gained by Filling a Cavity?

    PubMed Central

    Kawasaki, Yuko; Chufan, Eduardo E.; Lafont, Virginie; Hidaka, Koushi; Kiso, Yoshiaki; Amzel, L. Mario; Freire, Ernesto

    2011-01-01

    Binding affinity optimization is critical during drug development. Here we evaluate the thermodynamic consequences of filling a binding cavity with functionalities of increasing van der Waals radii (-H, -F, -Cl and CH3) that improve the geometric fit without participating in hydrogen bonding or other specific interactions. We observe a binding affinity increase of two orders of magnitude. There appears to be three phases in the process. The first phase is associated with the formation of stable van der Waals interactions. This phase is characterized by a gain in binding enthalpy and a loss in binding entropy, attributed to a loss of conformational degrees of freedom. For the specific case presented in this paper, the enthalpy gain amounts to −1.5 kcal/mol while the entropic losses amount to +0.9 kcal/mol resulting in a net 3.5-fold affinity gain. The second phase is characterized by simultaneous enthalpic and entropic gains. This phase improves the binding affinity 25-fold. The third phase represents the collapse of the trend and is triggered by the introduction of chemical functionalities larger than the binding cavity itself (CH(CH3)2). It is characterized by large enthalpy and affinity losses. The thermodynamic signatures associated with each phase provide guidelines for lead optimization. PMID:20028396

  6. Algal autolysate medium to label proteins for NMR in mammalian cells.

    PubMed

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia; Neri, Sara; Fragai, Marco

    2016-04-01

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

  7. Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

    PubMed

    Horejsí, V; Tichá, M; Kocourek, J

    1977-09-29

    Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

  8. Expression of the high-affinity choline transporter CHT1 in rat and human arteries.

    PubMed

    Lips, Katrin S; Pfeil, Uwe; Reiners, Katja; Rimasch, Christoph; Kuchelmeister, Klaus; Braun-Dullaeus, Ruediger C; Haberberger, Rainer V; Schmidt, Rupert; Kummer, Wolfgang

    2003-12-01

    The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.

  9. Affinity in electrophoresis.

    PubMed

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  10. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

    PubMed

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-09-01

    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  11. Solitons and the energy-momentum tensor for affine Toda theory

    NASA Astrophysics Data System (ADS)

    Olive, D. I.; Turok, N.; Underwood, J. W. R.

    1993-07-01

    Following Leznov and Saveliev, we present the general solution to Toda field theories of conformal, affine or conformal affine type, associated with a simple Lie algebra g. These depend on a free massless field and on a group element. By putting the former to zero, soliton solutions to the affine Toda theories with imaginary coupling constant result with the soliton data encoded in the group element. As this requires a reformulation of the affine Kac-Moody algebra closely related to that already used to formulate the physical properties of the particle excitations, including their scattering matrices, a unified treatment of particles and solitons emerges. The physical energy—momentum tensor for a general solution is broken into a total derivative plus a part dependent only on the derivatives of the free field. Despite the non-linearity of the field equations and their complex nature the energy and momentum of the N-soliton solution is shown to be real, equalling the sum of contributions from the individual solitons. There are rank-g species of soliton, with masses given by a generalisation of a formula due to Hollowood, being proportional to the components of the left Perron-Frobenius eigenvector of the Cartan matrix of g.

  12. Corynebacterium endocarditis species-specific risk factors and outcomes

    PubMed Central

    Belmares, Jaime; Detterline, Stephanie; Pak, Janet B; Parada, Jorge P

    2007-01-01

    Background Corynebacterium species are recognized as uncommon agents of endocarditis, but little is known regarding species-specific risk factors and outcomes in Corynebacterium endocarditis. Methods Case report and Medline search of English language journals for cases of Corynebacterium endocarditis. Inclusion criteria required that cases be identified as endocarditis, having persistent Corynebacterium bacteremia, murmurs described by the authors as identifying the affected valve, or vegetations found by echocardiography or in surgical or autopsy specimens. Cases also required patient-specific information on risk factors and outcomes (age, gender, prior prosthetic valve, other prior nosocomial risk factors (infected valve, involvement of native versus prosthetic valve, need for valve replacement, and death) to be included in the analysis. Publications of Corynebacterium endocarditis which reported aggregate data were excluded. Univariate analysis was conducted with chi-square and t-tests, as appropriate, with p = 0.05 considered significant. Results 129 cases of Corynebacterium endocarditis involving nine species met inclusion criteria. Corynebacterium endocarditis typically infects the left heart of adult males and nearly one third of patients have underlying valvular disease. One quarter of patients required valve replacement and one half of patients died. Toxigenic C. diphtheriae is associated with pediatric infections (p < 0.001). Only C. amycolatum has a predilection for women (p = 0.024), while C. pseudodiphtheriticum infections are most frequent in men (p = 0.023). C. striatum, C. jeikeium and C. hemolyticum are associated with nosocomial risk factors (p < 0.001, 0.028, and 0.024, respectively). No species was found to have a predilection for any particular heart valve. C. pseudodiphtheriticum is associated with a previous prosthetic valve replacement (p = 0.004). C. jeikeium infections are more likely to require valve replacement (p = 0.026). Infections

  13. Consumer knowledge and attitudes toward nutritional labels.

    PubMed

    Cannoosamy, Komeela; Pugo-Gunsam, Prity; Jeewon, Rajesh

    2014-01-01

    To determine Mauritian consumers' attitudes toward nutritional labels based on the Kano model and to identify determinants of the use and understanding of nutrition labels. The researchers also used a Kano model questionnaire to determine consumers' attitudes toward nutrition labeling. Four hundred consumers residing in Mauritius. Information was elicited via a questionnaire that assessed nutritional knowledge and information about the use and understanding of nutritional labels and demographic factors. Nutritional label use and understanding, nutrition knowledge, and association of demographic factors with label use. Statistical tests performed included 1-way ANOVA and independent samples t tests. Statistically significant relationships (P < .05) were found for nutritional knowledge and nutritional label usage with demographic factors. All demographic factors with the exception of gender were significantly associated (P < .05) with nutritional label understanding. Based on the outcome of the Kano survey, calorie content, trans fat content, protein content, and cholesterol content were found to be must-be attributes: that is, attributes that, when not present, result in consumer dissatisfaction. Age, education, income, household size, and nutrition knowledge had an impact on nutritional label use. Health promoters should aim to increase the use of nutritional labels. Copyright © 2014 Society for Nutrition Education and Behavior. Published by Elsevier Inc. All rights reserved.

  14. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

    PubMed

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

    2002-07-01

    Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

  15. [125I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors

    PubMed Central

    Dumont, Yvan; Quirion, Rémi

    2000-01-01

    GR231118 (also known as 1229U91 and GW1229), a purported Y1 antagonist and Y4 agonist was radiolabelled using the chloramine T method. [125I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [125I]-GR231118 binds with very high affinity (Kd of 0.09–0.24 nM) in transfected HEK293 cells with the rat Y1 and Y4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y2 or Y5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [125I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y1, but not rat Y4, receptor cDNA. Autoradiographic studies demonstrated that [125I]-GR231118 binding sites were fully inhibited by the Y1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [125I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [125I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y4 receptor subtype. In summary, [125I]-GR231118 is a new radiolabelled probe to investigate the Y1 and Y4 receptors; its major advantage being its high affinity. Using highly selective Y1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [125I]-GR231118 to the Y1 receptor allowing for the characterization and visualization of the purported Y4 subtype. PMID:10694200

  16. THE 1985 NAPAP EMISSIONS INVENTORY: DEVELOPMENT OF SPECIES ALLOCATION FACTORS

    EPA Science Inventory

    The report describes the methodologies and data bases used to develop species allocation factors and data processing software used to develop the 1985 National Acid Precipitation Assessment Program (NAPAP) Modelers' Emissions Inventory (Version 2). Species allocation factors were...

  17. Identifying the principal driving factors of water ecosystem dependence and the corresponding indicator species in a pilot City, China

    NASA Astrophysics Data System (ADS)

    Zhao, C. S.; Shao, N. F.; Yang, S. T.; Xiang, H.; Lou, H. Z.; Sun, Y.; Yang, Z. Y.; Zhang, Y.; Yu, X. Y.; Zhang, C. B.; Yu, Q.

    2018-01-01

    The world's aquatic ecosystems yield numerous vital services, which are essential to human existence but have deteriorated seriously in recent years. By studying the mechanisms of interaction between ecosystems and habitat processes, the constraining factors can be identified, and this knowledge can be used to improve the success rate of ecological restoration initiatives. At present, there is insufficient data on the link between hydrological, water quality factors and the changes in the structure of aquatic communities to allow any meaningful study of driving factors of aquatic ecosystems. In this study, the typical monitoring stations were selected by fuzzy clustering analysis based on the spatial and temporal distribution characteristics of water ecology in Jinan City, the first pilot city for the construction of civilized aquatic ecosystems in China. The dominant species identification model was used to identify the dominant species of the aquatic community. The driving effect of hydrological and water quality factors on dominant species was analyzed by Canonical Correspondence Analysis. Then, the principal factors of aquatic ecosystem dependence were selected. The results showed that there were 10 typical monitoring stations out of 59 monitoring sites, which were representative of aquatic ecosystems, 9 dominant fish species, and 20 dominant invertebrate species. The selection of factors for aquatic ecosystem dependence in Jinan were highly influenced by its regional conditions. Chemical environmental parameters influence the temporal and spatial variation of invertebrate much more than that of fish in Jinan City. However, the methodologies coupling typical monitoring stations selection, dominant species determination and driving factors identification were certified to be a cost-effective way, which can provide in-deep theoretical and technical directions for the restoration of aquatic ecosystems elsewhere.

  18. Recommendations for representative ballast water sampling

    NASA Astrophysics Data System (ADS)

    Gollasch, Stephan; David, Matej

    2017-05-01

    Until now, the purpose of ballast water sampling studies was predominantly limited to general scientific interest to determine the variety of species arriving in ballast water in a recipient port. Knowing the variety of species arriving in ballast water also contributes to the assessment of relative species introduction vector importance. Further, some sampling campaigns addressed awareness raising or the determination of organism numbers per water volume to evaluate the species introduction risk by analysing the propagule pressure of species. A new aspect of ballast water sampling, which this contribution addresses, is compliance monitoring and enforcement of ballast water management standards as set by, e.g., the IMO Ballast Water Management Convention. To achieve this, sampling methods which result in representative ballast water samples are essential. We recommend such methods based on practical tests conducted on two commercial vessels also considering results from our previous studies. The results show that different sampling approaches influence the results regarding viable organism concentrations in ballast water samples. It was observed that the sampling duration (i.e., length of the sampling process), timing (i.e., in which point in time of the discharge the sample is taken), the number of samples and the sampled water quantity are the main factors influencing the concentrations of viable organisms in a ballast water sample. Based on our findings we provide recommendations for representative ballast water sampling.

  19. An assessment of pharmacists' readiness for paperless labeling: a national survey.

    PubMed

    Ho, Yun-Xian; Chen, Qingxia; Nian, Hui; Johnson, Kevin B

    2014-01-01

    To assess the state of readiness for the adoption of paperless labeling among a nationally representative sample of pharmacies, including chain pharmacies, independent retail pharmacies, hospitals, and other rural or urban dispensing sites. Both quantitative and qualitative analyses were used to analyze responses to a cross-sectional survey disseminated to American Pharmacists Association pharmacists nationwide. The survey assessed factors related to pharmacists' attitudinal readiness (ie, perceptions of impact) and pharmacies' structural readiness (eg, availability of electronic resources, internet access) for the paperless labeling initiative. We received a total of 436 survey responses (6% response rate) from pharmacists representing 44 US states and territories. Across the spectrum of settings we studied, pharmacists had work access to computers, printers, fax machines and access to the internet or intranet. Approximately 79% of respondents believed that the initiative would improve the adequacy of drug information available in their work site and 95% believed it would either not change (33%) or would improve (62%) communication to patients. Overall, respondents' comments supported advancing the initiative; however, some comments revealed reservations regarding corporate or pharmacy buy-in, success of implementation, and ease of adoption. This is the first nationwide study to report about pharmacists' perspectives on paperless labeling. In general, pharmacists believe they are ready and that their pharmacies are well equipped for the transition to paperless labeling. Further exploration of perspectives from product label manufacturers and corporate pharmacy offices is needed to understand fully what will be necessary to complete this transition.

  20. A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo.

    PubMed

    Naudin, Clément; Schumski, Ariane; Salo-Ahen, Outi M H; Herwald, Heiko; Smeds, Emanuel

    2017-05-01

    Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well-characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA-based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost-effective method that can be used to prevent unnecessary animal experiments. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  1. Atrial natriuretic factor receptor heterogeneity in rat tissues

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Andresen, J.W.; Kuno, T.; Kamisaki, Y.

    1986-03-01

    Rat /sup 125/I-atrial natriuretic factor (ANF, 8-33) was used to identify ANF receptors in membrane preparations from rat adrenal gland and lung. When solubilized with Lubrol-PX, the receptors retained a binding profile and properties that correspond to the high affinity and specificity found in crude membranes. Single peaks of binding activity were observed in gel permeation HPLC and density gradient centrifugation analysis of the solubilized preparations. However, when membranes and solubilized preparations were labeled with /sup 125/I-ANF, treated with crosslinking reagent (disuccinimidyl suberate), and analyzed by SDS gel electrophoresis several specifically labeled bands (120,000, 70,000, and 60,000 daltons) were identifiedmore » by autoradiography. The relative distribution of the specifically labeled proteins varied significantly between rat adrenal gland and lung. In adrenal glands the 120K dalton band was the most prominent specifically labeled protein, while the 60K and 70K dalton proteins were labeled to a lesser degree. In lung membranes the lower molecular weight proteins were more prominent. These results suggest the presence of multiple ANF receptor subtypes, the distribution of which varies among tissues. Chromatographic separation and further characterization of these receptors are currently in progress, and preliminary purification studies support this hypothesis.« less

  2. Novel Curvularia species from clinical specimens.

    PubMed

    Madrid, H; da Cunha, K C; Gené, J; Dijksterhuis, J; Cano, J; Sutton, D A; Guarro, J; Crous, P W

    2014-12-01

    The fungal genus Curvularia includes numerous plant pathogens and some emerging opportunistic pathogens of humans. In a previous study we used morphology and sequences of the nuclear ribosomal internal transcribed spacer region (ITS) and the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene to identify species within a set of 99 clinical Curvularia isolates from the USA. Seventy-two isolates could be identified while the remaining 27 isolates belonged in three unclassified clades that were tentatively labelled Curvularia sp. I, II and III. In the present study, we further assess the taxonomic placement of these isolates using sequences of ITS, gpd, the large subunit rDNA, and the second largest subunit of RNA polymerase II. DNA sequence comparisons with a set of 87 isolates representing 33 Curvularia spp. and members of the closely-related genera Bipolaris and Exserohilum revealed that Curvularia sp. I, II and III represent novel lineages in Curvularia. These lineages are morphologically different from the currently accepted species. In the phylogenetic tree, Curvularia sp. I and sp. III were each split into two distinct lineages. Morphology and phylogeny supported the proposal of five new species, to be named C. americana, C. chlamydospora, C. hominis, C. muehlenbeckiae and C. pseudolunata. The concatenated 4-locus phylogeny revealed the existence of six clades in Curvularia, which are associated with particular morphological features. They were named after representative species, namely americana, eragrostidis, hominis, lunata, spicifera and trifolii.

  3. Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent.

    PubMed

    Kim, Eun-Mi; Joung, Min-Hee; Lee, Chang-Moon; Jeong, Hwan-Jeong; Lim, Seok Tae; Sohn, Myung-Hee; Kim, Dong Wook

    2010-07-15

    The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the 'click-to-chelate' protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12, [(99m)Tc(CO)(3)]13, and [(99m)Tc(CO)(3)]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)(3)]12, [Re(CO)(3)]13, and [Re(CO)(3)]14, were shown to have high binding affinities (0.13 microM, 0.06 microM, and 0.16 microM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12-14) to c-Met receptor positive U87MG cells. 2010 Elsevier Ltd. All rights reserved.

  4. Non-affine deformations in polymer hydrogels

    PubMed Central

    Wen, Qi; Basu, Anindita; Janmey, Paul A.; Yodh, A. G.

    2012-01-01

    Most theories of soft matter elasticity assume that the local strain in a sample after deformation is identical everywhere and equal to the macroscopic strain, or equivalently that the deformation is affine. We discuss the elasticity of hydrogels of crosslinked polymers with special attention to affine and non-affine theories of elasticity. Experimental procedures to measure non-affine deformations are also described. Entropic theories, which account for gel elasticity based on stretching out individual polymer chains, predict affine deformations. In contrast, simulations of network deformation that result in bending of the stiff constituent filaments generally predict non-affine behavior. Results from experiments show significant non-affine deformation in hydrogels even when they are formed by flexible polymers for which bending would appear to be negligible compared to stretching. However, this finding is not necessarily an experimental proof of the non-affine model for elasticity. We emphasize the insights gained from experiments using confocal rheoscope and show that, in addition to filament bending, sample micro-inhomogeneity can be a significant alternative source of non-affine deformation. PMID:23002395

  5. Intracellular processing of epidermal growth factor. I. Acidification of 125I-epidermal growth factor in intracellular organelles.

    PubMed

    Matrisian, L M; Planck, S R; Magun, B E

    1984-03-10

    We previously reported that 125I-labeled epidermal growth factor is processed intracellularly to acidic macromolecules in Rat-1 fibroblasts. The present study defines the precursor-product relationship and localization of the processing steps to subcellular organelles by the use of a single isoelectric species of 125I-epidermal growth factor and Percoll gradient fractionation. The native pI 4.55 125I-epidermal growth factor was rapidly processed to a pI 4.2 species on or near the cell surface and in organelles corresponding to clathrin-coated vesicles, Golgi, and endoplasmic reticulum. This species was then processed to a pI 4.35 species in similar organelles. The pI 4.2 and 4.35 species were converted to a pI 4.0 species in dense, lysosome-like organelles. This species was ultimately degraded and exocytosed from the cell as low molecular weight products.

  6. Ceratopetalum (Cunoniaceae) fruits of Australasian affinity from the early Eocene Laguna del Hunco flora, Patagonia, Argentina

    PubMed Central

    Hermsen, Elizabeth J

    2017-01-01

    Abstract Background and Aims Radially symmetrical, five-winged fossil fruits from the highly diverse early Eocene Laguna del Hunco flora of Chubut Province, Patagonia, Argentina, are named, described and illustrated. The main goals are to assess the affinities of the fossils and to place them in an evolutionary, palaeoecological and biogeographic context. Methods Specimens of fossil fruits were collected from the Tufolitas Laguna del Hunco. They were prepared, photographed and compared with similar extant and fossil fruits using published literature. Their structure was also evaluated by comparing them with that of modern Ceratopetalum (Cunoniaceae) fruits through examination of herbarium specimens. Key Results The Laguna del Hunco fossil fruits share the diagnostic features that characterize modern and fossil Ceratopetalum (symmetry, number of fruit wings, presence of a conspicuous floral nectary and overall venation pattern). The pattern of the minor wing (sepal) veins observed in the Patagonian fossil fruits is different from that of modern and previously described fossil Ceratopetalum fruits; therefore, a new fossil species is recognized. An apomorphy (absence of petals) suggests that the fossils belong within crown-group Ceratopetalum. Conclusions The Patagonian fossil fruits are the oldest known record for Ceratopetalum. Because the affinities, provenance and age of the fossils are so well established, this new Ceratopetalum fossil species is an excellent candidate for use as a calibration point in divergence dating studies of the family Cunoniaceae. It represents the only record of Ceratopetalum outside Australasia, and further corroborates the biogeographic connection between the Laguna del Hunco flora and ancient and modern floras of the Australasian region. PMID:28110267

  7. Optimization of Time-Resolved Fluorescence Assay for Detection of Eu-DOTA-labeled Ligand-Receptor Interactions

    PubMed Central

    De Silva, Channa R.; Vagner, Josef; Lynch, Ronald; Gillies, Robert J.; Hruby, Victor J.

    2010-01-01

    Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improved sensitivity and affordability in high throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as DTPA derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIA) have not yet been successfully used with more stable chelators, e.g. DOTA derivatives, due to the incomplete release of lanthanide(III) ions from the complex. Here, a modified and an optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA labeled peptides. Complete release of Eu(III) ions from DOTA labeled ligands was observed using hydrochloric acid (2.0 M) prior to the luminescent enhancement step. NDP-α-MSH labeled with Eu(III)-DOTA was synthesized and the binding affinity to cells overexpressing the human melanocortin-4 receptors (hMC4R) was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA labeled heterobivalent peptide to the cells expressing both hMC4R and CCK-2 (Cholecystokinin) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries. PMID:19852924

  8. [An examination of the determinants of social withdrawal and affinity for social withdrawal].

    PubMed

    Watanabe, Asami; Matsui, Yutaka; Takatsuka, Yusuke

    2010-12-01

    This study examined the determinants of social withdrawal using data from a survey by the Tokyo Metropolitan Government Office for Youth Affairs and Public Safety (2008). In addition, this study identified young people who showed an affinity for social withdrawal although they were not in a state of withdrawal, and examined the determinants of an affinity for social withdrawal. The results of stepwise discriminant analysis showed that factors such as social phobia, depression, violence, and emotional bonds with family differentiated between the general youth group and the social withdrawal group and the "affinity group". Social phobia, violence, and refusal to be interfered in self-decision making differentiated between the social withdrawal group and the "affinity group". This study shows that an "affinity group" should be cared as well as an actual withdrawal group.

  9. Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays

    EPA Science Inventory

    The development of a predictive model based upon a single aquatic species inevitably raises the question of whether this information is valid for other species. To partially address this question, relative binding affinities (RBA) for six alkylphenols (para-substituted, n- and b...

  10. Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition.

    PubMed

    Lambert, Jean-Philippe; Ivosev, Gordana; Couzens, Amber L; Larsen, Brett; Taipale, Mikko; Lin, Zhen-Yuan; Zhong, Quan; Lindquist, Susan; Vidal, Marc; Aebersold, Ruedi; Pawson, Tony; Bonner, Ron; Tate, Stephen; Gingras, Anne-Claude

    2013-12-01

    Characterizing changes in protein-protein interactions associated with sequence variants (e.g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies.

  11. A genetically encoded and gate for cell-targeted metabolic labeling of proteins.

    PubMed

    Mahdavi, Alborz; Segall-Shapiro, Thomas H; Kou, Songzi; Jindal, Granton A; Hoff, Kevin G; Liu, Shirley; Chitsaz, Mohsen; Ismagilov, Rustem F; Silberg, Jonathan J; Tirrell, David A

    2013-02-27

    We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNA(Met). Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.

  12. A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins

    PubMed Central

    Mahdavi, Alborz; Segall-Shapiro, Thomas H.; Kou, Songzi; Jindal, Granton A.; Hoff, Kevin G.; Liu, Shirley; Chitsaz, Mohsen; Ismagilov, Rustem F.; Silberg, Jonathan J.; Tirrell, David A.

    2013-01-01

    We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within five minutes after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals. PMID:23406315

  13. Smilax (Smilacaceae) from the Miocene of western Eurasia with Caribbean biogeographic affinities.

    PubMed

    Denk, Thomas; Velitzelos, Dimitrios; Güner, H Tuncay; Ferrufino-Acosta, Lilian

    2015-03-01

    • Recent molecular studies provide a phylogenetic framework and some dated nodes for the monocot genus Smilax. The Caribbean Havanensis group of Smilax is part of a well-supported "New World clade" with a few disjunct taxa in the Old World. Although the fossil record of the genus is rich, it has been difficult to assign fossil taxa to extant groups based on their preserved morphological characters.• Leaf fossils from Europe and Asia Minor were studied comparatively and put into a phylogenetic and biogeographic context using a molecular phylogeny of the genus.• Fossils from the early Miocene of Anatolia represent a new species of Smilax with systematic affinities with the Havanensis group. The leaf type encountered in the fossil species is exclusively found in species of the Havanensis group among all modern Smilax. Scattered fossils of this type from the Miocene of Greece and Austria, previously referred to Quercus (Fagaceae), Ilex (Aquifoliaceae), and Mahonia (Berberidaceae) also belong to the new species.• The new Smilax provides first fossil evidence of the Havanensis group and proves that this group had a western Eurasian distribution during the Miocene. The age of the fossils is in good agreement with the (molecular-based) purported split between the Havanensis and Hispida groups within Smilax. The Miocene Smilax provides evidence that all four subclades within the "New World clade" had a disjunct intercontinental distribution during parts of the Neogene involving trans-Atlantic crossings (via floating islands or the North Atlantic land bridge) and the Beringia land bridge. © 2015 Botanical Society of America, Inc.

  14. Factors Influencing Labeling Nonconsensual Sex as Sexual Assault.

    PubMed

    Yndo, Monica C; Zawacki, Tina

    2017-03-01

    The current study examined the effects of physical attractiveness and sexual interest cues on men's sexual perceptions of women and whether increases in sexual perceptions of a woman would lead to decreases in labeling of subsequent nonconsensual sex as sexual assault. Two hundred thirty-three male college students ( M age = 19.17, SD = 1.22) read a vignette describing a hypothetical social interaction between a man and a woman; within the vignette, the female character's physical attractiveness (attractive vs. less attractive) and the degree to which the female character behaved interested in the male character (uninterested vs. ambiguous) were manipulated. The vignette ends with the male character physically forcing sexual intercourse with the female character. After reading the vignette, participants' labeling of the nonconsensual sex as sexual assault was addressed. Participants' perceptions of the female character's sexual interest in the male character prior to the nonconsensual sex was assessed as a dependent variable during stopping points in the vignette, prior to sexual assault. Both physical attractiveness and interest cues had a significant positive influence on men's perception of the female character as sexually interested. In addition, perceptions of sexual interest had a direct negative effect on sexual assault labeling. These results indicate that increases in physical attractiveness and interest cues increase perceptions of sexual interest, in turn decreasing the labeling of nonconsensual sex as sexual assault. This experimental research contributes to the literature on misperception of sexual interest and sexual assault labeling. These findings provide implications for intervention programs and for forensic issues related to sexual assault.

  15. Lp-mixed affine surface area

    NASA Astrophysics Data System (ADS)

    Wang, Weidong; Leng, Gangsong

    2007-11-01

    According to the three notions of mixed affine surface area, Lp-affine surface area and Lp-mixed affine surface area proposed by Lutwak, in this article, we give the concept of ith Lp-mixed affine surface area such that the first and second notions of Lutwak are its special cases. Further, some Lutwak's results are extended associated with this concept. Besides, applying this concept, we establish an inequality for the volumes and dual quermassintegrals of a class of star bodies.

  16. Linking hard and soft traits: Physiology, morphology and anatomy interact to determine habitat affinities to soil water availability in herbaceous dicots.

    PubMed

    Belluau, Michaël; Shipley, Bill

    2018-01-01

    Species' habitat affinities along environmental gradients should be determined by a combination of physiological (hard) and morpho-anatomical (soft) traits. Using a gradient of soil water availability, we address three questions: How well can we predict habitat affinities from hard traits, from soft traits, and from a combination of the two? How well can we predict species' physiological responses to drought (hard traits) from their soft traits? Can we model a causal sequence as soft traits → hard traits → species distributions? We chose 25 species of herbaceous dicots whose affinities for soil moisture have already been linked to 5 physiological traits (stomatal conductance and net photosynthesis measured at soil field capacity, water use efficiency, stomatal conductance and soil water potential measured when leaves begin to wilt). Under controlled conditions in soils at field capacity, we measured five soft traits (leaf dry matter content, specific leaf area, leaf nitrogen content, stomatal area, specific root length). Soft traits alone were poor predictors (R2 = 0.129) while hard traits explained 48% of species habitat affinities. Moreover, hard traits were significantly related to combinations of soft traits. From a priori biological knowledge and hypothesized ecological links we built a path model showing a sequential pattern soft traits → hard traits → species distributions and accounting for 59.6% (p = 0.782) of habitat wetness. Both direct and indirect causal relationships existed between soft traits, hard traits and species' habitat preferences. The poor predictive abilities of soft traits alone were due to the existence of antagonistic and synergistic direct and indirect effects of soft traits on habitat preferences mediated by the hard traits. To obtain a more realistic model applicable to a population level, it has to be tested in an experiment including species competition for water supply.

  17. Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

    PubMed Central

    Hu, L.; Borleske, B. L.; Colman, R. F.

    1997-01-01

    Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes. PMID:9007975

  18. Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

    PubMed

    Hu, L; Borleske, B L; Colman, R F

    1997-01-01

    Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.

  19. Food Labels Survey

    MedlinePlus

    ... R EPORTS ® N ATIONAL R ESEARCH C ENTER Survey Research Report Food Labels Survey 2016 Nationally-Representative Phone Survey April 6, 2016 Consumer Reports® National Research Center Introduction In February, 2016, the Consumer Reports® ...

  20. 16 CFR Appendix K to Part 305 - Representative Average Unit Energy Costs

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 1 2014-01-01 2014-01-01 false Representative Average Unit Energy Costs K... CONGRESS ENERGY AND WATER USE LABELING FOR CONSUMER PRODUCTS UNDER THE ENERGY POLICY AND CONSERVATION ACT (âENERGY LABELING RULEâ) Pt. 305, App. K Appendix K to Part 305—Representative Average Unit Energy...

  1. Effect of 2,3-diphosphoglycerate on oxygen affinity of blood in sickle cell anemia

    PubMed Central

    Charache, Samuel; Grisolia, Santiago; Fiedler, Adam J.; Hellegers, Andre E.

    1970-01-01

    Blood of patients with sickle cell anemia (SS) exhibits decreased affinity for oxygen, although the oxygen affinity of hemoglobin S is the same as that of hemoglobin A. SS red cells contain more 2,3-diphosphoglycerate (DPG) than normal erythrocytes. The oxygen affinity of hemolyzed red cells is decreased by added DPG, and hemolysates prepared from SS red cells do not differ from normal hemolysates in this regard. Reduction of oxygen affinity to the levels found in intact SS red cells required DPG concentrations in excess of those found in most SS patients. The same was true of oxygen affinity of patients with pyruvate kinase deficiency. Other organic phosphates, as well as inorganic ions, are known to alter the oxygen affinity of dilute solutions of hemoglobin. These substances, the state of aggregation of hemoglobin molecules, and cytoarchitectural factors probably play roles in determining oxygen affinity of both normal and SS red cells. PMID:5443181

  2. Modulating the selectivity of affinity absorbents to multi-phosphopeptides by a competitive substitution strategy.

    PubMed

    Liu, Zheyi; Wang, Fangjun; Chen, Jin; Zhou, Ye; Zou, Hanfa

    2016-08-26

    Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ion coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells. Data are available via ProteomeXchange with identifier PXD004252. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

    1990-02-20

    High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

  4. Complete mitochondrial genome sequences of Atlantic representatives of the invasive Pacific coral species Tubastraea coccinea and T. tagusensis (Scleractinia, Dendrophylliidae): Implications for species identification.

    PubMed

    Capel, K C C; Migotto, A E; Zilberberg, C; Lin, M F; Forsman, Z; Miller, D J; Kitahara, M V

    2016-09-30

    Members of the azooxanthellate coral genus Tubastraea are invasive species with particular concern because they have become established and are fierce competitors in the invaded areas in many parts of the world. Pacific Tubastraea species are spreading fast throughout the Atlantic Ocean, occupying over 95% of the available substrate in some areas and out-competing native endemic species. Approximately half of all known coral species are azooxanthellate but these are seriously under-represented compared to zooxanthellate corals in terms of the availability of mitochondrial (mt) genome data. In the present study, the complete mt DNA sequences of Atlantic individuals of the invasive scleractinian species Tubastraea coccinea and Tubastraea tagusensis were determined and compared to the GenBank reference sequence available for a Pacific "T. coccinea" individual. At 19,094bp (compared to 19,070bp for the GenBank specimen), the mt genomes assembled for the Atlantic T. coccinea and T. tagusensis were among the longest sequence determined to date for "Complex" scleractinians. Comparisons of genomes data showed that the "T. coccinea" sequence deposited on GenBank was more closely related to that from Dendrophyllia arbuscula than to the Atlantic Tubastraea spp., in terms of genome length and base pair similarities. This was confirmed by phylogenetic analysis, suggesting that the former was misidentified and might actually be a member from the genus Dendrophyllia. In addition, although in general the COX1 locus has a slow evolutionary rate in Scleractinia, it was the most variable region of the Tubastraea mt genome and can be used as markers for genus or species identification. Given the limited data available for azooxanthellate corals, the results presented here represent an important contribution to our understanding of phylogenetic relationships and the evolutionary history of the Scleractinia. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Tritiated-nicotine- and /sup 125/I-alpha-bungarotoxin-labeled nicotinic receptors in the interpeduncular nucleus of rats. II. Effects of habenular destruction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Clarke, P.B.; Hamill, G.S.; Nadi, N.S.

    1986-09-15

    The cholinergic innervation of the interpeduncular nucleus (IPN) is wholly extrinsic and is greatly attenuated by bilateral habenular destruction. We describe changes in the labeling of putative nicotinic receptors within this nucleus at 3, 5, or 11 days after bilateral habenular lesions. Adjacent tissue sections of the rat IPN were utilized for /sup 3/H-nicotine and /sup 125/I-alpha-bungarotoxin (/sup 125/I-BTX) receptor autoradiography. Compared to sham-operated controls, habenular destruction significantly reduced autoradiographic /sup 3/H-nicotine labeling in rostral (-25%), intermediate (-13%), and lateral subnuclei (-36%). Labeling in the central subnucleus was unchanged. Loss of labeling was maximal at the shortest survival time (3more » days) and did not change thereafter. In order to establish whether this loss was due to a reduction in the number or the affinity of /sup 3/H-nicotine-binding sites, a membrane assay was performed on microdissected IPN tissue from rats that had received surgery 3 days previously. Bilateral habenular lesions produced a 35% reduction of high-affinity /sup 3/H-nicotine-binding sites, with no change in binding affinity. Bilateral habenular lesions reduced /sup 125/I-BTX labeling in the intermediate subnuclei, and a slight increase occurred in the rostral subnucleus. In the lateral subnuclei, /sup 125/I-BTX labeling was significantly reduced (27%) at 3 days but not at later survival times. In view of the known synaptic morphology of the habenulointerpeduncular tract, it is concluded that a subpopulation of /sup 3/H-nicotine binding sites within the IPN is located on afferent axons and/or terminals. This subpopulation, located within rostral, intermediate, and lateral subnuclei, may correspond to presynaptic nicotinic cholinergic receptors. Sites that bind /sup 125/I-BTX may include a presynaptic subpopulation located in the lateral and possibly the intermediate subnuclei.« less

  6. Paleobiogeographic affinities of emsian (late early devonian) gastropods from farewell terrane (west-central Alaska)

    USGS Publications Warehouse

    Fryda, J.; Blodgett, R.B.

    2008-01-01

    The vast majority of Emsian gastropods from Limestone Mountain, Medfra B-4 quadrangle, west-central Alaska (Farewell terrane) belong to species with lecithotrophic larval strategy. The present data show that there is no significant difference in the paleobiogeo-graphic distribution of Emsian gastropod genera with lecithotrophic and planktotrophic larval strategies. Numerical analysis of the faunal affinities of the Emsian gastropod fauna from the Farewell terrane reveals that this terrane has much stronger faunal connections to regions like Variscan Europe, eastern Australia, and the Alexander terrane of southeast Alaska than to cratonic North America (Laurentia). The Canadian Arctic Islands is the only region of cratonic North America (Laurentia) that shows significant faunal affinities to the Emsian gastropod faunas of the Farewell terrane. The analysis also indicates a close faunal link between the Farewell and Alexander terranes. Published paleontological and geological data suggest that the Farewell and Alexander terranes represents tectonic entities that have been rifted away from the Siberia, Baltica, or the paleo-Pacific margin of Australia. The results of the present numerical analysis are not in conflict with any of these possibilities. However, the principle of spatial continuity of the wandering path prefers Siberia as the most probable "parental" paleocontinent for the derivation of both the Farewell and Alexander terranes. ?? 2008 The Geological Society of America.

  7. Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Browne, E.S.; Bhalla, V.K.

    1991-02-01

    Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10{sup {minus} 10} M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable {sup 125}I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylatedmore » hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the {sup 125}I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.« less

  8. Fluorescent vancomycin and terephthalate comodified europium-doped layered double hydroxides nanoparticles: synthesis and application for bacteria labelling

    NASA Astrophysics Data System (ADS)

    Sun, Jianchao; Fan, Hai; Wang, Nan; Ai, Shiyun

    2014-09-01

    Vancomycin (Van)- and terephthalate (TA)-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles were successfully prepared by a two-step method, in which, TA acted as a sensitizer to enhance the fluorescent property and Van was modified on the surface of LDH to act as an affinity reagent to bacteria. The obtained products were characterized by X-ray diffraction, transmission electron microscope and fluorescent spectroscopy. The results demonstrated that the prepared Van- and TA-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles with diameter of 50 nm in size showed highly efficient fluorescent property. Furthermore, due to the high affinity of Van to bacteria, the prepared Van-TA-Eu-LDHs nanoparticles showed efficient bacteria labelling by fluorescent property. The prepared nanoparticles may have wide applications in the biological fields, such as biomolecular labelling and cell imaging.

  9. Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

    PubMed Central

    2014-01-01

    The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

  10. Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming.

    PubMed

    Capuano, Cristina; Battella, Simone; Pighi, Chiara; Franchitti, Lavinia; Turriziani, Ombretta; Morrone, Stefania; Santoni, Angela; Galandrini, Ricciarda; Palmieri, Gabriella

    2018-01-01

    Natural killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C + FcεRIγ - long-lived "memory" NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV-seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterized yet. Here, we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansions. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy.

  11. Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

    PubMed

    Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

    2018-05-22

    Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

  12. Affine generalization of the Komar complex of general relativity

    NASA Astrophysics Data System (ADS)

    Mielke, Eckehard W.

    2001-02-01

    On the basis of the ``on shell'' Noether identities of the metric-affine gauge approach of gravity, an affine superpotential is derived which comprises the energy- and angular-momentum content of exact solutions. In the special case of general relativity (GR) or its teleparallel equivalent, the Komar or Freud complex, respectively, are recovered. Applying this to the spontaneously broken anti-de Sitter gauge model of McDowell and Mansouri with an induced Euler term automatically yields the correct mass and spin of the Kerr-AdS solution of GR with a (induced) cosmological constant without the factor two discrepancy of the Komar formula.

  13. Differential affinities of molindone, metoclopramide and domperidone for classes of [3H]spiroperidol binding sites in rat striatum: evidence for pharmacologically distinct classes of receptors.

    PubMed

    Rosenfeld, M R; Dvorkin, B; Klein, P N; Makman, M H

    1982-03-04

    Rat striatum contains two populations of dopaminergic [3H]spiroperidol binding sites. The two populations are similar in their affinities for chlorpromazine and dopamine. Only one population, that with a somewhat higher affinity for spiroperidol itself, exhibits high affinity for the selective D2 antagonists molindone, metoclopramide and domperidone. Hence, this population may represent D2 receptor sites. The other larger population may represent either a separate class of receptor sites or a different form of D2 receptor sites.

  14. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    PubMed

    Marquèze-Pouey, Béatrice; Mailfert, Sébastien; Rouger, Vincent; Goaillard, Jean-Marc; Marguet, Didier

    2014-01-01

    Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  15. (3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Norman, A.B.; Battaglia, G.; Creese, I.

    1985-12-01

    In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 3/H) WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 3/H)LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5more » mM MgSO4, the Bmax of (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 3/H) LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 3/H)LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 3/H) LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of (/sup 3/H)WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of (/sup 3/H)WB4101 binding.« less

  16. 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

  17. Adsorption of endotoxins on Ca2+ -iminodiacetic acid by metal ion affinity chromatography.

    PubMed

    Lopes, André Moreni; Romeu, Jorge Sánchez; Meireles, Rolando Páez; Perera, Gabriel Marquez; Morales, Rolando Perdomo; Pessoa, Adalberto; Cárdenas, Lourdes Zumalacárregui

    2012-11-01

    Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by-products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ -iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+ was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than 100 EU/mL and 100 000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.

  18. Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling

    PubMed Central

    Fujishiro, Mitsuhiro; Okamura, Tomohisa; Fujio, Keishi; Okazaki, Hiroaki; Nomura, Seitaro; Takeda, Norifumi; Harada, Mutsuo; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Yamamoto, Kazuhiko; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays. PMID:26020647

  19. Hirudinella ventricosa (Pallas, 1774) Baird, 1853 represents a species complex based on ribosomal DNA.

    PubMed

    Calhoun, Dana M; Curran, Stephen S; Pulis, Eric E; Provaznik, Jennifer M; Franks, James S

    2013-10-01

    Digeneans in the genus Hirudinella de Blainville, 1828 (Hirudinellidae) from three species of pelagic fishes, Acanthocybium solandri (Cuvier), Makaira nigricans Lacépède and Thunnus albacares (Bonnaterre), and one benthic fish, Mulloidichthys martinicus (Cuvier), from the Gulf of Mexico are investigated using comparison of ribosomal DNA. Four species are identified based on molecular differences: Hirudinella ventricosa (Pallas, 1774) Baird, 1853 from A. solandri, Hirudinella ahi Yamaguti, 1970 from T. albacares, and two unidentified but distinct species of Hirudinella, herein referred to as Hirudinella sp. A (from both M. nigricans and M. martinicus) and Hirudinella sp. B from M. nigricans. Additionally, H. ahi, based tentatively on morphological identification, is reported from Thunnus thynnus (Linnaeus). This represents the first record of a hirudinellid from M. martinicus and the first record of H. ahi from T. thynnus. A phylogeny of some Hemiurata Skrjabin & Guschanskaja, 1954 using partial fragments of the 28S rDNA sequences is consistent with earlier phylogenies and the position of the Hirudinellidae Dollfus, 1932 is well-supported as a derived group most closely related to the Syncoeliidae Looss, 1899.

  20. Genetic, physiologic and ecogeographic factors contributing to variation in Homo sapiens: Homo floresiensis reconsidered.

    PubMed

    Richards, Gary D

    2006-11-01

    A new species, Homo floresiensis, was recently named for Pleistocene hominid remains on Flores, Indonesia. Significant controversy has arisen regarding this species. To address controversial issues and refocus investigations, I examine the affinities of these remains with Homo sapiens. Clarification of problematic issues is sought through an integration of genetic and physiological data on brain ontogeny and evolution. Clarification of the taxonomic value of various 'primitive' traits is possible given these data. Based on this evidence and using a H. sapiens morphological template, models are developed to account for the combination of features displayed in the Flores fossils. Given this overview, I find substantial support for the hypothesis that the remains represent a variant of H. sapiens possessing a combined growth hormone-insulin-like growth factor I axis modification and mutation of the MCPH gene family. Further work will be required to determine the extent to which this variant characterized the population.

  1. An assessment of pharmacists’ readiness for paperless labeling: a national survey

    PubMed Central

    Ho, Yun-Xian; Chen, Qingxia; Nian, Hui; Johnson, Kevin B

    2014-01-01

    Objective To assess the state of readiness for the adoption of paperless labeling among a nationally representative sample of pharmacies, including chain pharmacies, independent retail pharmacies, hospitals, and other rural or urban dispensing sites. Methods Both quantitative and qualitative analyses were used to analyze responses to a cross-sectional survey disseminated to American Pharmacists Association pharmacists nationwide. The survey assessed factors related to pharmacists’ attitudinal readiness (ie, perceptions of impact) and pharmacies’ structural readiness (eg, availability of electronic resources, internet access) for the paperless labeling initiative. Results We received a total of 436 survey responses (6% response rate) from pharmacists representing 44 US states and territories. Across the spectrum of settings we studied, pharmacists had work access to computers, printers, fax machines and access to the internet or intranet. Approximately 79% of respondents believed that the initiative would improve the adequacy of drug information available in their work site and 95% believed it would either not change (33%) or would improve (62%) communication to patients. Overall, respondents’ comments supported advancing the initiative; however, some comments revealed reservations regarding corporate or pharmacy buy-in, success of implementation, and ease of adoption. Conclusions This is the first nationwide study to report about pharmacists’ perspectives on paperless labeling. In general, pharmacists believe they are ready and that their pharmacies are well equipped for the transition to paperless labeling. Further exploration of perspectives from product label manufacturers and corporate pharmacy offices is needed to understand fully what will be necessary to complete this transition. PMID:23523874

  2. A rare and unusual new bittiine genus with two new species from the South Pacific (Cerithiidae, Gastropoda).

    PubMed

    Strong, Ellen E; Bouchet, Philippe

    2018-01-01

    A new genus, Limatium gen. n. , and two new species, L. pagodula sp. n. and L. aureum sp. n. are described, found on outer slopes of barrier reefs and fringing reefs in the South Pacific. They are rare for cerithiids, which typically occur in large populations. The two new species are represented by 108 specimens sampled over a period of 30 years, only 16 of which were collected alive. Three subadults from the Philippines and Vanuatu likely represent a third species. In addition to their rarity, Limatium species are atypical for cerithiids in their smooth, polished, honey to golden brown shells with distinctive white fascioles extending suture to suture. The radula presents a unique morphology that does not readily suggest an affinity to any of the cerithiid subfamilies. Two live-collected specimens, one of each species and designated as holotypes, were preserved in 95% ethanol and sequenced. Bayesian analysis of partial COI and 16S rDNA sequences demonstrates a placement in the Bittiinae, further extending our morphological concept of the subfamily.

  3. Genetic factors in Threatened Species Recovery Plans on three continents

    EPA Science Inventory

    Threatened species' recovery planning is applied globally to stem the current species extinction crisis. Evidence supports a key role of genetic processes, such as inbreeding depression, in determining species viability. We examined whether genetic factors are considered in threa...

  4. Mitochondrial genomes of Australian chicken Eimeria support the presence of ten species with low genetic diversity among strains.

    PubMed

    Morgan, Jess A T; Godwin, Rosamond M

    2017-08-30

    Modern molecular approaches have vastly improved diagnostic capabilities for differentiating among species of chicken infecting Eimeria. Consolidating information from multiple genetic markers, adding additional poultry Eimeria species and increasing the size of available data-sets is improving the resolving power of the DNA, and consequently our understanding of the genus. This study adds information from 25 complete mitochondrial DNA genomes from Australian chicken Eimeria isolates representing all 10 species known to occur in Australia, including OTU-X, -Y and -Z. The resulting phylogeny provides a comprehensive view of species relatedness highlighting where the OTUs align with respect to others members of the genus. All three OTUs fall within the Eimeria clade that contains only chicken-infecting species with close affinities to E. maxima, E. brunetti and E. mitis. Mitochondrial genetic diversity was low among Australian isolates likely reflecting their recent introduction to the country post-European settlement. The lack of observed genetic diversity is a promising outcome as it suggests that the currently used live vaccines should continue to offer widespread protection against Eimeria outbreaks in all states and territories. Flocks were frequently found to host multiple strains of the same species, a factor that should be considered when studying disease epidemiology in the field. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  5. Affinity of antigen encounter and other early B-cell signals determine B-cell fate

    PubMed Central

    Benson, Micah J; Erickson, Loren D; Gleeson, Michael W; Noelle, Randolph J

    2010-01-01

    Three possible effector fates await the naïve follicular B cell following antigen stimulation in thymus-dependent reactions. Short-lived plasma cells produce an initial burst of germline-encoded protective antibodies, and long-lived plasma cells and memory B cells arise from the germinal center and function to enhance and sustain the humoral immune response. The inherent B-cell receptor affinity of naïve follicular B cells and the contribution of other early B-cell signals pre-determines the pattern of transcription factor expression and the differentiation path taken by these cells. High initial B-cell receptor affinity shunts naïve follicular B-cell clones towards the short-lived plasma cell fate, whereas modest-affinity clones are skewed towards a plasma cell fate and low-affinity clones are recruited into the germinal center and are selected for both long-lived plasma cells and memory B cell pathways. In the germinal center reaction, increased levels of the transcription factor interferon regulatory factor-4 drive the molecular program that dictates differentiation into the long-lived plasma cell phenotype but has no impact on the memory B cell compartment. We hypothesize that graded interferon regulatory factor-4 levels driven by signals to B cells, including B-cell receptor signal strength, are responsible for this branch point in the B-cell terminal differentiation pathway. PMID:17433651

  6. Lutzomyia umbratilis, the Main Vector of Leishmania guyanensis, Represents a Novel Species Complex?

    PubMed Central

    Scarpassa, Vera Margarete; Alencar, Ronildo Baiatone

    2012-01-01

    Background Lutzomyia umbratilis is an important Leishmania guyanensis vector in South America. Previous studies have suggested differences in the vector competence between L. umbratilis populations situated on opposite banks of the Amazonas and Negro Rivers in the central Amazonian Brazil region, likely indicating a species complex. However, few studies have been performed on these populations and the taxonomic status of L. umbratilis remains unclear. Methodology/Principal Findings Phylogeographic structure was estimated for six L. umbratilis samples from the central Amazonian region in Brazil by analyzing mtDNA using 1181 bp of the COI gene to assess whether the populations on opposite banks of these rivers consist of incipient or distinct species. The genetic diversity was fairly high and the results revealed two distinct clades ( = lineages) with 1% sequence divergence. Clade I consisted of four samples from the left bank of the Amazonas and Negro Rivers, whereas clade II comprised two samples from the right bank of Negro River. No haplotypes were shared between samples of two clades. Samples within clades exhibited low to moderate genetic differentiation (F ST = −0.0390–0.1841), whereas samples between clades exhibited very high differentiation (F ST = 0.7100–0.8497) and fixed differences. These lineages have diverged approximately 0.22 Mya in the middle Pleistocene. Demographic expansion was detected for the lineages I and II approximately 30,448 and 15,859 years ago, respectively, in the late Pleistocene. Conclusions/Significance The two genetic lineages may represent an advanced speciation stage suggestive of incipient or distinct species within L. umbratilis. These findings suggest that the Amazonas and Negro Rivers may be acting as effective barriers, thus preventing gene flow between populations on opposite sides. Such findings have important implications for epidemiological studies, especially those related to vector competence and

  7. High-affinity copper transport and Snq2 export permease of saccharomyces cerevisiae modulate cytotoxicity of PR-10 from Theobroma cacao.

    PubMed

    Pungartnik, Cristina; da Silva, Aline Clara; de Melo, Sarah Alves; Gramacho, Karina Peres; de Mattos Cascardo, Júlio Cézar; Brendel, Martin; Micheli, Fabienne; da Silva Gesteira, Abelmon

    2009-01-01

    A pathogenesis-related (PR) protein from Theobroma cacao (TcPR-10) was identified from a cacao-Moniliophthora perniciosa interaction cDNA library. Nucleotide and amino acid sequences showed homology with other PR-10 proteins having P loop motif and Betv1 domain. Recombinant TcPR-10 showed in vitro and in vivo ribonuclease activity, and antifungal activity against the basidiomycete cacao pathogen M. perniciosa and the yeast Saccharomyces cerevisiae. Fluorescein isothiocyanate-labeled TcPR-10 was internalized by M. perniciosa hyphae and S. cerevisiae cells and inhibited growth of both fungi. Energy and temperature-dependent internalization of the TcPR-10 suggested an active importation into the fungal cells. Chronical exposure to TcPR-10 of 29 yeast mutants with single gene defects in DNA repair, general membrane transport, metal transport, and antioxidant defenses was tested. Two yeast mutants were hyperresistant compared with their respective isogenic wild type: ctr3Delta mutant, lacking the high-affinity plasma membrane copper transporter and mac1Delta, the copper-sensing transcription factor involved in regulation of high-affinity copper transport. Acute exposure of exponentially growing yeast cells revealed that TcPR-10 resistance is also enhanced in the Snq2 export permease-lacking mutant which has reduced intracellular presence of TcPR-10.

  8. Preferential uptake of soil nitrogen forms by grassland plant species.

    PubMed

    Weigelt, Alexandra; Bol, Roland; Bardgett, Richard D

    2005-02-01

    In this study, we assessed whether a range of temperate grassland species showed preferential uptake for different chemical forms of N, including inorganic N and a range of amino acids that commonly occur in temperate grassland soil. Preferential uptake of dual-labelled (13C and 15N) glycine, serine, arginine and phenylalanine, as compared to inorganic N, was tested using plants growing in pots with natural field soil. We selected five grass species representing a gradient from fertilised, productive pastures to extensive, low productivity pastures (Lolium perenne, Holcus lanatus, Anthoxanthum odoratum, Deschampsia flexuosa, and Nardus stricta). Our data show that all grass species were able to take up directly a diversity of soil amino acids of varying complexity. Moreover, we present evidence of marked inter-species differences in preferential use of chemical forms of N of varying complexity. L. perenne was relatively more effective at using inorganic N and glycine compared to the most complex amino acid phenylalanine, whereas N. stricta showed a significant preference for serine over inorganic N. Total plant N acquisition, measured as root and shoot concentration of labelled compounds, also revealed pronounced inter-species differences which were related to plant growth rate: plants with higher biomass production were found to take up more inorganic N. Our findings indicate that species-specific differences in direct uptake of different N forms combined with total N acquisition could explain changes in competitive dominance of grass species in grasslands of differing fertility.

  9. 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…

  10. Rational design of peptide affinity ligands for the purification of therapeutic enzymes.

    PubMed

    Trasatti, John P; Woo, James; Ladiwala, Asif; Cramer, Steven; Karande, Pankaj

    2018-04-25

    Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

  11. Food consumption and nutritional labeling among immigrants to Israel from the former Soviet Union.

    PubMed

    Gesser-Edelsburg, Anat; Endevelt, Ronit; Zemach, Mina; Tirosh-Kamienchick, Yaara

    2015-04-01

    Nutritional labeling helps consumers make healthier choices regarding food product purchases. In this study, we examined the difference between immigrants from the former Soviet Union who emigrated to Israel beginning in 1990 (IIFSU) and the general population of Israel regarding food consumption broadly and the use of nutritional labeling specifically. A representative sample of each population (n = 592) was composed and interviewed. According to the findings, compared to the general population, the IIFSU attribute less importance to health factors in purchasing food products and information about the ingredients contained in food products; they tend not to follow nutritional labels; and report less on the need for nutritional integrative labeling. Following from this, in the second part of the study, we investigated which of the socio-economic variables is most dominant in shaping attitudes towards food consumption and nutritional labeling. Only immigration and age were found in correlation with attitudes related to healthy food consumption. In contrast, gender, education and religious observance did not affect food selection. Immigration was recognized as the main factor with more clout than the other variables. In conclusion, it is crucial to clarify immigrants' perceptions of the concept of "health" and "proper nutrition" in formulating health promotion programs.

  12. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Perkins, J; Balhorn, R; Cosman, M

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus ormore » botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.« less

  13. Determination of alkaline phosphatase based on affinity adsorption solid-substrate room temperature phosphorimetry using rhodamine 6G-dibromoluciferin luminescent nanoparticle to label lectin and prediction of diseases.

    PubMed

    Liu, Jia-Ming; Liu, Zhen-Bo; Hu, Li-Xiang; He, Hang-Xia; Yang, Min-Lan; Zhou, Ping; Chen, Xin-Hua; Zheng, Min-Min; Zeng, Xiao-Yi; Xu, Yue-Long

    2006-10-15

    In the presence of heavy atom perturber LiAc, the silicon dioxide nanoparticle containing rhodamine 6G (R) and dibromoluciferin (D) (R-D-SiO(2)) can emit strong and stable solid-substrate room temperature phosphorescence signal of R (lambda(ex)/lambda(em)=481/648 nm) and D (lambda(ex)/lambda(em)=457/622 nm) on the surface of acetyl cellulose membrane (ACM). R-D-SiO(2) is used to label triticum vulgare lectin (WGA). Then two types of affinity adsorption reactions, R-D-SiO(2)-WGA- alkaline phosphatase (ALP) (direct method) and WGA-ALP-WGA-R-D-SiO(2) (sandwich method), are carried out on ACM. The conditions and the analytical characteristics for the determination of ALP using affinity adsorption solid-substrate room temperature phosphorimetry (AA-SS-RTP) were studied. For a 0.40-microl drop of sample, results show that the detection limits of the sandwich method are 0.16 ag spot(-1)(457/622 nm) and 0.17 ag spot(-1)(481/648 nm), and the detection limits of the direct method are 0.41 ag spot(-1) (457/622 nm) and 0.44 ag spot(-1) (481/648 nm). The contents of ALP in human serum correlated well with those obtained by enzyme-linked immunoassay. This study shows that AA-SS-RTP whether by the sandwich method or the direct method, can combine very well the characteristics of both high sensitivity of SS-RTP and specificity of the immunoreaction. Simultaneously, whether the phosphorescence excitation/emission wavelength of either R or D in R-D-SiO(2) is chosen to determine ALP, this can promote the agility and widen the adaptability of AA-SS-RTP.

  14. Super-Chelators for Advanced Protein Labeling in Living Cells.

    PubMed

    Gatterdam, Karl; Joest, Eike F; Dietz, Marina S; Heilemann, Mike; Tampé, Robert

    2018-05-14

    Live-cell labeling, super-resolution microscopy, single-molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N-nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA-based small interaction pairs described so far. Coupled to bright organic fluorophores with fine-tuned photophysical properties, the super-chelator probes were delivered into human cells by chemically gated nanopores. These super-chelators permit kinetic profiling, multiplexed labeling of His 6 - and His 12 -tagged proteins as well as single-molecule-based super-resolution imaging. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Accurate and sensitive quantification of protein-DNA binding affinity.

    PubMed

    Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

    2018-04-17

    Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

  16. Accurate and sensitive quantification of protein-DNA binding affinity

    PubMed Central

    Rastogi, Chaitanya; Rube, H. Tomas; Kribelbauer, Judith F.; Crocker, Justin; Loker, Ryan E.; Martini, Gabriella D.; Laptenko, Oleg; Freed-Pastor, William A.; Prives, Carol; Stern, David L.; Mann, Richard S.; Bussemaker, Harmen J.

    2018-01-01

    Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. PMID:29610332

  17. Ceratopetalum (Cunoniaceae) fruits of Australasian affinity from the early Eocene Laguna del Hunco flora, Patagonia, Argentina.

    PubMed

    Gandolfo, María A; Hermsen, Elizabeth J

    2017-03-01

    Radially symmetrical, five-winged fossil fruits from the highly diverse early Eocene Laguna del Hunco flora of Chubut Province, Patagonia, Argentina, are named, described and illustrated. The main goals are to assess the affinities of the fossils and to place them in an evolutionary, palaeoecological and biogeographic context. Specimens of fossil fruits were collected from the Tufolitas Laguna del Hunco. They were prepared, photographed and compared with similar extant and fossil fruits using published literature. Their structure was also evaluated by comparing them with that of modern Ceratopetalum (Cunoniaceae) fruits through examination of herbarium specimens. The Laguna del Hunco fossil fruits share the diagnostic features that characterize modern and fossil Ceratopetalum (symmetry, number of fruit wings, presence of a conspicuous floral nectary and overall venation pattern). The pattern of the minor wing (sepal) veins observed in the Patagonian fossil fruits is different from that of modern and previously described fossil Ceratopetalum fruits; therefore, a new fossil species is recognized. An apomorphy (absence of petals) suggests that the fossils belong within crown-group Ceratopetalum . The Patagonian fossil fruits are the oldest known record for Ceratopetalum . Because the affinities, provenance and age of the fossils are so well established, this new Ceratopetalum fossil species is an excellent candidate for use as a calibration point in divergence dating studies of the family Cunoniaceae. It represents the only record of Ceratopetalum outside Australasia, and further corroborates the biogeographic connection between the Laguna del Hunco flora and ancient and modern floras of the Australasian region. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company.

  18. PRIORITIZING FUTURE RESEACH ON OFF-LABEL PRESCRIBING: RESULTS OF A QUANTITATIVE EVALUATION

    PubMed Central

    Walton, Surrey M.; Schumock, Glen T.; Lee, Ky-Van; Alexander, G. Caleb; Meltzer, David; Stafford, Randall S.

    2015-01-01

    Background Drug use for indications not approved by the Food and Drug Administration exceeds 20% of prescribing. Available compendia indicate that a minority of off-label uses are well supported by evidence. Policy makers, however, lack information to identify where systematic reviews of the evidence or other research would be most valuable. Methods We developed a quantitative model for prioritizing individual drugs for future research on off-label uses. The base model incorporated three key factors, 1) the volume of off-label use with inadequate evidence, 2) safety, and 3) cost and market considerations. Nationally representative prescribing data were used to estimate the number of off-label drug uses by indication from 1/2005 through 6/2007 in the United States, and these indications were then categorized according to the adequacy of scientific support. Black box warnings and safety alerts were used to quantify drug safety. Drug cost, date of market entry, and marketing expenditures were used to quantify cost and market considerations. Each drug was assigned a relative value for each factor, and the factors were then weighted in the final model to produce a priority score. Sensitivity analyses were conducted by varying the weightings and model parameters. Results Drugs that were consistently ranked highly in both our base model and sensitivity analyses included quetiapine, warfarin, escitalopram, risperidone, montelukast, bupropion, sertraline, venlafaxine, celecoxib, lisinopril, duloxetine, trazodone, olanzapine, and epoetin alfa. Conclusion Future research into off-label drug use should focus on drugs used frequently with inadequate supporting evidence, particularly if further concerns are raised by known safety issues, high drug cost, recent market entry, and extensive marketing. Based on quantitative measures of these factors, we have prioritized drugs where targeted research and policy activities have high potential value. PMID:19025425

  19. The factors controlling species density in herbaceous plant communities: An assessment

    USGS Publications Warehouse

    Grace, J.B.

    1999-01-01

    This paper evaluates both the ideas and empirical evidence pertaining to the control of species density in herbaceous plant communities. While most theoretical discussions of species density have emphasized the importance of habitat productivity and disturbance regimes, many other factors (e.g. species pools, plant litter accumulation, plant morphology) have been proposed to be important. A review of literature presenting observations on the density of species in small plots (in the vicinity of a few square meters or less), as well as experimental studies, suggests several generalizations: (1) Available data are consistent with an underlying unimodal relationship between species density and total community biomass. While variance in species density is often poorly explained by predictor variables, there is strong evidence that high levels of community biomass are antagonistic to high species density. (2) Community biomass is just one of several factors affecting variations in species density. Multivariate analyses typically explain more than twice as much variance in species density as can be explained by community biomass alone. (3) Disturbance has important and sometimes complex effects on species density. In general, the evidence is consistent with the intermediate disturbance hypothesis but exceptions exist and effects can be complex. (4) Gradients in the species pool can have important influences on patterns of species density. Evidence is mounting that a considerable amount of the observed variability in species density within a landscape or region may result from environmental effects on the species pool. (5) Several additional factors deserve greater consideration, including time lags, species composition, plant morphology, plant density and soil microbial effects. Based on the available evidence, a conceptual model of the primary factors controlling species density is presented here. This model suggests that species density is controlled by the effects of

  20. Data structures and apparatuses for representing knowledge

    DOEpatents

    Hohimer, Ryan E; Thomson, Judi R; Harvey, William J; Paulson, Patrick R; Whiting, Mark A; Tratz, Stephen C; Chappell, Alan R; Butner, Robert S

    2014-02-18

    Data structures and apparatuses to represent knowledge are disclosed. The processes can comprise labeling elements in a knowledge signature according to concepts in an ontology and populating the elements with confidence values. The data structures can comprise knowledge signatures stored on computer-readable media. The knowledge signatures comprise a matrix structure having elements labeled according to concepts in an ontology, wherein the value of the element represents a confidence that the concept is present in an information space. The apparatus can comprise a knowledge representation unit having at least one ontology stored on a computer-readable medium, at least one data-receiving device, and a processor configured to generate knowledge signatures by comparing datasets obtained by the data-receiving devices to the ontologies.

  1. SNP2TFBS - a database of regulatory SNPs affecting predicted transcription factor binding site affinity.

    PubMed

    Kumar, Sunil; Ambrosini, Giovanna; Bucher, Philipp

    2017-01-04

    SNP2TFBS is a computational resource intended to support researchers investigating the molecular mechanisms underlying regulatory variation in the human genome. The database essentially consists of a collection of text files providing specific annotations for human single nucleotide polymorphisms (SNPs), namely whether they are predicted to abolish, create or change the affinity of one or several transcription factor (TF) binding sites. A SNP's effect on TF binding is estimated based on a position weight matrix (PWM) model for the binding specificity of the corresponding factor. These data files are regenerated at regular intervals by an automatic procedure that takes as input a reference genome, a comprehensive SNP catalogue and a collection of PWMs. SNP2TFBS is also accessible over a web interface, enabling users to view the information provided for an individual SNP, to extract SNPs based on various search criteria, to annotate uploaded sets of SNPs or to display statistics about the frequencies of binding sites affected by selected SNPs. Homepage: http://ccg.vital-it.ch/snp2tfbs/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Utilization of nanoparticle labels for signal amplification in ultrasensitive electrochemical affinity biosensors: a review.

    PubMed

    Ding, Liang; Bond, Alan M; Zhai, Jianping; Zhang, Jie

    2013-10-03

    Nanoparticles with desirable properties not exhibited by the bulk material can be readily synthesized because of rapid technological developments in the fields of materials science and nanotechnology. In particular their highly attractive electrochemical properties and electrocatalytic activity have facilitated achievement of the high level of signal amplification needed for the development of ultrasensitive electrochemical affinity biosensors for the detection of proteins and DNA. This review article explains the basic principles of nanoparticle based electrochemical biosensors, highlights the recent advances in the development of nanoparticle based signal amplification strategies, and provides a critical assessment of the likely drawbacks associated with each strategy. Finally, future perspectives for achieving advanced signal simplification in nanoparticles based biosensors are considered. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Determinants of the Differential Antizyme-Binding Affinity of Ornithine Decarboxylase

    PubMed Central

    Liu, Yen-Chin; Hsu, Den-Hua; Huang, Chi-Liang; Liu, Yi-Liang; Liu, Guang-Yaw; Hung, Hui-Chih

    2011-01-01

    Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The K d value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the K d value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the K d was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC K d, which suggests that residues 119 and 137 play a role in AZ binding. PMID:22073206

  4. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    PubMed

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses. © 2012 John Wiley & Sons A/S.

  5. Taxonomic evaluation of selected Ganoderma species and database sequence validation

    PubMed Central

    Jargalmaa, Suldbold; Eimes, John A.; Park, Myung Soo; Park, Jae Young; Oh, Seung-Yoon

    2017-01-01

    Species in the genus Ganoderma include several ecologically important and pathogenic fungal species whose medicinal and economic value is substantial. Due to the highly similar morphological features within the Ganoderma, identification of species has relied heavily on DNA sequencing using BLAST searches, which are only reliable if the GenBank submissions are accurately labeled. In this study, we examined 113 specimens collected from 1969 to 2016 from various regions in Korea using morphological features and multigene analysis (internal transcribed spacer, translation elongation factor 1-α, and the second largest subunit of RNA polymerase II). These specimens were identified as four Ganoderma species: G. sichuanense, G. cf. adspersum, G. cf. applanatum, and G. cf. gibbosum. With the exception of G. sichuanense, these species were difficult to distinguish based solely on morphological features. However, phylogenetic analysis at three different loci yielded concordant phylogenetic information, and supported the four species distinctions with high bootstrap support. A survey of over 600 Ganoderma sequences available on GenBank revealed that 65% of sequences were either misidentified or ambiguously labeled. Here, we suggest corrected annotations for GenBank sequences based on our phylogenetic validation and provide updated global distribution patterns for these Ganoderma species. PMID:28761785

  6. Effects of dietary esfenvalerate exposures on three aquatic insect species representing different functional feeding groups.

    PubMed

    Palmquist, Katherine R; Jenkins, Jeffrey J; Jepson, Paul C

    2008-08-01

    Given the chemical properties of synthetic pyrethroids, it is probable that compounds, including esfenvalerate, that enter surface waters may become incorporated into aquatic insect food sources. We examined the effect of dietary esfenvalerate uptake in aquatic insects representing different functional feeding groups. We used three field-collected aquatic insect species: A grazing scraper, Cinygmula reticulata McDunnough (Ephemeroptera: Heptageniidae); an omnivorous filter feeder, Brachycentrus americanus Banks (Trichoptera: Brachycentridae); and a predator, Hesperoperla pacifica Banks (Plecoptera: Perlidae). Laboratory-cultured algae were preexposed for 24 h to esfenvalerate concentrations of 0, 0.025, 0.05, and 0.1 microg/L and provided to two C. reticulata age classes (small and final-instar nymphs). Reduction in small nymph growth was observed following three weeks of feeding on algae exposed to 0.05 and 0.1 microg/L of esfenvalerate, and the highest dietary exposure reduced egg production in final-instar nymphs. The diet for B. americanus and H. pacifica consisted of dead third-instar Chironomus tentans larvae preexposed for 24 h to esfenvalerate concentrations ranging between 0.1 and 1.0 microg/L. Consumption of larvae exposed to 0.5 to 1.0 microg/L of esfenvalerate caused case abandonment and mortality in B. americanus caddisfly larvae. Although H. pacifica nymphs readily consumed esfenvalerate-exposed larvae, no adverse effects were observed during the present study. Furthermore, no evidence of esfenvalerate-induced feeding deterrence was found in any of the species tested, suggesting that aquatic insects may not be able to distinguish between pyrethroid-contaminated and uncontaminated food sources. These findings indicate that feeding deterrence is not a factor in regulating aquatic insect dietary exposures to synthetic pyrethroids.

  7. Influence of heme environment structure on dioxygen affinity for the dual function Amphitrite ornata hemoglobin/dehaloperoxidase. Insights into the evolutional structure-function adaptations

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sun, Shengfang; Sono, Masanori; Wang, Chunxue

    Sea worm, Amphitrite ornata, has evolved its globin (an O 2 carrier) also to serves as a dehaloperoxidase (DHP) to detoxify haloaromatic pollutants generated by competing species. A previous mutagenesis study by our groups on both DHP and sperm whale myoglobin (SW Mb) revealed some structural factors that influence the dehaloperoxidase activities (significantly lower for Mb) of both proteins. Using an isocyanide/O 2 partition constant measurement method in this study, we have examined the effects of these structural factors on the O 2 equilibrium constants (K O2) of DHP, SW Mb, and their mutants. A clear trend of decreasing Omore » 2 affinity and increasing catalytic activity along with the increase in the distal His N ε–heme iron distance is observed. An H93K/T95H Mb double mutant mimicking the DHP proximal His positioning exhibited markedly enhanced O 2 affinity, confirming the essential effect of proximal His rotation on the globin function of DHP. For DHP, the L100F, T56G and M86E variants showed the effects of distal volume, distal His flexibility and proximal electronic push, respectively, on the O 2 affinity. This study provides insights into how DHP has evolved its heme environment to gain significantly enhanced peroxidase capability without compromising its primary function as an O 2 carrier.« less

  8. Darwin's diagram of divergence of taxa as a causal model for the origin of species.

    PubMed

    Bouzat, Juan L

    2014-03-01

    On the basis that Darwin's theory of evolution encompasses two logically independent processes (common descent and natural selection), the only figure in On the Origin of Species (the Diagram of Divergence of Taxa) is often interpreted as illustrative of only one of these processes: the branching patterns representing common ancestry. Here, I argue that Darwin's Diagram of Divergence of Taxa represents a broad conceptual model of Darwin's theory, illustrating the causal efficacy of natural selection in producing well-defined varieties and ultimately species. The Tree Diagram encompasses the idea that natural selection explains common descent and the origin of organic diversity, thus representing a comprehensive model of Darwin's theory on the origin of species. I describe Darwin's Tree Diagram in relation to his argumentative strategy under the vera causa principle, and suggest that the testing of his theory based on the evidence from the geological record, the geographical distribution of organisms, and the mutual affinities of organic beings can be framed under the hypothetico-deductive method. Darwin's Diagram of Divergence of Taxa therefore represents a broad conceptual model that helps understanding the causal construction of Darwin's theory of evolution, the structure of his argumentative strategy, and the nature of his scientific methodology.

  9. Instance annotation for multi-instance multi-label learning

    Treesearch

    F. Briggs; X.Z. Fern; R. Raich; Q. Lou

    2013-01-01

    Multi-instance multi-label learning (MIML) is a framework for supervised classification where the objects to be classified are bags of instances associated with multiple labels. For example, an image can be represented as a bag of segments and associated with a list of objects it contains. Prior work on MIML has focused on predicting label sets for previously unseen...

  10. Extraction and labeling high-resolution images from PDF documents

    NASA Astrophysics Data System (ADS)

    Chachra, Suchet K.; Xue, Zhiyun; Antani, Sameer; Demner-Fushman, Dina; Thoma, George R.

    2013-12-01

    Accuracy of content-based image retrieval is affected by image resolution among other factors. Higher resolution images enable extraction of image features that more accurately represent the image content. In order to improve the relevance of search results for our biomedical image search engine, Open-I, we have developed techniques to extract and label high-resolution versions of figures from biomedical articles supplied in the PDF format. Open-I uses the open-access subset of biomedical articles from the PubMed Central repository hosted by the National Library of Medicine. Articles are available in XML and in publisher supplied PDF formats. As these PDF documents contain little or no meta-data to identify the embedded images, the task includes labeling images according to their figure number in the article after they have been successfully extracted. For this purpose we use the labeled small size images provided with the XML web version of the article. This paper describes the image extraction process and two alternative approaches to perform image labeling that measure the similarity between two images based upon the image intensity projection on the coordinate axes and similarity based upon the normalized cross-correlation between the intensities of two images. Using image identification based on image intensity projection, we were able to achieve a precision of 92.84% and a recall of 82.18% in labeling of the extracted images.

  11. Qualitative variation of photolabelled benzodiazepine receptors in different species.

    PubMed

    Hebebrand, J; Friedl, W; Lentes, K U; Propping, P

    1986-01-01

    In order to examine whether species differences of benzodiazepine receptor subunits exist, we compared the fluorographic pattern of photoaffinity labelled subunits after SDS-PAGE in five species: fish, frog, chicken, mouse and calf. Each species showed a distinct pattern of specifically labelled proteins. We conclude that species variation of benzodiazepine receptor does indeed exist.

  12. Relationships between chemical structure and affinity for acetylcholine receptors

    PubMed Central

    Abramson, F. B.; Barlow, R. B.; Mustafa, M. G.; Stephenson, R. P.

    1969-01-01

    contributions made by one phenyl and one cyclohexyl group. 6. The factors which influence the binding of molecules to receptors are discussed. There is no evidence that the separation between the onium group and the group in the receptor with which it interacts is greater in compounds with high affinity nor is there any evidence, from the study of the series which contain agonists and partial agonists, that ability to activate receptors depends upon the onium group being able to come close to this charged group in the receptors. PMID:5343350

  13. Inhibition of anthrax lethal factor by ssDNA aptamers.

    PubMed

    Lahousse, Mieke; Park, Hae-Chul; Lee, Sang-Choon; Ha, Na-Reum; Jung, In-Pil; Schlesinger, Sara R; Shackelford, Kaylin; Yoon, Moon-Young; Kim, Sung-Kun

    2018-05-15

    Anthrax is caused by Bacillus anthracis, a bacterium that is able to secrete the toxins protective antigen, edema factor and lethal factor. Due to the high level of secretion from the bacteria and its severe virulence, lethal factor (LF) has been sought as a biomarker for detecting bacterial infection and as an effective target to neutralize toxicity. In this study, we found three aptamers, and binding affinity was determined by fluorescently labeled aptamers. One of the aptamers exhibited high affinity, with a K d value of 11.0 ± 2.7 nM, along with low cross reactivity relative to bovine serum albumin and protective antigen. The therapeutic functionality of the aptamer was examined by assessing the inhibition of LF protease activity against a mitogen-activated protein kinase kinase. The aptamer appears to be an effective inhibitor of LF with an IC 50 value of 15 ± 1.5 μM and approximately 85% cell viability, suggesting that this aptamer provides a potential clue for not only development of a sensitive diagnostic device of B. anthracis infection but also the design of novel inhibitors of LF. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Object matching using a locally affine invariant and linear programming techniques.

    PubMed

    Li, Hongsheng; Huang, Xiaolei; He, Lei

    2013-02-01

    In this paper, we introduce a new matching method based on a novel locally affine-invariant geometric constraint and linear programming techniques. To model and solve the matching problem in a linear programming formulation, all geometric constraints should be able to be exactly or approximately reformulated into a linear form. This is a major difficulty for this kind of matching algorithm. We propose a novel locally affine-invariant constraint which can be exactly linearized and requires a lot fewer auxiliary variables than other linear programming-based methods do. The key idea behind it is that each point in the template point set can be exactly represented by an affine combination of its neighboring points, whose weights can be solved easily by least squares. Errors of reconstructing each matched point using such weights are used to penalize the disagreement of geometric relationships between the template points and the matched points. The resulting overall objective function can be solved efficiently by linear programming techniques. Our experimental results on both rigid and nonrigid object matching show the effectiveness of the proposed algorithm.

  15. Dye-ligand affinity systems.

    PubMed

    Denizli, A; Pişkin, E

    2001-10-30

    Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.

  16. High Affinity Binding of Indium and Ruthenium Ions by Gastrins

    PubMed Central

    Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

  17. Superposition and alignment of labeled point clouds.

    PubMed

    Fober, Thomas; Glinca, Serghei; Klebe, Gerhard; Hüllermeier, Eyke

    2011-01-01

    Geometric objects are often represented approximately in terms of a finite set of points in three-dimensional euclidean space. In this paper, we extend this representation to what we call labeled point clouds. A labeled point cloud is a finite set of points, where each point is not only associated with a position in three-dimensional space, but also with a discrete class label that represents a specific property. This type of model is especially suitable for modeling biomolecules such as proteins and protein binding sites, where a label may represent an atom type or a physico-chemical property. Proceeding from this representation, we address the question of how to compare two labeled points clouds in terms of their similarity. Using fuzzy modeling techniques, we develop a suitable similarity measure as well as an efficient evolutionary algorithm to compute it. Moreover, we consider the problem of establishing an alignment of the structures in the sense of a one-to-one correspondence between their basic constituents. From a biological point of view, alignments of this kind are of great interest, since mutually corresponding molecular constituents offer important information about evolution and heredity, and can also serve as a means to explain a degree of similarity. In this paper, we therefore develop a method for computing pairwise or multiple alignments of labeled point clouds. To this end, we proceed from an optimal superposition of the corresponding point clouds and construct an alignment which is as much as possible in agreement with the neighborhood structure established by this superposition. We apply our methods to the structural analysis of protein binding sites.

  18. Labeling of Chromosomes in Cell Development and the Appearance of Monozygotic Twins.

    PubMed

    Jim, Carol; Berkovich, Simon

    2015-01-01

    Understanding the mechanism behind the structure of the internal cellular clock can lead to advances in the knowledge of origins of pairs of monozygotic twins and higher order multiples as well as other biological phenomena. To gain insight into this mechanism, we analyze possible cell labeling schemes that model an organism's development. Our findings lead us to predict that monozygotic quadruplets are not quadruplets in the traditional sense but rather two pairs of monozygotic twins where the pairs slightly differ-a situation we coin quadruplet twins. From the considered model, the probability of monozygotic twins is found to be (1/2) (K) , and we discover that the probability of monozygotic quadruplets, or triplets as in the case of the death of an embryo, is (1/8) (K) , where K is a species-specific integer representing the number of pairs of homologous chromosomes. The parameter K may determine cancerization with a probability threshold that is approximately inversely proportional to the Hayflick limit. Exposure to some cancerization factors such as small levels of ionizing radiation and chemical pollution may not produce cancer.

  19. Labeling of Chromosomes in Cell Development and the Appearance of Monozygotic Twins

    PubMed Central

    Berkovich, Simon

    2015-01-01

    Understanding the mechanism behind the structure of the internal cellular clock can lead to advances in the knowledge of origins of pairs of monozygotic twins and higher order multiples as well as other biological phenomena. To gain insight into this mechanism, we analyze possible cell labeling schemes that model an organism's development. Our findings lead us to predict that monozygotic quadruplets are not quadruplets in the traditional sense but rather two pairs of monozygotic twins where the pairs slightly differ—a situation we coin quadruplet twins. From the considered model, the probability of monozygotic twins is found to be (1/2)K, and we discover that the probability of monozygotic quadruplets, or triplets as in the case of the death of an embryo, is (1/8)K, where K is a species-specific integer representing the number of pairs of homologous chromosomes. The parameter K may determine cancerization with a probability threshold that is approximately inversely proportional to the Hayflick limit. Exposure to some cancerization factors such as small levels of ionizing radiation and chemical pollution may not produce cancer. PMID:26185760

  20. Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

    PubMed

    Kawamoto, Norio; Kamemura, Norio; Kido, Hiroshi; Fukao, Toshiyuki

    2017-06-01

    Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Species-specific identification of Dekkera/Brettanomyces yeasts by fluorescently labeled DNA probes targeting the 26S rRNA.

    PubMed

    Röder, Christoph; König, Helmut; Fröhlich, Jürgen

    2007-09-01

    Sequencing of the complete 26S rRNA genes of all Dekkera/Brettanomyces species colonizing different beverages revealed the potential for a specific primer and probe design to support diagnostic PCR approaches and FISH. By analysis of the complete 26S rRNA genes of all five currently known Dekkera/Brettanomyces species (Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. nanus and B. naardenensis), several regions with high nucleotide sequence variability yet distinct from the D1/D2 domains were identified. FISH species-specific probes targeting the 26S rRNA gene's most variable regions were designed. Accessibility of probe targets for hybridization was facilitated by the construction of partially complementary 'side'-labeled probes, based on secondary structure models of the rRNA sequences. The specificity and routine applicability of the FISH-based method for yeast identification were tested by analyzing different wine isolates. Investigation of the prevalence of Dekkera/Brettanomyces yeasts in the German viticultural regions Wonnegau, Nierstein and Bingen (Rhinehesse, Rhineland-Palatinate) resulted in the isolation of 37 D. bruxellensis strains from 291 wine samples.

  2. Method for performing site-specific affinity fractionation for use in DNA sequencing

    DOEpatents

    Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

    1999-01-01

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

  3. Method for performing site-specific affinity fractionation for use in DNA sequencing

    DOEpatents

    Mirzabekov, A.D.; Lysov, Y.P.; Dubley, S.A.

    1999-05-18

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between the cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting the extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to the extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from the array. 14 figs.

  4. Label it or ban it? Public perceptions of nano-food labels and propositions for banning nano-food applications

    NASA Astrophysics Data System (ADS)

    Chuah, Agnes S. F.; Leong, Alisius D.; Cummings, Christopher L.; Ho, Shirley S.

    2018-02-01

    The future of nano-food largely hinges on public perceptions and willingness to accept this novel technology. The present study utilizes the scientific literacy model and psychometric paradigm as the key theoretical frameworks to examine the factors influencing public support for labeling and banning of nano-food in Singapore. Using data collected from a nationally representative survey of 1001 respondents, the findings demonstrated that attitudes toward technology, preference for natural product, science knowledge, and risk perception were found to substantially affect public support for both labeling and banning of nano-food. Conversely, attention to food safety news on traditional media and attention to nano-news on new media were only associated with public support for labeling of nano-food. Similarly, benefit perception was only significantly associated with public support for banning of nano-food. Theoretically, these findings support the growing body of literature that argues for the significant role played by predispositions, media use, science knowledge, and risk and benefit perceptions on attitude formation toward nano-food. It serves as the pioneering piece to address the aspect of banning in the field of nano-food. Practically, insights drawn from this study could aid relevant stakeholders in enlisting effecting strategies to convey the benefits of nano-food while mitigating the risk perceptions among the public.

  5. Near-infrared fluorescence probes for enzymes based on binding affinity modulation of squarylium dye scaffold.

    PubMed

    Oushiki, Daihi; Kojima, Hirotatsu; Takahashi, Yuki; Komatsu, Toru; Terai, Takuya; Hanaoka, Kenjiro; Nishikawa, Makiya; Takakura, Yoshinobu; Nagano, Tetsuo

    2012-05-15

    We present a novel design strategy for near-infrared (NIR) fluorescence probes utilizing dye-protein interaction as a trigger for fluorescence enhancement. The design principle involves modification of a polymethine dye with cleavable functional groups that reduce the dye's protein-binding affinity. When these functional groups are removed by specific interaction with the target enzymes, the dye's protein affinity is restored, protein binding occurs, and the dye's fluorescence is strongly enhanced. To validate this strategy, we first designed and synthesized an alkaline phosphatase (ALP) sensor by introducing phosphate into the squarylium dye scaffold; this sensor was able to detect ALP-labeled secondary antibodies in Western blotting analysis. Second, we synthesized a probe for β-galactosidase (widely used as a reporter of gene expression) by means of β-galactosyl substitution of the squarylium scaffold; this sensor was able to visualize β-galactosidase activity both in vitro and in vivo. Our strategy should be applicable to obtain NIR fluorescence probes for a wide range of target enzymes.

  6. A rapid and fluorogenic TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells.

    PubMed

    Liu, Wei; Li, Fu; Chen, Xi; Hou, Jian; Yi, Long; Wu, Yao-Wen

    2014-03-26

    Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

  7. Specific labelling of serotonin 5-HT(1B) receptors in rat frontal cortex with the novel, phenylpiperazine derivative, [3H]GR125,743. A pharmacological characterization.

    PubMed

    Millan, M J; Newman-Tancredi, A; Lochon, S; Touzard, M; Aubry, S; Audinot, V

    2002-04-01

    Although several tritiated agonists have been used for radiolabelling serotonin (5-hydroxytryptamine, 5-HT)(1B) receptors in rats, data with a selective, radiolabelled antagonist have not been presented. Inasmuch as [3H]GR125,743 specifically labels cloned, human and native guinea pig 5-HT(1B) receptors and has been employed for characterization of cerebral 5-HT(1B) receptor in the latter species [Eur. J. Pharmacol. 327 (1997) 247.], the present study evaluated its utility for characterization of native, cerebral 5-HT(1B) sites in the rat. In homogenates of frontal cortex, [3H]GR125,743 (0.8 nM) showed rapid association (t(1/2)=3.4 min), >90% specific binding and high affinity (K(d)=0.6 nM) for a homogeneous population of receptors with a density (B(max)) of 160 fmol/mg protein. In competition binding studies, affinities were determined for 15 chemically diverse 5-HT(1B) agonists, including 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl]ethylamine (L694,247; pK(i), 10.4), 5-carboxamidotryptamine (5-CT; 9.7), 3-[3-(2-dimethylamino-ethyl)-1H-indol-6-yl]-N-(4-methoxybenzyl)acrylamide (GR46,611; 9.6), 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU24,969; 9.5), dihydroergotamine (DHE; 8.6), 5-H-pyrrolo[3,2-b]pyridin-5-one,1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl (CP93,129; 8.4), anpirtoline (7.9), sumatriptan (7.4), 1-[2-(3-fluorophenyl)ethyl]-4-[3-[5-(1,2,4-triazol-4-yl)-1H-indol-3-yl]propyl]piperazine (L775,606; 6.4) and (minus sign)-1(S)-[2-[4-(4-methoxyphenyl)piperazin-1-yl]ethyl]-N-methyl-3,4-dihydro-1H-2-benzopyran-6-carboxamide (PNU109,291; <5.0). Similarly, affinities were established for 13 chemically diverse antagonists, including N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR125,743; pK(i), 9.1), (-)cyanopindolol (9.0), (-)-tertatolol (8.2), N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiozol-3-yl)biphenyl-4-carboxamide (GR127,935; 8.2), N-[3

  8. Affinity-aware checkpoint restart

    DOE PAGES

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  9. Affinity-aware checkpoint restart

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Saini, Ajay; Rezaei, Arash; Mueller, Frank

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  10. Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

    PubMed

    Battersby, J E; Snedecor, B; Chen, C; Champion, K M; Riddle, L; Vanderlaan, M

    2001-08-24

    An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

  11. Quantitative cumulative biodistribution of antibodies in mice: effect of modulating binding affinity to the neonatal Fc receptor.

    PubMed

    Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

    2014-01-01

    The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.

  12. RELATIVE BINDING AFFINITY OF ENDOCRINE DISRUPTING CHEMICALS TO ESTROGEN RECEPTOR IN TWO SPECIES OF FRESHWATER FISH

    EPA Science Inventory

    The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

  13. GAD autoantibody affinity in adult patients with latent autoimmune diabetes, the study participants of a GAD65 vaccination trial.

    PubMed

    Krause, Stephanie; Landherr, Ulrike; Agardh, Carl-David; Hausmann, Simone; Link, Katarina; Hansen, Jesse M; Lynch, Kristian F; Powell, Michael; Furmaniak, Jadwiga; Rees-Smith, Bernard; Bonifacio, Ezio; Ziegler, Anette G; Lernmark, Ake; Achenbach, Peter

    2014-06-01

    Patients with latent autoimmune diabetes in adults (LADA) express autoantibodies against the 65-kDa isoform of GAD (GADA). Intervention with recombinant human GAD65 formulated with aluminium hydroxide (GAD-alum) given twice subcutaneously to LADA patients at intervals of 4 weeks was safe and did not compromise β-cell function in a Phase II clinical trial. GADA affinity has been shown to predict progression to type 1 diabetes. Here, we asked whether GADA affinity was affected by the GAD65 antigen-specific vaccination and/or associated with β-cell function in participants of this trial. GADA affinity was measured in sera of 46 LADA patients obtained prior to the first week and 20 weeks after the second injection with GAD-alum or placebo using competitive binding experiments with [125I]-labeled and unlabeled human GAD65. At baseline, GADA affinities ranged from 1.9 × 10(7) to 5.0 × 10(12) L/mol (median 2.8 × 10(10) L/mol) and were correlated with GADA titers (r = 0.47; P = 0.0009), fasting (r = -0.37; P = 0.01) and stimulated (r = -0.40; P = 0.006) C-peptide concentrations, and HbA1c (r = 0.39; P = 0.007). No significant changes in affinity were observed from baseline to week 24. Patients with GADA affinities in the lower first quartile (<4 × 10(9) L/mol) had better preserved fasting C-peptide concentrations at baseline than those with higher affinities (mean 1.02 vs. 0.66 nmol/L; P = 0.004) and retained higher concentrations over 30 months of follow-up (mean 1.26 vs. 0.62 nmol/L; P = 0.01). Intervention with GAD-alum in LADA patients had no effect on GADA affinity. Our data suggest that patients with low GADA affinity have a prolonged preservation of residual β-cell function. © 2014 by the American Diabetes Association.

  14. Factors affecting species distribution predictions: A simulation modeling experiment

    Treesearch

    Gordon C. Reese; Kenneth R. Wilson; Jennifer A. Hoeting; Curtis H. Flather

    2005-01-01

    Geospatial species sample data (e.g., records with location information from natural history museums or annual surveys) are rarely collected optimally, yet are increasingly used for decisions concerning our biological heritage. Using computer simulations, we examined factors that could affect the performance of autologistic regression (ALR) models that predict species...

  15. 2017 Guralp Affinity Digitizer Evaluation.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Merchant, Bion J.

    Sandia National Laboratories has tested and evaluated two Guralp Affinity digitizers. The Affinity digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self- noise, dynamic range, system noise, response, passband, and timing. The Affinity digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

  16. Using lidocaine and benzocaine to link sodium channel molecular conformations to state-dependent antiarrhythmic drug affinity.

    PubMed

    Hanck, Dorothy A; Nikitina, Elena; McNulty, Megan M; Fozzard, Harry A; Lipkind, Gregory M; Sheets, Michael F

    2009-08-28

    Lidocaine and other antiarrhythmic drugs bind in the inner pore of voltage-gated Na channels and affect gating use-dependently. A phenylalanine in domain IV, S6 (Phe1759 in Na(V)1.5), modeled to face the inner pore just below the selectivity filter, is critical in use-dependent drug block. Measurement of gating currents and concentration-dependent availability curves to determine the role of Phe1759 in coupling of drug binding to the gating changes. The measurements showed that replacement of Phe1759 with a nonaromatic residue permits clear separation of action of lidocaine and benzocaine into 2 components that can be related to channel conformations. One component represents the drug acting as a voltage-independent, low-affinity blocker of closed channels (designated as lipophilic block), and the second represents high-affinity, voltage-dependent block of open/inactivated channels linked to stabilization of the S4s in domains III and IV (designated as voltage-sensor inhibition) by Phe1759. A homology model for how lidocaine and benzocaine bind in the closed and open/inactivated channel conformation is proposed. These 2 components, lipophilic block and voltage-sensor inhibition, can explain the differences in estimates between tonic and open-state/inactivated-state affinities, and they identify how differences in affinity for the 2 binding conformations can control use-dependence, the hallmark of successful antiarrhythmic drugs.

  17. Surface plasmon resonance label-free monitoring of antibody antigen interactions in real time

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kausaite, A.; van Dijk, M.; Castrop, J.

    2007-01-01

    Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without labeling. The system can therefore be used to determine both affinity and rate constants for interactions between various types of molecules. Here, we describe the application of a surface plasmon resonance biosensor for label-free investigation of the interaction between an immobilized antigen bovine serum albumin (BSA) and antibodymore » rabbit anti-cow albumin IgG1 (anti-BSA). The formation of a self-assembled monolayer (SAM) over a gold surface is introduced into this laboratory training protocol as an effective immobilization method, which is very promising in biosensing systems based on detection of affinity interactions. In the next step, covalent attachment via artificially formed amide bonds is applied for the immobilization of proteins on the formed SAM surface. These experiments provide suitable experience for postgraduate students to help them understand immobilization of biologically active materials via SAMs, fundamentals of surface plasmon resonance biosensor applications, and determination of non-covalent biomolecular interactions. The experiment is designed for master and/or Ph.D. students. In some particular cases, this protocol might be adoptable for bachelor students that already have completed an extended biochemistry program that included a background in immunology.« less

  18. Scale factor management in the studies of affine models of shockproof garment elements

    NASA Astrophysics Data System (ADS)

    Denisov, Oleg; Pleshko, Mikhail; Ponomareva, Irina; Merenyashev, Vitaliy

    2018-03-01

    New samples of protective garment for performing construction work at height require numerous tests in conditions close to real conditions of extreme vital activity. The article presents some results of shockproof garment element studies and a description of a patented prototype. The tests were carried out on a model which geometric dimensions were convenient for manufacturing it in a limited batch. In addition, the used laboratory equipment (for example, a unique power pendulum), blanks made of a titanium-nickel alloy with a shape memory effect also imposed their limitations. The problem of the adequacy of the obtained experimental results transfer to mass-produced products was solved using tools of the classical similarity theory. Scale factor management influence in the affine modeling of the shockproof element, studied on the basis of the equiatomic titanium-nickel alloy with the shape memory effect, allowed us to assume, with a sufficient degree of reliability, the technical possibility of extrapolating the results of experimental studies to full-scale objects for the formation of the initial data of the mathematical model of shockproof garment dynamics elastoplastic deformation (while observing the similarity of the features of external loading).

  19. 16 CFR 305.10 - Ranges of comparability on the required labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... cost. The Representative Average Unit Energy Cost figures to be used on labels as required by § 305.11... Commission shall publish revised Representative Average Unit Energy Cost figures in the Federal Register in... ACTS OF CONGRESS ENERGY AND WATER USE LABELING FOR CONSUMER PRODUCTS UNDER THE ENERGY POLICY AND...

  20. Species-Level Identification of Orthopoxviruses with an Oligonucleotide Microchip

    PubMed Central

    Lapa, Sergey; Mikheev, Maxim; Shchelkunov, Sergei; Mikhailovich, Vladimir; Sobolev, Alexander; Blinov, Vladimir; Babkin, Igor; Guskov, Alexander; Sokunova, Elena; Zasedatelev, Alexander; Sandakhchiev, Lev; Mirzabekov, Andrei

    2002-01-01

    A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within the crmB gene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used). PMID:11880388

  1. Richness, systematics, and distribution of molluscs associated with the macroalga Gigartina skottsbergii in the Strait of Magellan, Chile: A biogeographic affinity study.

    PubMed

    Rosenfeld, Sebastián; Aldea, Cristian; Mansilla, Andrés; Marambio, Johanna; Ojeda, Jaime

    2015-01-01

    Knowledge about the marine malacofauna in the Magellan Region has been gained from many scientific expeditions that were carried out during the 19th century. However, despite the information that exists about molluscs in the Magellan Region, there is a lack of studies about assemblages of molluscs co-occurring with macroalgae, especially commercially exploitable algae such as Gigartina skottsbergii, a species that currently represents the largest portion of carrageenans within the Chilean industry. The objective of this study is to inform about the richness, systematics, and distribution of the species of molluscs associated with natural beds in the Strait of Magellan. A total of 120 samples from quadrates of 0.25 m(2) were obtained by SCUBA diving at two sites within the Strait of Magellan. Sampling occurred seasonally between autumn 2010 and summer 2011: 15 quadrates were collected at each site and season. A total of 852 individuals, corresponding to 42 species of molluscs belonging to Polyplacophora (9 species), Gastropoda (24), and Bivalvia (9), were identified. The species richness recorded represents a value above the average richness of those reported in studies carried out in the last 40 years in sublittoral bottoms of the Strait of Magellan. The biogeographic affinity indicates that the majority of those species (38%) present an endemic Magellanic distribution, while the rest have a wide distribution in the Magellanic-Pacific, Magellanic-Atlantic, and Magellanic-Southern Ocean. The molluscs from the Magellan Region serve as study models for biogeographic relationships that can explain long-reaching patterns and are meaningful in evaluating possible ecosystemic changes generated by natural causes or related to human activities.

  2. Richness, systematics, and distribution of molluscs associated with the macroalga Gigartina skottsbergii in the Strait of Magellan, Chile: A biogeographic affinity study

    PubMed Central

    Rosenfeld, Sebastián; Aldea, Cristian; Mansilla, Andrés; Marambio, Johanna; Ojeda, Jaime

    2015-01-01

    Abstract Knowledge about the marine malacofauna in the Magellan Region has been gained from many scientific expeditions that were carried out during the 19th century. However, despite the information that exists about molluscs in the Magellan Region, there is a lack of studies about assemblages of molluscs co-occurring with macroalgae, especially commercially exploitable algae such as Gigartina skottsbergii, a species that currently represents the largest portion of carrageenans within the Chilean industry. The objective of this study is to inform about the richness, systematics, and distribution of the species of molluscs associated with natural beds in the Strait of Magellan. A total of 120 samples from quadrates of 0.25 m2 were obtained by SCUBA diving at two sites within the Strait of Magellan. Sampling occurred seasonally between autumn 2010 and summer 2011: 15 quadrates were collected at each site and season. A total of 852 individuals, corresponding to 42 species of molluscs belonging to Polyplacophora (9 species), Gastropoda (24), and Bivalvia (9), were identified. The species richness recorded represents a value above the average richness of those reported in studies carried out in the last 40 years in sublittoral bottoms of the Strait of Magellan. The biogeographic affinity indicates that the majority of those species (38%) present an endemic Magellanic distribution, while the rest have a wide distribution in the Magellanic-Pacific, Magellanic-Atlantic, and Magellanic-Southern Ocean. The molluscs from the Magellan Region serve as study models for biogeographic relationships that can explain long-reaching patterns and are meaningful in evaluating possible ecosystemic changes generated by natural causes or related to human activities. PMID:26448707

  3. Modification degrees at specific sites on heparan sulphate: an approach to measure chemical modifications on biological molecules with stable isotope labelling

    PubMed Central

    Wu, Zhengliang L.; Lech, Miroslaw

    2005-01-01

    Chemical modification of biological molecules is a general mechanism for cellular regulation. A quantitative approach has been developed to measure the extent of modification on HS (heparan sulphates). Sulphation on HS by sulphotransferases leads to variable sulphation levels, which allows cells to tune their affinities to various extracellular proteins, including growth factors. With stable isotope labelling and HPLC-coupled MS, modification degrees at various O-sulphation sites could be determined. A bovine kidney HS sample was first saturated in vitro with 34S by an OST (O-sulphotransferase), then digested with nitrous acid and analysed with HPLC-coupled MS. The 34S-labelled oligosaccharides were identified based on their unique isotope clusters. The modification degrees at the sulphotransferase recognition sites were obtained by calculating the intensities of isotopic peaks in the isotope clusters. The modification degrees at 3-OST-1 and 6-OST-1 sites were examined in detail. This approach can also be used to study other types of chemical modifications on biological molecules. PMID:15743272

  4. Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

    PubMed

    Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

    2006-07-01

    High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

  5. Enrichment of high affinity subclasses and glycoforms from serum-derived IgG using FcγRs as affinity ligands.

    PubMed

    Boesch, Austin W; Kappel, James H; Mahan, Alison E; Chu, Thach H; Crowley, Andrew R; Osei-Owusu, Nana Y; Alter, Galit; Ackerman, Margaret E

    2018-05-01

    As antibodies continue to gain predominance in drug discovery and development pipelines, efforts to control and optimize their activity in vivo have matured to incorporate sophisticated abilities to manipulate engagement of specific Fc binding partners. Such efforts to promote diverse functional outcomes include modulating IgG-Fc affinity for FcγRs to alternatively potentiate or reduce effector functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. While a number of natural and engineered Fc features capable of eliciting variable effector functions have been demonstrated in vitro and in vivo, elucidation of these important functional relationships has taken significant effort through use of diverse genetic, cellular and enzymatic techniques. As an orthogonal approach, we demonstrate use of FcγR as chromatographic affinity ligands to enrich and therefore simultaneously identify favored binding species from a complex mixture of serum-derived pooled polycloncal human IgG, a load material that contains the natural repertoire of Fc variants and post-translational modifications. The FcγR-enriched IgG was characterized for subclass and glycoform composition and the impact of this bioseparation step on antibody activity was measured in cell-based effector function assays including Natural Killer cell activation and monocyte phagocytosis. This work demonstrates a tractable means to rapidly distinguish complex functional relationships between two or more interacting biological agents by leveraging affinity chromatography followed by secondary analysis with high-resolution biophysical and functional assays and emphasizes a platform capable of surveying diverse natural post-translational modifications that may not be easily produced with high purity or easily accessible with recombinant expression techniques. © 2018 Wiley Periodicals, Inc.

  6. Brief communication: a cranial morphometric assessment of the taxonomic affinities of Trachypithecus auratus (E. Geoffroy, 1812 primates: Colobinae) with a reassessment of the T. auratus type specimen.

    PubMed

    Ingicco, Thomas; Balzeau, Antoine; Callou, Cécile; Fitriana, Yuliana Sulistya

    2011-10-01

    The debate over the taxonomic position and affinities of Trachypithecus auratus has been ongoing since its identification by E. Geoffroy Saint-Hilaire in 1812. The type specimen of this species is housed in Muséum national d'Histoire naturelle in Paris (MNHN-ZM 2005-912). This point is debated due to the complex and fluctuating taxonomy of Southeast Asian Colobinae (Brandon-Jones et al.: Int J Primatol 25 (2004) 97-164) and to the fact that this individual is represented by a mounted skeleton. By means of 3D medical imaging methodologies we describe for the first time the cranial anatomy of the specimen MNHN-ZM 2005-912 and compare it with other Trachypithecus species, in order to test the molecular systematic hypotheses for affinities among the T. auratus-T. cristatus group. We ascertain the taxonomic attribution of this individual to the species Trachypithecus auratus species. The most diagnostic characters shared by the type specimen and Trachypithecus auratus compared to other species of Trachypithecus are the rounded orbits and the straight facial profile. We then try to clarify the inconsistencies concerning the geographical provenance of the type. The island of Java appears to be the most probable locality from a cluster analysis based on linear morphometry. After this approach and a discriminant analysis, a northeastern Javanese provenance of this specimen, as proposed by Brandon-Jones et al. (Int J Primatol 25 (2004) 97-164) is dubious. Finally we provide 3D models of the skull and the endocast, and a list of cranial landmark coordinates of the holotype for future research. Copyright © 2011 Wiley-Liss, Inc.

  7. Dialkoxyquinazolines: Screening Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.

    2005-09-01

    The epidermal growth factor receptor (EGFR), a long-standingdrug development target, is also a desirable target for imaging. Sixteendialkoxyquinazoline analogs, suitable for labeling with positron-emittingisotopes, have been synthesized and evaluated in a battery of in vitroassays to ascertain their chemical and biological properties. Thesecharacteristics provided the basis for the adoption of a selection schemato identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of thecompounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFRtyrosine kinase. All of the analogs inhibited ligand-induced EGFRtyrosine phosphorylation (IC50 = 0.8 - 20more » nM). The HPLC-estimatedoctanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline aswell as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the bestcombination of characteristics that warrant radioisotope labeling andfurther evaluation in tumor-bearing mice.« less

  8. Improvement of Aptamer Affinity by Dimerization

    PubMed Central

    Hasegawa, Hijiri; Taira, Ken-ichi; Sode, Koji; Ikebukuro, Kazunori

    2008-01-01

    To increase the affinities of aptamers for their targets, we designed an aptamer dimer for thrombin and VEGF. This design is based on the avidity of the antibody, which enables the aptamer to connect easily since it is a single-strand nucleic acid. In this study, we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer. Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimer has a Kd value which is 1/10 of that of the monomers from which it is composed. Also, the designed aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers against vascular endothelial growth factor (VEGF165), which is a homodimeric protein. As in the case of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower Kd value than that of the monomer. This study demonstrated that the dimerization of aptamers effectively improves the affinities of those aptamers for their targets. PMID:27879754

  9. Environmental factors influence both abundance and genetic diversity in a widespread bird species

    PubMed Central

    Liu, Yang; Webber, Simone; Bowgen, Katharine; Schmaltz, Lucie; Bradley, Katharine; Halvarsson, Peter; Abdelgadir, Mohanad; Griesser, Michael

    2013-01-01

    Genetic diversity is one of the key evolutionary variables that correlate with population size, being of critical importance for population viability and the persistence of species. Genetic diversity can also have important ecological consequences within populations, and in turn, ecological factors may drive patterns of genetic diversity. However, the relationship between the genetic diversity of a population and how this interacts with ecological processes has so far only been investigated in a few studies. Here, we investigate the link between ecological factors, local population size, and allelic diversity, using a field study of a common bird species, the house sparrow (Passer domesticus). We studied sparrows outside the breeding season in a confined small valley dominated by dispersed farms and small-scale agriculture in southern France. Population surveys at 36 locations revealed that sparrows were more abundant in locations with high food availability. We then captured and genotyped 891 house sparrows at 10 microsatellite loci from a subset of these locations (N = 12). Population genetic analyses revealed weak genetic structure, where each locality represented a distinct substructure within the study area. We found that food availability was the main factor among others tested to influence the genetic structure between locations. These results suggest that ecological factors can have strong impacts on both population size per se and intrapopulation genetic variation even at a small scale. On a more general level, our data indicate that a patchy environment and low dispersal rate can result in fine-scale patterns of genetic diversity. Given the importance of genetic diversity for population viability, combining ecological and genetic data can help to identify factors limiting population size and determine the conservation potential of populations. PMID:24363897

  10. Two new species of Nemouridae (Plecoptera) from Vietnam.

    PubMed

    Fochetti, Romolo; Ceci, Massimo

    2017-05-23

    Two new species belonging to the family Nemouridae, Nemoura kontumensis sp. nov. and Amphinemura konplongensis sp. nov. are described from the Central Highlands of Vietnam. Remarks on the affinities with related species are given.

  11. Relationship between the species-representative phenotype and intraspecific variation in Ranunculaceae floral organ and Asteraceae flower numbers

    PubMed Central

    Kitazawa, Miho S.; Fujimoto, Koichi

    2016-01-01

    Background and Aims Phenotypic variation in floral morphologies contributes to speciation by testing various morphologies that might have higher adaptivity, leading eventually to phylogenetic diversity. Species diversity has been recognized, however, by modal morphologies where the variation is averaged out, so little is known about the relationship between the variation and the diversity. Methods We analysed quantitatively the intraspecific variation of the organ numbers within flowers of Ranunculaceae, a family which branched near the monocot–eudicot separation, and the numbers of flowers within the capitula of Asteraceae, one of the most diverse families of eudicots. We used four elementary statistical quantities: mean, standard deviation (s.d.), degree of symmetry (skewness) and steepness (kurtosis). Key Results While these four quantities vary among populations, we found a common relationship between s.d. and the mean number of petals and sepals in Ranunculaceae and number of flowers per capitulum in Asteraceae. The s.d. is equal to the square root of the difference between the mean and specific number, showing robustness: for example, 3 in Ficaria sepals, 5 in Ranunculus petals and Anemone tepals, and 13 in Farfugium ray florets. This square-root relationship was not applicable to Eranthis petals which show little correlation between the s.d. and mean, and the stamens and carpels of Ranunculaceae whose s.d. is proportional to the mean. The specific values found in the square-root relationship provide a novel way to find the species-representative phenotype among varied morphologies. Conclusions The representative phenotype is, in most cases, unique to the species or genus level, despite intraspecific differences of average phenotype among populations. The type of variation shown by the statistical quantities indicates not only the robustness of the morphologies but also how flowering plants changed during evolution among representative phenotypes that

  12. Evaluation of a metalloporphyrin (THPPMnCl) for necrosis-affinity in rat models of necrosis.

    PubMed

    Li, Yue; Liu, Xuejiao; Zhang, Dongjian; Lou, Bin; Peng, Fei; Wang, Xiaoning; Shan, Xin; Jiang, Cuihua; Gao, Meng; Sun, Ziping; Ni, Yicheng; Huang, Dejian; Zhang, Jian

    2015-12-01

    The combination of an (13I)I-labeled necrosis-targeting agent (NTA) with a vascular disrupting agent is a novel and potentially powerful technique for tumor necrosis treatment (TNT). The purpose of this study was to evaluate a NTA candidate, THPPMnCl, using (131)I isotope for tracing its biodistribution and necrosis affinity. (131)I-THPPMnCl was intravenously injected in rat models with liver, muscle, and tumor necrosis and myocardial infarction (MI), followed by investigations with macroscopic autoradiography, triphenyltetrazolium chloride (TTC) histochemical staining, fluorescence microscopy and H&E stained histology for up to 9 days. (131)I-THPPMnCl displayed a long-term affinity for all types of necrosis and accumulation in the mononuclear phagocytic system especially in the liver. Autoradiograms and TTC staining showed a good targetability of (131)I-THPPMnCl for MI. These findings indicate the potential of THPPMnCl for non-invasive imaging assessment of necrosis, such as in MI. However, (13I)I-THPPMnCl is unlikely suitable for TNT due to its long-term retention in normal tissues.

  13. Toward improved pregnancy labelling.

    PubMed

    Koren, Gideon; Sakaguchi, Sachi; Klieger, Chagit; Kazmin, Alex; Osadchy, Alla; Yazdani-Brojeni, Parvaneh; Matok, Ilan

    2010-01-01

    Information about the use of a medication in pregnancy is part of overall drug labelling as prepared by the pharmaceutical company and approved by the regulators. It is aimed at assisting clinicians in prescribing, however, very few drugs are labelled for specific indications in pregnancy, since there is rarely information about the use of a drug in this condition. Recently the FDA has drafted new guidelines for the labeling of drugs in pregnancy and breastfeeding, to replace the A,B,C,D,X system that was used for more than 30 years. Here we document the use of the new system through 3 different medications; each representing a different clinical situation in pregnancy--acute infection, chronic pain, and drug use during labor. Advantages and challenges in the new system are being highlighted.

  14. The Mobile Phone Affinity Scale: Enhancement and Refinement

    PubMed Central

    Rosen, Rochelle K

    2016-01-01

    Background Existing instruments that assess individuals’ relationships with mobile phones tend to focus on negative constructs such as addiction or dependence, and appear to assume that high mobile phone use reflects pathology. Mobile phones can be beneficial for health behavior change, disease management, work productivity, and social connections, so there is a need for an instrument that provides a more balanced assessment of the various aspects of individuals’ relationships with mobile phones. Objective The purpose of this research was to develop, revise, and validate the Mobile Phone Affinity Scale, a multi-scale instrument designed to assess key factors associated with mobile phone use. Methods Participants (N=1058, mean age 33) were recruited from Amazon Mechanical Turk between March and April of 2016 to complete a survey that assessed participants’ mobile phone attitudes and use, anxious and depressive symptoms, and resilience. Results Confirmatory factor analysis supported a 6-factor model. The final measure consisted of 24 items, with 4 items on each of 6 factors: Connectedness, Productivity, Empowerment, Anxious Attachment, Addiction, and Continuous Use. The subscales demonstrated strong internal consistency (Cronbach alpha range=0.76-0.88, mean 0.83), and high item factor loadings (range=0.57-0.87, mean 0.75). Tests for validity further demonstrated support for the individual subscales. Conclusions Mobile phone affinity may have an important impact in the development and effectiveness of mobile health interventions, and continued research is needed to assess its predictive ability in health behavior change interventions delivered via mobile phones. PMID:27979792

  15. SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Klein, M.; Canoll, P.D.; Musacchio, J.M.

    1991-01-01

    The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drugmore » has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.« less

  16. Antisymmetric tensor generalizations of affine vector fields.

    PubMed

    Houri, Tsuyoshi; Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-02-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank- p antisymmetric affine tensor fields in n -dimensions is bounded by ( n + 1)!/ p !( n - p )!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes.

  17. Processes, data structures, and apparatuses for representing knowledge

    DOEpatents

    Hohimer, Ryan E [West Richland, WA; Thomson, Judi R [Guelph, CA; Harvey, William J [Richland, WA; Paulson, Patrick R [Pasco, WA; Whiting, Mark A [Richland, WA; Tratz, Stephen C [Richland, WA; Chappell, Alan R [Seattle, WA; Butner, R Scott [Richland, WA

    2011-09-20

    Processes, data structures, and apparatuses to represent knowledge are disclosed. The processes can comprise labeling elements in a knowledge signature according to concepts in an ontology and populating the elements with confidence values. The data structures can comprise knowledge signatures stored on computer-readable media. The knowledge signatures comprise a matrix structure having elements labeled according to concepts in an ontology, wherein the value of the element represents a confidence that the concept is present in an information space. The apparatus can comprise a knowledge representation unit having at least one ontology stored on a computer-readable medium, at least one data-receiving device, and a processor configured to generate knowledge signatures by comparing datasets obtained by the data-receiving devices to the ontologies.

  18. Microbial status and product labelling of 58 original tattoo inks.

    PubMed

    Høgsberg, T; Saunte, D M; Frimodt-Møller, N; Serup, J

    2013-01-01

    European Council resolutions on tattoo ink introduce sterility and preservation of inks to protect customers. Inks used in Denmark are typically purchased over the internet from international suppliers and manufacturers from the US and the UK. In Denmark tattoo inks are regulated and labelled according to REACH as if they were plain chemicals. The objective of this study was to check the microbial product safety of unopened and opened tattoo ink stock bottles. Packaging, labelling, preservation, sterility and contamination with micro-organisms were studied. Physical inspection and culture of bacteria and fungi. Six of 58 unopened stock bottles (10%) were contaminated with bacteria and one of six samples (17%) of previously used stock bottles was contaminated. The bacterial species represented bacteria considered pathogenic in humans as well as non-pathogenic environmental bacteria. Yeast or moulds were detected in none of the samples. A total of 31% of the manufacturers informed only about the brand name. No information about content, sterility, risks or expiry date was indicated on the label. A total of 42% claimed sterility of their inks. A total of 54% labelled a maximum period of durability of typically 2-3 years. The physical sealing was leaking in 28% of the products. The European Council resolutions regarding safety of tattoo inks are not effective. Stock bottles of tattoo ink may contain bacteria pathogenic to humans and environmental bacteria, and packaging, labelling and preservation of inks are of inadequate quality. Claim of sterility can be erroneous. © 2011 The Authors. Journal of the European Academy of Dermatology and Venereology © 2011 European Academy of Dermatology and Venereology.

  19. 64Cu-Labeled Repebody Molecules for Imaging of Epidermal Growth Factor Receptor-Expressing Tumors.

    PubMed

    Pyo, Ayoung; Yun, Misun; Kim, Hyeon Sik; Kim, Tae-Yoon; Lee, Joong-Jae; Kim, Jung Young; Lee, Sunwoo; Kwon, Seong Young; Bom, Hee-Seung; Kim, Hak-Sung; Kim, Dong-Yeon; Min, Jung-Joon

    2018-02-01

    The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptors and is overexpressed in many tumor types. A repebody is a newly designed nonantibody protein scaffold for tumor targeting that contains leucine-rich repeat modules. In this study, 3 64 Cu-labeled anti-EGFR repebodies with different chelators were synthesized, and their biologic characteristics were assessed in cultured cells and tumor-bearing mice. Methods: Repebodies were synthesized with the chelators 2-( p -isothiocyanatobenzyl)-1,4,7-triazacyclononane- N,N',N,″- triacetic acid trihydrochloride ([ p -SCN-Bn]-NOTA), 2,2',2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA- N -hydroxysuccinimide ester), or 1-( p -isothiocyanatobenzyl)diethylenetriamine pentaacetic acid trihydrochloride ([ p -SCN-Bn]-DTPA) in 1.0 M NaHCO 3 buffer (pH 9.2) for 24 h. Purified NOTA-, DOTA-, and DTPA-conjugated repebody were radiolabeled with 64 Cu in 0.1 M NH 4 OAc buffer (pH 5.5). To compare the EGFR-binding affinities of the repebodies, cellular uptake studies were performed with the human non-small cell lung cancer cell line H1650 (high expression of EGFR) and the human colon adenocarcinoma cell line SW620 (low expression of EGFR). Biodistribution and small-animal PET imaging studies were performed using H1650 tumor-bearing mice. Results: Radiochemical yields of the 64 Cu-labeled repebodies were approximately 70%-80%. Cellular uptake of the NOTA-, DOTA-, and DTPA-repebodies was over 4-fold higher in H1650 cells than in SW620 cells at 1 h. The 3 repebodies had accumulated specifically in H1650 tumor-bearing nude mice by 1 h after intravenous injection and were retained for over 24 h, as measured by the percentage injected dose per gram of tissue (%ID/g). Tumor uptake of all repebodies increased from 1 to 6 h (at 1 h, 6.28, 8.46, and 6.91 %ID/g for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 9.4, 8.28, and 10.1 %ID

  20. Personality Types and Affinity for Computers

    DTIC Science & Technology

    1991-03-01

    differences on personality dimensions between the respondents, and to explore the relationship between these differences and computer affinity. The results...between the respondents, and to explore the relationship between these differences and computer affinity. The results revealed no significant differences...type to this measure of computer affinity. 2 II. LITERATURZ REVIEW The interest of this study was the relationship between a person’s psychological

  1. Current Status of Pediatric Labeling in China and the near Future Efforts Needed for the Country

    PubMed Central

    Li, Zhiping; Wang, Yi; Wu, Dan; Gao, Xuan; Wang, Zhiyun

    2014-01-01

    Background: Children are recognized as “therapeutic orphan” in many parts of the world, one expression of this is the lack of adequate pediatric labeling information. Some research studies have been done to investigate the pediatric labeling condition in the U.S. and other countries, but no national studies had been carried out in China. This survey was conducted aiming to inquire the current situation of pediatric labeling in China. Methods: We investigated 6020 child-applied medicines from 15 representative Chinese hospitals, and analyzed the information according to the dosage forms, therapeutic category, and label information integrity. Results: Among all these medicines, only 238 (3.95%) are pediatric products, the rest are adult formulations with an extended use in children. The major pediatric formulations were injection (45.95%), tablet (23.69%), and capsule (4.93%), respectively. Alimentary tract/metabolism medicine (24.70%) and infections medicines (20.60%) had the most species. In prescription drugs, only 210 of 5187 (4%) medicines had adequate pediatric labeling information. The main cause of this deficiency was lack of evidence derived from pediatric clinical trials. Conclusion: The dilemma of “therapeutic orphan” requires significant attention. Inadequate labeling information and lack of pediatric clinical trials were two prominent issues in China. It calls for more efforts from pharmaceutical industries, regulatory agencies, and legislature in China to collaborate and find solution to improve the situation. PMID:24724075

  2. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

    Treesearch

    Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

    2001-01-01

    BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

  3. Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation.

    PubMed

    Zhou, Zhengyuan; Vaidyanathan, Ganesan; McDougald, Darryl; Kang, Choong Mo; Balyasnikova, Irina; Devoogdt, Nick; Ta, Angeline N; McNaughton, Brian R; Zalutsky, Michael R

    2017-12-01

    Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts. sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[ 18 F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([ 18 F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [ 18 F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [ 18 F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[ 18 F]RL-I conjugates. [ 18 F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97-100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [ 125 I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95-99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [ 18 F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively

  4. 2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification

    PubMed Central

    2016-01-01

    Photoaffinity labels are powerful tools for dissecting ligand–protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C–H/X–H bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. Herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (ACT), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding affinity and specificity of the parent drug. Compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies ACT showed higher photo-cross-linking yields toward their protein targets in vitro based on mass spectrometry analysis. In the in situ target identification studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine. We expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery. PMID:27740749

  5. 2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification.

    PubMed

    Herner, András; Marjanovic, Jasmina; Lewandowski, Tracey M; Marin, Violeta; Patterson, Melanie; Miesbauer, Laura; Ready, Damien; Williams, Jon; Vasudevan, Anil; Lin, Qing

    2016-11-09

    Photoaffinity labels are powerful tools for dissecting ligand-protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C-H/X-H bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. Herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (ACT), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding affinity and specificity of the parent drug. Compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies ACT showed higher photo-cross-linking yields toward their protein targets in vitro based on mass spectrometry analysis. In the in situ target identification studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine. We expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery.

  6. Smooth affine shear tight frames: digitization and applications

    NASA Astrophysics Data System (ADS)

    Zhuang, Xiaosheng

    2015-08-01

    In this paper, we mainly discuss one of the recent developed directional multiscale representation systems: smooth affine shear tight frames. A directional wavelet tight frame is generated by isotropic dilations and translations of directional wavelet generators, while an affine shear tight frame is generated by anisotropic dilations, shears, and translations of shearlet generators. These two tight frames are actually connected in the sense that the affine shear tight frame can be obtained from a directional wavelet tight frame through subsampling. Consequently, an affine shear tight frame indeed has an underlying filter bank from the MRA structure of its associated directional wavelet tight frame. We call such filter banks affine shear filter banks, which can be designed completely in the frequency domain. We discuss the digitization of affine shear filter banks and their implementations: the forward and backward digital affine shear transforms. Redundancy rate and computational complexity of digital affine shear transforms are also investigated in this paper. Numerical experiments and comparisons in image/video processing show the advantages of digital affine shear transforms over many other state-of-art directional multiscale representation systems.

  7. Using mass spectrometry to study the photo-affinity labeling of protein tyrosine phosphatase 1B

    NASA Astrophysics Data System (ADS)

    Leriche, Tammy; Skorey, Kathryn; Roy, Patrick; McKay, Dan; Bateman, Kevin P.

    2004-11-01

    Protein tyrosine phosphatase 1B (PTP1B) is a potential target for the treatment of Type II diabetes and several companies are developing small molecule inhibitors of this enzyme. Part of the characterization of these compounds as PTP1B inhibitors is the understanding of how they bind in the enzyme active site. The use of photo-activated inhibitors that target the active site can provide such insight. This paper describes the characterization of a photoprobe directed at the active site of PTP1B. Mass spectrometry revealed the specific binding of the probe to the intact protein. Digestion of the labeled protein followed by LC-MS and LC-MS/MS was used to show that the photoprobe binds to a specific active site amino acid. This was confirmed by comparison with the X-ray structure of PTP1B with a PTP1B inhibitor. The probe labels a conserved acidic residue (Asp) that is required for catalytic activity. This photoprobe may prove to be a useful tool for the development of a PTP1B inhibitor or for the study of PTPs in general.

  8. The practice and perception of precautionary allergen labelling by the Australasian food manufacturing industry.

    PubMed

    Zurzolo, G A; Peters, R L; Koplin, J J; de Courten, M; Mathai, M L; Tye-Din, J A; Tang, M L K; Campbell, D E; Ponsonby, A-L; Prescott, S L; Gurrin, L; Dharmage, S C; Allen, K J

    2017-07-01

    The precautionary allergen labelling (PAL) and Voluntary Incidental Trace Allergen Labelling (VITAL ® ) tools were designed by industry to assist consumers with selecting safe foods for consumption. However, a sizeable proportion of food products bear no label, and it is unclear whether these products are free from allergens and therefore safe to consume or have simply not undergone a risk assessment and therefore remain unlabelled for that reason. To assess the prevalence of unlabelled products that have undergone a risk assessment process and to examine the factors influencing industry's uptake of the VITAL ® process. A web-based questionnaire was distributed to Australasian food and grocery manufacturers. One hundred and thirty-seven Australasian manufacturers were contacted, and 59 questionnaires were returned (response rate: 43%). The respondents represented 454 different manufacturing sites. Manufacturers reported that 23% (95% CI 19-28) of products (n=102/434) that had been through the VITAL ® risk assessment process had no PAL statement on the label. 34% (95% CI 30-38), (n=204/600) of products that had undergone another (non-VITAL ® ) risk assessment process had no PAL statement. In examining the factors that influenced industry's uptake of the VITAL ® process, 25 manufacturers reported on factors that influenced the uptake of the VITAL ® process, 76% (CI 95% 55-91) reported that VITAL ® was an effective tool because it was based on science; 52% (CI 95% 31-72) reported that it was too time-consuming and 36% (CI 95% 18-57) identified a concern with it not being endorsed by the government. Currently, we estimate that at least 30% of products may have been through a risk assessment process and yet bear no PAL statement on the label. Permissive labelling could be incorporated onto these products if they have been assessed to be safe for consumption. © 2017 John Wiley & Sons Ltd.

  9. Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification.

    PubMed

    Ou, Keli; Kesuma, Djohan; Ganesan, Kumaresan; Yu, Kun; Soon, Sou Yen; Lee, Suet Ying; Goh, Xin Pei; Hooi, Michelle; Chen, Wei; Jikuya, Hiroyuki; Ichikawa, Tetsuo; Kuyama, Hiroki; Matsuo, Ei-ichi; Nishimura, Osamu; Tan, Patrick

    2006-09-01

    The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.

  10. Yttrium-90 and indium-111 labelling, receptor binding and biodistribution of [DOTA0,d-Phe1,Tyr3]octreotide, a promising somatostatin analogue for radionuclide therapy.

    PubMed

    de Jong, M; Bakker, W H; Krenning, E P; Breeman, W A; van der Pluijm, M E; Bernard, B F; Visser, T J; Jermann, E; Béhé, M; Powell, P; Mäcke, H R

    1997-04-01

    In vitro octreotide receptor binding of [111In-DOTA0,d-Phe1, Tyr3]octreotide (111In-DOTATOC) and the in vivo metabolism of 90Y- or 111In-labelled DOTATOC were investigated in rats in comparison with [111In-DTPA0]octreotide [111In-DTPAOC). 111In-DOTATOC was found to have an affinity similar to octreotide itself for the octreotide receptor in rat cerebral cortex microsomes. Twenty-four hours after injection of 90Y- or 111In-labelled DOTATOC, uptake of radioactivity in the octreotide receptor-expressing tissues pancreas, pituitary, adrenals and tumour was a factor of 2-6 that after injection of 111In-DTPAOC. Uptake of labelled DOTATOC in pituitary, pancreas, adrenals and tumour was almost completely blocked by pretreatment with 0.5 mg unlabelled octreotide, indicating specific binding to the octreotide receptors. These findings strongly indicate that 90Y-DOTATOC is a promising radiopharmaceutical for radiotherapy and that 111In-DOTATOC is of potential value for diagnosis of patients with octreotide receptor-positive lesions, such as most neuroendocrine tumours.

  11. Study and development of label-free optical biosensors for biomedical applications

    NASA Astrophysics Data System (ADS)

    Choi, Charles J.

    For the majority of assays currently performed, fluorescent or colorimetric chemical labels are commonly attached to the molecules under study so that they may be readily visualized. The methods of using labels to track biomolecular binding events are very sensitive and effective, and are employed as standardized assay protocol across research labs worldwide. However, using labels induces experimental uncertainties due to the effect of the label on molecular conformation, active binding sites, or inability to find an appropriate label that functions equivalently for all molecules in an experiment. Therefore, the ability to perform highly sensitive biochemical detection without the use of fluorescent labels would further simplify assay protocols and would provide quantitative kinetic data, while removing experimental artifacts from fluorescent quenching, shelf-life, and background fluorescence phenomena. In view of the advantages mentioned above, the study and development of optical label-free sensor technologies have been undertaken here. In general, label-free photonic crystal (PC) biosensors and metal nanodome array surface-enhanced Raman scattering (SERS) substrates, both of which are fabricated by nanoreplica molding process, have been used as the method to attack the problem. Chapter 1 shows the work on PC label-free biosensor incorporated microfluidic network for bioassay performance enhancement and kinetic reaction rate constant determination. Chapter 2 describes the work on theoretical and experimental comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays. Chapter 3 shows the work on integration of PC biosensor with actuate-to-open valve microfluidic chip for pL-volume combinatorial mixing and screening application. In Chapter 4, the development and characterization of SERS nanodome array is shown. Lastly, Chapter 5 describes SERS nanodome sensor incorporated tubing for point-of-care monitoring of

  12. Growth and mortality of larval Myctophum affine (Myctophidae, Teleostei).

    PubMed

    Namiki, C; Katsuragawa, M; Zani-Teixeira, M L

    2015-04-01

    The growth and mortality rates of Myctophum affine larvae were analysed based on samples collected during the austral summer and winter of 2002 from south-eastern Brazilian waters. The larvae ranged in size from 2·75 to 14·00 mm standard length (L(S)). Daily increment counts from 82 sagittal otoliths showed that the age of M. affine ranged from 2 to 28 days. Three models were applied to estimate the growth rate: linear regression, exponential model and Laird-Gompertz model. The exponential model best fitted the data, and L(0) values from exponential and Laird-Gompertz models were close to the smallest larva reported in the literature (c. 2·5 mm L(S)). The average growth rate (0·33 mm day(-1)) was intermediate among lanternfishes. The mortality rate (12%) during the larval period was below average compared with other marine fish species but similar to some epipelagic fishes that occur in the area. © 2015 The Fisheries Society of the British Isles.

  13. 16 CFR 309.20 - Labeling requirements for new covered vehicles.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    .... (b) Layout. Figures 4, 5, and 5.1 are prototype labels that demonstrate the proper layout. All positioning, spacing, type size, and line widths shall be similar to and consistent with the prototype labels... and 71/2 inches (19.05 cm) long. Figure 4 of appendix A represents the prototype for the labels for...

  14. Food label use and its relation to dietary intake among US adults.

    PubMed

    Ollberding, Nicholas Jay; Wolf, Randi L; Contento, Isobel

    2010-08-01

    Rates of diet-related chronic disease combined with the lack of current data on patterns of food label use by the US population warrant re-examination of the use and potential influence of this public health tool. The purpose of this study was to describe the prevalence of food label use and the association between food label use and nutrient intake in a nationally representative sample of US adults who participated in the 2005-2006 National Health and Nutrition Examination Survey. Data on food label use were collected during the interview portion of the survey, and nutrient intake was estimated using the average of two 24-hour dietary recalls. In this sample, 61.6% of participants reported using the Nutrition Facts panel, 51.6% looked at the list of ingredients, 47.2% looked at serving size, and 43.8% reviewed health claims at least sometimes when deciding to purchase a food product. There were significant differences (P<0.05) in food label use across all demographic characteristics examined. Significant differences (P<0.05) in mean nutrient intake of total energy, total fat, saturated fat, cholesterol, sodium, dietary fiber, and sugars were observed between food label users and non-users with label users reporting healthier nutrient consumption. The greatest differences observed were for total energy and fat and for use of specific nutrient information on the food label. Despite food label use being associated with improved dietary factors, label use alone is not expected to be sufficient in modifying behavior ultimately leading to improved health outcomes. 2010 American Dietetic Association. Published by Elsevier Inc. All rights reserved.

  15. Using Lidocaine and Benzocaine to Link Sodium Channel Molecular Conformations to State-Dependent Antiarrhythmic Drug Affinity

    PubMed Central

    Hanck, Dorothy A.; Nikitina, Elena; McNulty, Megan M.; Fozzard, Harry A.; Lipkind, Gregory M.; Sheets, Michael F.

    2009-01-01

    Rationale Lidocaine and other antiarrhythmic drugs bind in the inner pore of voltage-gated Na channels and affect gating use-dependently. A phenylalanine in domain IV, S6 (Phe1759 in NaV1.5), modeled to face the inner pore just below the selectivity filter, is critical in use-dependent drug block. Objective Measurement of gating currents and concentration-dependent availability curves to determine the role of Phe1759 in coupling of drug binding to the gating changes. Methods & Results The measurements showed that replacement of Phe1759 with a non-aromatic residue permits clear separation of action of lidocaine and benzocaine into two components that can be related to channel conformations. One component represents the drug acting as a voltage-independent, low-affinity blocker of closed channels (designated as lipophilic block), and the second represents high-affinity, voltage-dependent block of open/inactivated channels linked to stabilization of the S4's in domains III and IV (designated as voltage-sensor inhibition) by Phe1759. A homology model for how lidocaine and benzocaine bind in the closed and open/inactivated channel conformation is proposed. Conclusions These two components, lipophilic block and voltage-sensor inhibition, can explain the differences in estimates between tonic and open-state/inactivated-state affinities, and they identify how differences in affinity for the two binding conformations can control use-dependence, the hallmark of successful antiarrhythmic drugs. PMID:19661462

  16. Synthesis and preliminary biological evaluations of fluorescent or 149Promethium labeled Trastuzumab-polyethylenimine

    DOE PAGES

    Fitzsimmons, Jonathan; Nayak, Tapan; Cutler, Cathy; ...

    2015-12-30

    Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI), Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab wasmore » concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. In conclusion, the results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.« less

  17. Heparin affin regulatory peptide/pleiotrophin mediates fibroblast growth factor 2 stimulatory effects on human prostate cancer cells.

    PubMed

    Hatziapostolou, Maria; Polytarchou, Christos; Katsoris, Panagiotis; Courty, Jose; Papadimitriou, Evangelia

    2006-10-27

    Fibroblast growth factor 2 (FGF2) is a pleiotropic growth factor that has been implicated in prostate cancer formation and progression. In the present study we found that exogenous FGF2 significantly increased human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be an important mediator of FGF2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF2, through FGF receptors (FGFRs), significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full-length promoter of HARP gene. Using a combination of Western blot analyses, as well as genetic and pharmacological inhibitors, we found that activation of FGFR by FGF2 in LNCaP cells leads to NAD(P)H oxidase-dependent hydrogen peroxide production, phosphorylation of ERK1/2 and p38, activation of AP-1, increased expression and secretion of HARP, and, finally, increased cell proliferation and migration. These results establish the role and the mode of activity of FGF2 in LNCaP cells and support an interventional role of HARP in FGF2 effects, providing new insights on the interplay among growth factor pathways within prostate cancer cells.

  18. Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

    PubMed

    Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

    2017-08-01

    We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

  19. Physicians' reasons not to discontinue long-term used off-label antipsychotic drugs in people with intellectual disability.

    PubMed

    de Kuijper, G M; Hoekstra, P J

    2017-10-01

    People with intellectual disability (ID) frequently use antipsychotic drugs on an off-label base, often for many years. Physicians' decisions to discontinue these drugs not only depend on patient characteristics, like the presence of mental or behavioural disorders, but also on environmental factors, such as inappropriate living circumstances, and on attitudes, knowledge and beliefs of staff, clients and their representatives towards the effects of antipsychotic drug use. In this study, we therefore investigated the influence of participant and setting-related factors on decisions of physicians not to discontinue off-label prescribed antipsychotics. The study took place in living facilities of six service providers for people with ID spread over the Netherlands and staffed with support professionals, nurses, behavioural scientist and physicians and was part of an antipsychotics discontinuation trial. ID physicians had to decide whether the off-label use of antipsychotics could be discontinued. Medical and pharmaceutical records were used to establish the prevalence of antipsychotic drug use in the study population, along with duration of use and whether the use was off-label. Reasons of physicians not to discontinue the prescription of antipsychotics in those participants who used off-label antipsychotics for more than a year were collected and categorised as related to participant or setting characteristics, including lack of consent to discontinue, and staff members, participants or their legal representatives. Of the 3299 clients of the service providers, 977 used one or more antipsychotic drugs. The prevalence of antipsychotic drug use was 30%. Reasons for use were in 5% of cases, a chronic psychotic disorder classified according to Diagnostic System Mental Disorders, Fourth Edition, criteria, in 25%, present or past (suspected) non-schizophrenia-related psychotic symptoms and in 69%, challenging behaviours. Overall, physicians were willing to discontinue their

  20. Factors controlling bark decomposition and its role in wood decomposition in five tropical tree species

    PubMed Central

    Dossa, Gbadamassi G. O.; Paudel, Ekananda; Cao, Kunfang; Schaefer, Douglas; Harrison, Rhett D.

    2016-01-01

    Organic matter decomposition represents a vital ecosystem process by which nutrients are made available for plant uptake and is a major flux in the global carbon cycle. Previous studies have investigated decomposition of different plant parts, but few considered bark decomposition or its role in decomposition of wood. However, bark can comprise a large fraction of tree biomass. We used a common litter-bed approach to investigate factors affecting bark decomposition and its role in wood decomposition for five tree species in a secondary seasonal tropical rain forest in SW China. For bark, we implemented a litter bag experiment over 12 mo, using different mesh sizes to investigate effects of litter meso- and macro-fauna. For wood, we compared the decomposition of branches with and without bark over 24 mo. Bark in coarse mesh bags decomposed 1.11–1.76 times faster than bark in fine mesh bags. For wood decomposition, responses to bark removal were species dependent. Three species with slow wood decomposition rates showed significant negative effects of bark-removal, but there was no significant effect in the other two species. Future research should also separately examine bark and wood decomposition, and consider bark-removal experiments to better understand roles of bark in wood decomposition. PMID:27698461

  1. Factors controlling bark decomposition and its role in wood decomposition in five tropical tree species.

    PubMed

    Dossa, Gbadamassi G O; Paudel, Ekananda; Cao, Kunfang; Schaefer, Douglas; Harrison, Rhett D

    2016-10-04

    Organic matter decomposition represents a vital ecosystem process by which nutrients are made available for plant uptake and is a major flux in the global carbon cycle. Previous studies have investigated decomposition of different plant parts, but few considered bark decomposition or its role in decomposition of wood. However, bark can comprise a large fraction of tree biomass. We used a common litter-bed approach to investigate factors affecting bark decomposition and its role in wood decomposition for five tree species in a secondary seasonal tropical rain forest in SW China. For bark, we implemented a litter bag experiment over 12 mo, using different mesh sizes to investigate effects of litter meso- and macro-fauna. For wood, we compared the decomposition of branches with and without bark over 24 mo. Bark in coarse mesh bags decomposed 1.11-1.76 times faster than bark in fine mesh bags. For wood decomposition, responses to bark removal were species dependent. Three species with slow wood decomposition rates showed significant negative effects of bark-removal, but there was no significant effect in the other two species. Future research should also separately examine bark and wood decomposition, and consider bark-removal experiments to better understand roles of bark in wood decomposition.

  2. Single-step affinity purification for fungal proteomics.

    PubMed

    Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

    2010-05-01

    A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

  3. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa

    2011-05-20

    Highlights: {yields} Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. {yields} These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. {yields} The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. Thesemore » antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.« less

  4. Germylenes: structures, electron affinities, and singlet-triplet gaps of the conventional XGeCY(3) (X = H, F, Cl, Br, and I; Y = F and Cl) species and the unexpected cyclic XGeCY(3) (Y = Br and I) systems.

    PubMed

    Bundhun, Ashwini; Abdallah, Hassan H; Ramasami, Ponnadurai; Schaefer, Henry F

    2010-12-23

    A systematic investigation of the X-Ge-CY(3) (X = H, F, Cl, Br, and I; Y = F, Cl, Br, and I) species is carried out using density functional theory. The basis sets used for all atoms (except iodine) in this work are of double-ζ plus polarization quality with additional s- and p-type diffuse functions, and denoted DZP++. Vibrational frequency analyses are performed to evaluate zero-point energy corrections and to determine the nature of the stationary points located. Predicted are four different forms of neutral-anion separations: adiabatic electron affinity (EA(ad)), zero-point vibrational energy corrected EA(ad(ZPVE)), vertical electron affinity (EA(vert)), and vertical detachment energy (VDE). The electronegativity (χ) reactivity descriptor for the halogens (X = F, Cl, Br, and I) is used as a tool to assess the interrelated properties of these germylenes. The topological position of the halogen atom bound to the divalent germanium center is well correlated with the trend in the electron affinities and singlet-triplet gaps. For the expected XGeCY(3) structures (X = H, F, Cl, Br, and I; Y = F and Cl), the predicted trend in the electron affinities is well correlated with simpler germylene derivatives (J. Phys. Chem. A 2009, 113, 8080). The predicted EA(ad(ZPVE)) values with the BHLYP functional range from 1.66 eV (FGeCCl(3)) to 2.20 eV (IGeCF(3)), while the singlet-triplet splittings range from 1.28 eV (HGeCF(3)) to 2.22 eV (FGeCCl(3)). The XGeCY(3) (Y = Br and I) species are most often characterized by three-membered cyclic systems involving the divalent germanium atom, the carbon atom, and a halogen atom.

  5. Photoaffinity Labeling of Ras Converting Enzyme using Peptide Substrates that Incorporate Benzoylphenylalanine (Bpa) Residues: Improved Labeling and Structural Implications

    PubMed Central

    Kyro, Kelly; Manandhar, Surya P.; Mullen, Daniel; Schmidt, Walter K.; Distefano, Mark D.

    2012-01-01

    Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-L-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design. PMID:22079863

  6. Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration.

    PubMed

    Cappione, Amedeo; Mabuchi, Masaharu; Briggs, David; Nadler, Timothy

    2015-04-01

    Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Diagnostic labels assigned to patients with orthopedic conditions and the influence of the label on selection of interventions: a qualitative study of orthopaedic clinical specialists.

    PubMed

    Miller-Spoto, Marcia; Gombatto, Sara P

    2014-06-01

    A variety of diagnostic classification systems are used by physical therapists, but little information about how therapists assign diagnostic labels and how the labels are used to direct intervention is available. The purposes of this study were: (1) to examine the diagnostic labels assigned to patient problems by physical therapists who are board-certified Orthopaedic Clinical Specialists (OCSs) and (2) to determine whether the label influences selection of interventions. A cross-sectional survey was conducted. Two written cases were developed for patients with low back and shoulder pain. A survey was used to evaluate the diagnostic label assigned and the interventions considered important for each case. The cases and survey were sent to therapists who are board-certified OCSs. Respondents assigned a diagnostic label and rated the importance of intervention categories for each case. Each diagnostic label was coded based on the construct it represented. Percentage responses for each diagnostic label code and intervention category were calculated. Relative importance of intervention category based on diagnostic label was examined. For the low back pain and shoulder pain cases, respectively, "Combination" (48.5%, 34.9%) and "Pathology/Pathophysiology" (32.7%, 57.3%) diagnostic labels were most common. Strengthening (85.9%, 98.1%), stretching (86.8%, 84.9%), neuromuscular re-education (87.6%, 93.4%), functional training (91.4%, 88.6%), and mobilization/manipulation (85.1%, 86.8%) were considered the most important interventions. Relative importance of interventions did not differ based on diagnostic label (χ2=0.050-1.263, P=.261-.824). The low response rate may limit the generalizability of the findings. Also, examples provided for labels may have influenced responses, and some of the label codes may have represented overlapping constructs. There is little consistency with which OCS therapists assign diagnostic labels, and the label does not seem to influence

  8. Evaluation of off-label recombinant activated factor VII for multiple indications in children.

    PubMed

    Reiter, Pamela D; Valuck, Robert J; Taylor, Ruston S

    2007-07-01

    Despite a paucity of safety and efficacy data, the use of recombinant activated factor VII in children for off-label indications has now surpassed its use in hemophilia. A retrospective chart review was conducted of 46 subjects (age, 6.7 +/- 6 years; weight, 26 +/- 20 kg) who received recombinant activated factor VII for nonhemophiliac indications between January 1, 2004, and September 1, 2005. Indications for use included prevention (n = 6) or treatment (n = 40) of bleeding due to general surgery, hepatic failure, gastrointestinal bleeding, severe traumatic brain injury, bone marrow transplant, cardiac, acetaminophen overdose, and multiorgan system failure. Decreases in prothrombin time, partial thromboplastin time, and international normalized ratio were observed. No inappropriate thrombotic events were noted. Administration of recombinant activated factor VII was associated with a reduction in coagulation markers without obvious adverse thrombotic events at cost of $4189 per dose. These findings should be confirmed in a prospective trial.

  9. pMHC affinity controls duration of CD8+ T cell–DC interactions and imprints timing of effector differentiation versus expansion

    PubMed Central

    Sharpe, James; Zehn, Dietmar; Kreutzfeldt, Mario

    2016-01-01

    During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity–primed T cells acquired cytotoxic activity earlier than high affinity–primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity–stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. PMID:27799622

  10. Mobile Technology Affinity in Renal Transplant Recipients.

    PubMed

    Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

    Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Detection of glycoproteins in the Acanthamoeba plasma membrane

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Paatero, G.I.L.; Gahmberg, C.G.

    1988-11-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presencemore » of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.« less

  12. Off-label use of recombinant factor VIIa in U.S. hospitals: analysis of hospital records.

    PubMed

    Logan, Aaron C; Yank, Veronica; Stafford, Randall S

    2011-04-19

    Recombinant factor VIIa (rFVIIa) is approved for treatment of bleeding in patients who have hemophilia with inhibitors but has been applied to a wide range of off-label indications. To estimate patterns of off-label rFVIIa use in U.S. hospitals. Retrospective database analysis. Data were extracted from the Premier Perspectives database (Premier, Charlotte, North Carolina), which contains discharge records from a sample of academic and nonacademic U.S. hospitals. 12 644 hospitalizations for patients who received rFVIIa during a hospital stay. Hospital diagnoses and patient dispositions from 1 January 2000 to 31 December 2008. Statistical weights for each hospital were used to provide national estimates of rFVIIa use. From 2000 to 2008, off-label use of rFVIIa in hospitals increased more than 140-fold, such that in 2008, 97% (95% CI, 96% to 98%) of 18 311 in-hospital uses were off-label. In contrast, in-hospital use for hemophilia increased less than 4-fold and accounted for 2.7% (CI, 1.9% to 3.5%) of use in 2008. Adult and pediatric cardiovascular surgery (29% [CI, 21% to 33%]), body and brain trauma (29% [CI, 19% to 38%]), and intracranial hemorrhage (11% [CI, 7.7% to 14%]) were the most common indications for rFVIIa use. Across all indications, in-hospital mortality was 27% (CI, 19% to 34%) and 43% (CI, 26% to 59%) of patients were discharged to home. Accuracy and completeness of the discharge diagnoses and patient medication records in the database sample cannot be verified. Off-label use of rFVIIa in the hospital setting far exceeds use for approved indications. These patterns raise concern about the application of rFVIIa to conditions for which strong supporting evidence is lacking.

  13. The Development of Ethnic/Racial Self-Labeling: Individual Differences in Context.

    PubMed

    Cheon, Yuen Mi; Bayless, Sara Douglass; Wang, Yijie; Yip, Tiffany

    2018-03-15

    Ethnic/racial self-labeling represents one's knowledge of and preference for ethnic/racial group membership, which is related to, but distinguishable from, ethnic/racial identity. This study examined the development of ethnic/racial self-labeling over time by including the concept of elaboration among a diverse sample of 297 adolescents (Time 1 mean age 14.75, 67% female, 37.4% Asian or Asian American, 10.4% Black, African American, or West Indian, 23.2% Hispanic or Latinx, 24.2% White, 4.4% other). Growth mixture modeling revealed two distinct patterns-low and high self-labeling elaboration from freshman to sophomore year of high school. Based on logistic regression analyses, the level of self-labeling elaboration was generally low among the adolescents who were foreign-born, reported low levels of ethnic/racial identity exploration, or attended highly diverse schools. We also found a person-by-context interaction where the impact of school diversity varied for foreign-born and native-born adolescents (b = 12.81, SE = 6.30, p < 0.05) and by the level of ethnic/racial identity commitment (b = 14.32, SE = 6.65, p < 0.05). These findings suggest varying patterns in ethnic/racial self-labeling elaboration among adolescents from diverse backgrounds and their linkage to individual and contextual factors.

  14. Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Besemer, J.; Hujber, A.; Kuhn, B.

    1989-10-15

    The interaction of {sup 125}I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4{degree}C to pH 3 resistance at 37{degree}C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37{degree}C led to nonlinearmore » curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.« less

  15. A recombinant fungal lectin for labeling truncated glycans on human cancer cells.

    PubMed

    Audfray, Aymeric; Beldjoudi, Mona; Breiman, Adrien; Hurbin, Amandine; Boos, Irene; Unverzagt, Carlo; Bouras, Mourad; Lantuejoul, Sylvie; Coll, Jean-Luc; Varrot, Annabelle; Le Pendu, Jacques; Busser, Benoit; Imberty, Anne

    2015-01-01

    Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases.

  16. A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells

    PubMed Central

    Hurbin, Amandine; Boos, Irene; Unverzagt, Carlo; Bouras, Mourad; Lantuejoul, Sylvie; Coll, Jean-Luc; Varrot, Annabelle; Le Pendu, Jacques; Busser, Benoit; Imberty, Anne

    2015-01-01

    Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases. PMID:26042789

  17. A new real-time method for investigation of affinity properties and binding kinetics of magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Orlov, Alexey V.; Nikitin, Maxim P.; Bragina, Vera A.; Znoyko, Sergey L.; Zaikina, Marina N.; Ksenevich, Tatiana I.; Gorshkov, Boris G.; Nikitin, Petr I.

    2015-04-01

    A method for quantitative investigation of affinity constants of receptors immobilized on magnetic nanoparticles (MP) is developed based on spectral correlation interferometry (SCI). The SCI records with a picometer resolution the thickness changes of a layer of molecules or nanoparticles due to a biochemical reaction on a cover slip, averaged over the sensing area. The method is compatible with other types of sensing surfaces employed in biosensing. The measured values of kinetic association constants of magnetic nanoparticles are 4 orders of magnitude higher than those of molecular antibody association with antigen. The developed method also suggests highly sensitive detection of antigens in a wide dynamic range. The limit of detection of 92 pg/ml has been demonstrated for prostate-specific antigen (PSA) with 50-nm MP employed as labels, which produce 3-order amplification of the SCI signals. The calibration curve features high sensitivity (slope) of 3-fold signal raise per 10-fold increase of PSA concentration within 4-order dynamic range, which is an attractive compromise for precise quantitative and highly sensitive immunoassay. The proposed biosensing technique offers inexpensive disposable sensor chips of cover slips and represents an economically sound alternative to traditional immunoassays for disease diagnostics, detection of pathogens in food and environmental monitoring.

  18. Development of a nonlinear model for the prediction of response times of glucose affinity sensors using concanavalin A and dextran and the development of a differential osmotic glucose affinity sensor

    NASA Astrophysics Data System (ADS)

    Reis, Louis G.

    With the increasing prevalence of diabetes in the United States and worldwide, blood glucose monitoring must be accurate and reliable. Current enzymatic sensors have numerous disadvantages that make them unreliable and unfavorable among patients. Recent research in glucose affinity sensors correct some of the problems that enzymatic sensors experience. Dextran and concanavalin A are two of the more common components used in glucose affinity sensors. When these sensors were first explored, a model was derived to predict the response time of a glucose affinity sensor using concanavalin A and dextran. However, the model assumed the system was linear and fell short of calculating times representative of the response times determined through experimental tests with the sensors. In this work, a new model that uses the Stokes-Einstein Equation to demonstrate the nonlinear behavior of the glucose affinity assay was developed to predict the response times of similar glucose affinity sensors. In addition to the device tested by the original linear model, additional devices were identified and tested with the proposed model. The nonlinear model was designed to accommodate the many different variations between systems. The proposed model was able to accurately calculate response times for sensors using the concanavalin A-dextran affinity assay with respect to the experimentally reported times by the independent research groups. Parameter studies using the nonlinear model were able to identify possible setbacks that could compromise the response of thesystem. Specifically, the model showed that the improper use of asymmetrical membranes could increase the response time by as little as 20% or more as the device is miniaturized. The model also demonstrated that systems using the concanavalin Adextran assay would experience higher response times in the hypoglycemic range. This work attempted to replicate and improve an osmotic glucose affinity sensor. The system was designed to

  19. A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

    NASA Astrophysics Data System (ADS)

    Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

    1992-09-01

    A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

  20. [Off-label therapies in oncology].

    PubMed

    Telekes, András

    2009-02-22

    The unapproved (off-label) therapies represent a special problem in oncology since they are in the borderline of legal regulation and of free medical practice. Although in Hungary the off-label therapies were regarded as clinical trials without permission until the new regulation came into action at the end of October 2008, certain experts were even so arguing for its use because clinical practice changes more rapidly than the prescription label. Moreover, manufacturers are not obliged to submit Supplemental New Drug Application in spite of the fact that enough evidence is generated to do so. The regulation of off-label therapies should meet the conditions of free medical practice, evidence-based medicine, demand of patients for new chances and expectations of regulatory authorities. In this paper, following the criticism of the Hungarian status and the new regulation, as well as the review of international practice, the author outlines the frame of a potential regulation separately indicating the role of practicing physicians and authorities.

  1. Evidence that human immunoglobulin M rheumatoid factors can Be derived from the natural autoantibody pool and undergo an antigen driven immune response in which somatically mutated rheumatoid factors have lower affinities for immunoglobulin G Fc than their germline counterparts.

    PubMed

    Carayannopoulos, M O; Potter, K N; Li, Y; Natvig, J B; Capra, J D

    2000-04-01

    The question of whether immunoglobulin (Ig)M rheumatoid factors (RF) arise as the result of an abnormal expansion of already existing clones producing natural autoantibodies or emerge as new clones that are somatically mutated owing to an antigen driven immune response has never been conclusively answered. In this study, an inhibition ELISA was utilized to measure the affinities of recombinant antibodies using VH segments reverted back to their closest germline counterparts (germline revertants). In all cases, the somatically mutated parental RFs had a decreased affinity for immunoglobulin (Ig)G Fc compared to the germline revertant, indicating that the antibodies in the germline configuration had the higher affinities. This demonstrates that somatic mutation is not a prerequisite to generate disease associated antibodies. The presence of mutations in the parental IgM RFS suggests that these cells had been involved in a germinal centre reaction. As the germinal centre is the conventional site of the acquisition of mutations during an antigen driven response, these data suggest a role for germinal centres in the generation of the antibody diversity in addition to the selection of higher affinity antibodies. Assuming that only antigen selected cells survive deletion, these data support the hypothesis that IgM RFS can be derived from the natural autoantibody repertoire and result from an antigen driven response. Mechanisms controlling the survival of B cells based on the affinity/avidity of the immunoglobulin receptor are shown to be functional in patients with rheumatoid arthritis.

  2. Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

    PubMed Central

    Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

    1998-01-01

    The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding

  3. 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.

  4. Relationship between the species-representative phenotype and intraspecific variation in Ranunculaceae floral organ and Asteraceae flower numbers.

    PubMed

    Kitazawa, Miho S; Fujimoto, Koichi

    2016-04-01

    Phenotypic variation in floral morphologies contributes to speciation by testing various morphologies that might have higher adaptivity, leading eventually to phylogenetic diversity. Species diversity has been recognized, however, by modal morphologies where the variation is averaged out, so little is known about the relationship between the variation and the diversity. We analysed quantitatively the intraspecific variation of the organ numbers within flowers of Ranunculaceae, a family which branched near the monocot-eudicot separation, and the numbers of flowers within the capitula of Asteraceae, one of the most diverse families of eudicots. We used four elementary statistical quantities: mean, standard deviation (s.d.), degree of symmetry (skewness) and steepness (kurtosis). While these four quantities vary among populations, we found a common relationship between s.d. and the mean number of petals and sepals in Ranunculaceae and number of flowers per capitulum in Asteraceae. The s.d. is equal to the square root of the difference between the mean and specific number, showing robustness: for example, 3 in Ficaria sepals, 5 in Ranunculus petals and Anemone tepals, and 13 in Farfugium ray florets. This square-root relationship was not applicable to Eranthis petals which show little correlation between the s.d. and mean, and the stamens and carpels of Ranunculaceae whose s.d. is proportional to the mean. The specific values found in the square-root relationship provide a novel way to find the species-representative phenotype among varied morphologies. The representative phenotype is, in most cases, unique to the species or genus level, despite intraspecific differences of average phenotype among populations. The type of variation shown by the statistical quantities indicates not only the robustness of the morphologies but also how flowering plants changed during evolution among representative phenotypes that eventually led to phylogenetic diversification. © The

  5. Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline

    PubMed Central

    Schilling, Birgit; Rardin, Matthew J.; MacLean, Brendan X.; Zawadzka, Anna M.; Frewen, Barbara E.; Cusack, Michael P.; Sorensen, Dylan J.; Bereman, Michael S.; Jing, Enxuan; Wu, Christine C.; Verdin, Eric; Kahn, C. Ronald; MacCoss, Michael J.; Gibson, Bradford W.

    2012-01-01

    Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models. PMID:22454539

  6. Affinity Spaces and 21st Century Learning

    ERIC Educational Resources Information Center

    Gee, James Paul

    2017-01-01

    This article discusses video games as "attractors" to "affinity spaces." It argues that affinity spaces are key sites today where people teach and learn 21st Century skills. While affinity spaces are proliferating on the Internet as interest-and-passion-driven sites devoted to a common set of endeavors, they are not new, just…

  7. Use of DNA barcoding to reveal species composition of convenience seafood.

    PubMed

    Huxley-Jones, Elizabeth; Shaw, Jennifer L A; Fletcher, Carly; Parnell, Juliette; Watts, Phillip C

    2012-04-01

    Increased education of consumers can be an effective tool for conservation of commercially harvested marine species when product labeling is accurate and allows an informed choice. However, generic labeling (e.g., as white fish or surimi) and mislabeling of seafood prevents this and may erode consumer confidence in seafood product labels in general. We used DNA barcoding to identify the species composition of two types of convenience seafood (i.e., products processed for ease of consumption): fish fingers (long pieces of fish covered with bread crumbs or batter, n = 241) and seafood sticks (long pieces of cooked fish, n = 30). In products labeled as either white fish or surimi, four teleost species were present. Less than 1.5% of fish fingers with species-specific information were mislabeled. Results of other studies show substantially more mislabeling (e.g., >25%) of teleost products, which likely reflects the lower economic gains associated with mislabeling of convenience seafood compared with whole fillets. In addition to species identification, seafood product labels should be required to contain information about, for example, harvesting practices, and our data indicate that consumers can have reasonable confidence in the accuracy of the labels of convenience seafood and thus select brands on the basis of information about current fisheries practice. ©2012 Society for Conservation Biology.

  8. Functionality of the STNV translational enhancer domain correlates with affinity for two wheat germ factors

    PubMed Central

    Lipzig, Rosalinde van; Montagu, Marc Van; Cornelissen, Marc; Meulewaeter, Frank

    2001-01-01

    The satellite tobacco necrosis virus RNA is uncapped and requires a 3′ translational enhancer domain (TED) for translation. Both in the wheat germ extract and in tobacco, TED stimulates in cis translation of heterologous, uncapped RNAs. In this study we investigated to what extent translation stimulation by TED depends on binding to wheat germ factors. We show that in vitro TED binds at least seven wheat germ proteins. Translation and crosslinking assays, to which TED or TED derivatives with reduced functionality were included as competitor, showed that TED function correlates with binding to a 28 kDa protein (p28). One particular condition of competition revealed that p28 binding is not obligatory for TED function. Under this condition, a 30 kDa protein (p30) binds to TED. Importantly, affinity of p30 correlates with functionality of TED. These results strongly suggest that TED has the capacity to stimulate translation by recruiting the translational machinery either via binding to p28 or via binding to p30. PMID:11222757

  9. Microspore development of three coniferous species: affinity of nuclei for flavonoids.

    PubMed

    Feucht, Walter; Treutter, Dieter; Dithmar, Heike; Polster, Jürgen

    2008-12-01

    The nuclear localization of blue-staining flavanols was investigated histochemically throughout microsporogenesis in yellow cypress (Callitropsis nootkatensis (D. Don) Oerst., formerly Cupressus nootkatensis), juniper (Juniperus communis L.) and yew (Taxus baccata L.). During meiotic development, both the cytoplasm and nuclei of microspores of all species contained varying amounts of flavanols; however, the flavanols were largely confined to the nuclei in microspores just released from tetrads. Quantification by HPLC analysis indicated that, in all species, catechin and epicatechin were the dominant nuclear flavanols. At the early free microspore stage, the nuclear flavanols were barely detectable in all species, but they increased fivefold on incubation in the presence of 0.1 mM benzylaminopurine (BA) or zeatin. Histochemical studies revealed that, in addition to non-fluorescing flavanols, microspores contained yellow-fluorescing flavonoids, which yielded a distinct HPLC flavonoid profile for each species. In yellow cypress, the hydrolyzed flavonoids were identified as quercetin, apigenin, kaempferol and luteolin, whereas only quercetin and myricetin were found in microspores of juniper and in anthers of yew. Application of a UV-VIS titration technique revealed that the aglycone quercetin seems to interact more strongly with histone H3 than either glycoside rutin or kaempferol.

  10. Toxicity of fluoride to aquatic species and evaluation of toxicity modifying factors.

    PubMed

    Pearcy, Krysta; Elphick, James; Burnett-Seidel, Charlene

    2015-07-01

    The present study was performed to investigate the toxicity of fluoride to a variety of freshwater aquatic organisms and to establish whether water quality variables contribute substantively to modifying its toxicity. Water hardness, chloride, and alkalinity were tested as possible toxicity modifying factors for fluoride using acute toxicity tests with Hyalella azteca and Oncorhynchus mykiss. Chloride appeared to be the major toxicity modifying factor for fluoride in these acute toxicity tests. The chronic toxicity of fluoride was evaluated with a variety of species, including 3 fish (Pimephales promelas, O. mykiss, and Salvelinus namaycush), 3 invertebrates (Ceriodaphnia dubia, H. azteca, and Chironomus dilutus), 1 plant (Lemna minor), and 1 alga (Pseudokirchneriella subcapitata). Hyalella azteca was the most sensitive species overall, and O. mykiss was the most sensitive species of fish. The role of chloride as a toxicity modifying factor was inconsistent between species in the chronic toxicity tests. © 2015 SETAC.

  11. Post-transcriptional labeling by using Suzuki-Miyaura cross-coupling generates functional RNA probes.

    PubMed

    Walunj, Manisha B; Tanpure, Arun A; Srivatsan, Seergazhi G

    2018-06-20

    Pd-catalyzed C-C bond formation, an important vertebra in the spine of synthetic chemistry, is emerging as a valuable chemoselective transformation for post-synthetic functionalization of biomacromolecules. While methods are available for labeling protein and DNA, development of an analogous procedure to label RNA by cross-coupling reactions remains a major challenge. Herein, we describe a new Pd-mediated RNA oligonucleotide (ON) labeling method that involves post-transcriptional functionalization of iodouridine-labeled RNA transcripts by using Suzuki-Miyaura cross-coupling reaction. 5-Iodouridine triphosphate (IUTP) is efficiently incorporated into RNA ONs at one or more sites by T7 RNA polymerase. Further, using a catalytic system made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (ADHP) or dimethylamino-substituted ADHP (DMADHP), we established a modular method to functionalize iodouridine-labeled RNA ONs in the presence of various boronic acid and ester substrates under very mild conditions (37°C and pH 8.5). This method is highly chemoselective, and offers direct access to RNA ONs labeled with commonly used fluorescent and affinity tags and new fluorogenic environment-sensitive nucleoside probes in a ligand-controlled stereoselective fashion. Taken together, this simple approach of generating functional RNA ON probes by Suzuki-Miyaura coupling will be a very important addition to the resources and tools available for analyzing RNA motifs.

  12. Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution.

    PubMed

    Ahmad, Kareem M; Xiao, Yi; Soh, H Tom

    2012-12-01

    Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K(d)) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.

  13. Density Functional Study of Structures and Electron Affinities of BrO4F/BrO4F−

    PubMed Central

    Gong, Liangfa; Xiong, Jieming; Wu, Xinmin; Qi, Chuansong; Li, Wei; Guo, Wenli

    2009-01-01

    The structures, electron affinities and bond dissociation energies of BrO4F/BrO4F− species have been investigated with five density functional theory (DFT) methods with DZP++ basis sets. The planar F-Br…O2…O2 complexes possess 3A′ electronic state for neutral molecule and 4A′ state for the corresponding anion. Three types of the neutral-anion energy separations are the adiabatic electron affinity (EAad), the vertical electron affinity (EAvert), and the vertical detachment energy (VDE). The EAad value predicted by B3LYP method is 4.52 eV. The bond dissociation energies De (BrO4F → BrO4-mF + Om) (m = 1–4) and De− (BrO4F− → BrO4-mF− + Om and BrO4F− → BrO4-mF + Om−) are predicted. The adiabatic electron affinities (EAad) were predicted to be 4.52 eV for F-Br…O2…O2 (3A′←4A′) (B3LYP method). PMID:19742128

  14. Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.

    PubMed

    Gómez-Cortés, Pilar; Brenna, J Thomas; Sacks, Gavin L

    2012-06-19

    Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-α-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve

  15. A New F-18 Labeled PET Agent For Imaging Alzheimer's Plaques

    NASA Astrophysics Data System (ADS)

    Kulkarni, Padmakar V.; Vasdev, Neil; Hao, Guiyang; Arora, Veera; Long, Michael; Slavine, Nikolai; Chiguru, Srinivas; Qu, Bao Xi; Sun, Xiankai; Bennett, Michael; Antich, Peter P.; Bonte, Frederick J.

    2011-06-01

    Amyloid plaques and neurofibrillary tangles are hallmarks of Alzheimer's disease (AD). Advances in development of imaging agents have focused on targeting amyloid plaques. Notable success has been the development of C-11 labeled PIB (Pittsburgh Compound) and a number of studies have demonstrated the utility of this agent. However, the short half life of C-11 (t1/2: 20 min), is a limitation, thus has prompted the development of F-18 labeled agents. Most of these agents are derivatives of amyloid binding dyes; Congo red and Thioflavin. Some of these agents are in clinical trials with encouraging results. We have been exploring new class of agents based on 8-hydroxy quinoline, a weak metal chelator, targeting elevated levels of metals in plaques. Iodine-123 labeled clioquinol showed affinity for amyloid plaques however, it had limited brain uptake and was not successful in imaging in intact animals and humans. We have been successful in synthesizing F-18 labeled 8-hydroxy quinoline. Small animal PET/CT imaging studies with this agent showed high (7-10% ID/g), rapid brain uptake and fast washout of the agent from normal mice brains and delayed washout from transgenic Alzheimer's mice. These promising results encouraged us in further evaluation of this class of compounds for imaging AD plaques.

  16. DNA barcoding detected improper labelling and supersession of crab food served by restaurants in India.

    PubMed

    Vartak, Vivek Rohidas; Narasimmalu, Rajendran; Annam, Pavan Kumar; Singh, Dhirendra P; Lakra, Wazir S

    2015-01-01

    Detection of improper labelling of raw and processed seafood is of global importance for reducing commercial fraud and enhancing food safety. Crabs are crustaceans with intricate morphological as well as genetic divergence among species and are popular as seafood in restaurants. Owing to the high number of crab species available, it can be difficult to identify those included in particular food dishes, thus increasing the chance of supersession. DNA barcoding is an advanced technology for detecting improper food labelling and has been used successfully to authenticate seafood. This study identified 11 edible crab species from India by classical taxonomy and developed molecular barcodes with the cytochrome c oxidase I (COI) gene. These barcodes were used as reference barcodes for detecting any improper labelling of 50 restaurant crab samples. Neighbour-joining tree analysis with COI barcodes showed distinct clusters of restaurant samples with respective reference species. The study demonstrated 100% improper labelling of restaurant samples to cover up acts of inferior crab supersession. DNA barcoding successfully identified 11 edible crabs in accordance with classical taxonomy and discerned improper crab food labelling in restaurants of India. © 2014 Society of Chemical Industry.

  17. Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

    PubMed

    Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-15

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

  18. Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

    PubMed Central

    Basu, Koli; Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-01

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms. PMID:24457629

  19. A Computational Approach to Evaluate the Androgenic Affinity of Iprodione, Procymidone, Vinclozolin and Their Metabolites

    PubMed Central

    Fumagalli, Amos; Parravicini, Chiara; Marinovich, Marina; Eberini, Ivano

    2014-01-01

    Our research is aimed at devising and assessing a computational approach to evaluate the affinity of endocrine active substances (EASs) and their metabolites towards the ligand binding domain (LBD) of the androgen receptor (AR) in three distantly related species: human, rat, and zebrafish. We computed the affinity for all the selected molecules following a computational approach based on molecular modelling and docking. Three different classes of molecules with well-known endocrine activity (iprodione, procymidone, vinclozolin, and a selection of their metabolites) were evaluated. Our approach was demonstrated useful as the first step of chemical safety evaluation since ligand-target interaction is a necessary condition for exerting any biological effect. Moreover, a different sensitivity concerning AR LBD was computed for the tested species (rat being the least sensitive of the three). This evidence suggests that, in order not to over−/under-estimate the risks connected with the use of a chemical entity, further in vitro and/or in vivo tests should be carried out only after an accurate evaluation of the most suitable cellular system or animal species. The introduction of in silico approaches to evaluate hazard can accelerate discovery and innovation with a lower economic effort than with a fully wet strategy. PMID:25111804

  20. A computational approach to evaluate the androgenic affinity of iprodione, procymidone, vinclozolin and their metabolites.

    PubMed

    Galli, Corrado Lodovico; Sensi, Cristina; Fumagalli, Amos; Parravicini, Chiara; Marinovich, Marina; Eberini, Ivano

    2014-01-01

    Our research is aimed at devising and assessing a computational approach to evaluate the affinity of endocrine active substances (EASs) and their metabolites towards the ligand binding domain (LBD) of the androgen receptor (AR) in three distantly related species: human, rat, and zebrafish. We computed the affinity for all the selected molecules following a computational approach based on molecular modelling and docking. Three different classes of molecules with well-known endocrine activity (iprodione, procymidone, vinclozolin, and a selection of their metabolites) were evaluated. Our approach was demonstrated useful as the first step of chemical safety evaluation since ligand-target interaction is a necessary condition for exerting any biological effect. Moreover, a different sensitivity concerning AR LBD was computed for the tested species (rat being the least sensitive of the three). This evidence suggests that, in order not to over-/under-estimate the risks connected with the use of a chemical entity, further in vitro and/or in vivo tests should be carried out only after an accurate evaluation of the most suitable cellular system or animal species. The introduction of in silico approaches to evaluate hazard can accelerate discovery and innovation with a lower economic effort than with a fully wet strategy.

  1. Reactive Oxygen Species-Inducible ECF σ Factors of Bradyrhizobium japonicum

    PubMed Central

    Masloboeva, Nadezda; Reutimann, Luzia; Stiefel, Philipp; Follador, Rainer; Leimer, Nadja; Hennecke, Hauke; Mesa, Socorro; Fischer, Hans-Martin

    2012-01-01

    Extracytoplasmic function (ECF) σ factors control the transcription of genes involved in different cellular functions, such as stress responses, metal homeostasis, virulence-related traits, and cell envelope structure. The genome of Bradyrhizobium japonicum, the nitrogen-fixing soybean endosymbiont, encodes 17 putative ECF σ factors belonging to nine different ECF σ factor families. The genes for two of them, ecfQ (bll1028) and ecfF (blr3038), are highly induced in response to the reactive oxygen species hydrogen peroxide (H2O2) and singlet oxygen (1O2). The ecfF gene is followed by the predicted anti-σ factor gene osrA (blr3039). Mutants lacking EcfQ, EcfF plus OsrA, OsrA alone, or both σ factors plus OsrA were phenotypically characterized. While the symbiotic properties of all mutants were indistinguishable from the wild type, they showed increased sensitivity to singlet oxygen under free-living conditions. Possible target genes of EcfQ and EcfF were determined by microarray analyses, and candidate genes were compared with the H2O2-responsive regulon. These experiments disclosed that the two σ factors control rather small and, for the most part, distinct sets of genes, with about half of the genes representing 13% of the members of H2O2-responsive regulon. To get more insight into transcriptional regulation of both σ factors, the 5′ ends of ecfQ and ecfF mRNA were determined. The presence of conserved sequence motifs in the promoter region of ecfQ and genes encoding EcfQ-like σ factors in related α-proteobacteria suggests regulation via a yet unknown transcription factor. By contrast, we have evidence that ecfF is autoregulated by transcription from an EcfF-dependent consensus promoter, and its product is negatively regulated via protein-protein interaction with OsrA. Conserved cysteine residues 129 and 179 of OsrA are required for normal function of OsrA. Cysteine 179 is essential for release of EcfF from an EcfF-OsrA complex upon H2O2 stress while

  2. Tumor affinity of radiolabeled peanut agglutinin compared with that of Ga-67 citrate in animal models

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yokoyama, K.; Aburano, T.; Watanabe, N.

    1985-05-01

    Peanut agglutinin (PNA) binds avidly to the immunodominant group of the tumor associated T antigen. The purpose of this study was to evaluate oncodiagnostic potential of radiolabeled PNA in animal models. PNA was labeled with I-125 or I-131 by Iodogen and also with In-111 by cyclic DTPA anhydride. The biological activity of PNA was examined by a hemaglutination titer with a photometer before and after labeling. Animal tumor models used were Lewis Lung Cancer(LLC), B-16 Melanotic Melanoma(MM), Yoshida Sarcoma(YS), Ehrlich Ascites Tumor(EAT and Hepatoma AH109A(HAH). Inflammatory tissue induced by turpentine oil was used as an abscess model. Serial scintigraphic imagesmore » were obtained following IV injections of 100 ..mu..Ci of I-131 or In-111-DTPA-PNA. The tumor affinity of Ga-67 citrate was studied to compare that of radiolabeled PNA. Tissue biodistribution was studied in EAT bearing mice. All of these tumor models except HAH were clearly visible by radiolabeled PNA without subtraction techniques. In the models of LLC and EAT, PNA showed the better accumulation into the tumor tissue than Ga-67 citrate. In YS and MM, PNA represented almost the same accumulation as Ga-67 citrate. The localization of PNA into abscess tissue wasn't found although Ga-67 citrate markedly accumulated into abscess tissue as well as tumor tissue. The clearance of PNA from tumor was slower than those from any other organs. Tumor to muscle ratio was 5.1 at 48hrs. and tumor to blood ratio increased with time to 2.3 at 96hrs. These results suggested that radiolabeled PNA may have a potential in the detection of tumor.« less

  3. Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides.

    PubMed

    Zhou, Wen; Håkansson, Kristina

    2011-12-01

    We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C-2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA-labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared with 2-AA-labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

    PubMed

    Spear, Timothy T; Wang, Yuan; Foley, Kendra C; Murray, David C; Scurti, Gina M; Simms, Patricia E; Garrett-Mayer, Elizabeth; Hellman, Lance M; Baker, Brian M; Nishimura, Michael I

    2017-11-01

    T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

  5. Comparative Study of Three Methods for Affinity Measurements: Capillary Electrophoresis Coupled with UV Detection and Mass Spectrometry, and Direct Infusion Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Mironov, Gleb G.; Logie, Jennifer; Okhonin, Victor; Renaud, Justin B.; Mayer, Paul M.; Berezovski, Maxim V.

    2012-07-01

    We present affinity capillary electrophoresis and mass spectrometry (ACE-MS) as a comprehensive separation technique for label-free solution-based affinity analysis. The application of ACE-MS for measuring affinity constants between eight small molecule drugs [ibuprofen, s-flurbiprofen, diclofenac, phenylbutazone, naproxen, folic acid, resveratrol, and 4,4'-(propane-1,3-diyl) dibenzoic acid] and β-cyclodextrin is described. We couple on-line ACE with MS to combine the separation and kinetic capability of ACE together with the molecular weight and structural elucidation of MS in one system. To understand the full potential of ACE-MS, we compare it with two other methods: Direct infusion mass spectrometry (DIMS) and ACE with UV detection (ACE-UV). After the evaluation, DIMS provides less reliable equilibrium dissociation constants than separation-based ACE-UV and ACE-MS, and cannot be used solely for the study of noncovalent interactions. ACE-MS determines apparent dissociation constants for all reacting small molecules in a mixture, even in cases when drugs overlap with each other during separation. The ability of ACE-MS to interact, separate, and rapidly scan through m/z can facilitate the simultaneous affinity analysis of multiple interacting pairs, potentially leading to the high-throughput screening of drug candidates.

  6. The Mobile Phone Affinity Scale: Enhancement and Refinement.

    PubMed

    Bock, Beth C; Lantini, Ryan; Thind, Herpreet; Walaska, Kristen; Rosen, Rochelle K; Fava, Joseph L; Barnett, Nancy P; Scott-Sheldon, Lori Aj

    2016-12-15

    Existing instruments that assess individuals' relationships with mobile phones tend to focus on negative constructs such as addiction or dependence, and appear to assume that high mobile phone use reflects pathology. Mobile phones can be beneficial for health behavior change, disease management, work productivity, and social connections, so there is a need for an instrument that provides a more balanced assessment of the various aspects of individuals' relationships with mobile phones. The purpose of this research was to develop, revise, and validate the Mobile Phone Affinity Scale, a multi-scale instrument designed to assess key factors associated with mobile phone use. Participants (N=1058, mean age 33) were recruited from Amazon Mechanical Turk between March and April of 2016 to complete a survey that assessed participants' mobile phone attitudes and use, anxious and depressive symptoms, and resilience. Confirmatory factor analysis supported a 6-factor model. The final measure consisted of 24 items, with 4 items on each of 6 factors: Connectedness, Productivity, Empowerment, Anxious Attachment, Addiction, and Continuous Use. The subscales demonstrated strong internal consistency (Cronbach alpha range=0.76-0.88, mean 0.83), and high item factor loadings (range=0.57-0.87, mean 0.75). Tests for validity further demonstrated support for the individual subscales. Mobile phone affinity may have an important impact in the development and effectiveness of mobile health interventions, and continued research is needed to assess its predictive ability in health behavior change interventions delivered via mobile phones. ©Beth C Bock, Ryan Lantini, Herpreet Thind, Kristen Walaska, Rochelle K Rosen, Joseph L Fava, Nancy P Barnett, Lori AJ Scott-Sheldon. Originally published in JMIR Mhealth and Uhealth (http://mhealth.jmir.org), 15.12.2016.

  7. Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

    PubMed

    Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

    2011-01-19

    Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

  8. HaloTag technology for specific and covalent labeling of fusion proteins.

    PubMed

    Benink, Hélène A; Urh, Marjeta

    2015-01-01

    Appending proteins of interest to fluorescent protein tags such as GFP has revolutionized how proteins are studied in the cellular environment. Over the last few decades many varieties of fluorescent proteins have been generated, each bringing new capability to research. However, taking full advantage of standard fluorescent proteins with advanced and differential features requires significant effort on the part of the researcher. This approach necessitates that many genetic fusions be generated and confirmed to function properly in cells with the same protein of interest. To lessen this burden, a newer category of protein fusion tags termed "self-labeling protein tags" has been developed. This approach utilizes a single protein tag, the function of which can be altered by attaching various chemical moieties (fluorescent labels, affinity handles, etc.). In this way a single genetically encoded protein fusion can easily be given functional diversity and adaptability as supplied by synthetic chemistry. Here we present protein labeling methods using HaloTag technology; comprised of HaloTag protein and the collection of small molecules designed to bind it specifically and provide it with varied functionalities. For imaging purposes these small molecules, termed HaloTag ligands, contain distinct fluorophores. Due to covalent and rapid binding between HaloTag protein and its ligands, labeling is permanent and efficient. Many of these ligands have been optimized for permeability across cellular membranes allowing for live cell labeling and imaging analysis. Nonpermeable ligands have also been developed for specific labeling of surface proteins. Overall, HaloTag is a versatile technology that empowers the end user to label a protein of interest with the choice of different fluorophores while alleviating the need for generation of multiple genetic fusions.

  9. Analyses of mitochondrial amino acid sequence datasets support the proposal that specimens of Hypodontus macropi from three species of macropodid hosts represent distinct species

    PubMed Central

    2013-01-01

    Background Hypodontus macropi is a common intestinal nematode of a range of kangaroos and wallabies (macropodid marsupials). Based on previous multilocus enzyme electrophoresis (MEE) and nuclear ribosomal DNA sequence data sets, H. macropi has been proposed to be complex of species. To test this proposal using independent molecular data, we sequenced the whole mitochondrial (mt) genomes of individuals of H. macropi from three different species of hosts (Macropus robustus robustus, Thylogale billardierii and Macropus [Wallabia] bicolor) as well as that of Macropicola ocydromi (a related nematode), and undertook a comparative analysis of the amino acid sequence datasets derived from these genomes. Results The mt genomes sequenced by next-generation (454) technology from H. macropi from the three host species varied from 13,634 bp to 13,699 bp in size. Pairwise comparisons of the amino acid sequences predicted from these three mt genomes revealed differences of 5.8% to 18%. Phylogenetic analysis of the amino acid sequence data sets using Bayesian Inference (BI) showed that H. macropi from the three different host species formed distinct, well-supported clades. In addition, sliding window analysis of the mt genomes defined variable regions for future population genetic studies of H. macropi in different macropodid hosts and geographical regions around Australia. Conclusions The present analyses of inferred mt protein sequence datasets clearly supported the hypothesis that H. macropi from M. robustus robustus, M. bicolor and T. billardierii represent distinct species. PMID:24261823

  10. 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.

  11. Classical affine W-algebras associated to Lie superalgebras

    NASA Astrophysics Data System (ADS)

    Suh, Uhi Rinn

    2016-02-01

    In this paper, we prove classical affine W-algebras associated to Lie superalgebras (W-superalgebras), which can be constructed in two different ways: via affine classical Hamiltonian reductions and via taking quasi-classical limits of quantum affine W-superalgebras. Also, we show that a classical finite W-superalgebra can be obtained by a Zhu algebra of a classical affine W-superalgebra. Using the definition by Hamiltonian reductions, we find free generators of a classical W-superalgebra associated to a minimal nilpotent. Moreover, we compute generators of the classical W-algebra associated to spo(2|3) and its principal nilpotent. In the last part of this paper, we introduce a generalization of classical affine W-superalgebras called classical affine fractional W-superalgebras. We show these have Poisson vertex algebra structures and find generators of a fractional W-superalgebra associated to a minimal nilpotent.

  12. Affine connection form of Regge calculus

    NASA Astrophysics Data System (ADS)

    Khatsymovsky, V. M.

    2016-12-01

    Regge action is represented analogously to how the Palatini action for general relativity (GR) as some functional of the metric and a general connection as independent variables represents the Einstein-Hilbert action. The piecewise flat (or simplicial) spacetime of Regge calculus is equipped with some world coordinates and some piecewise affine metric which is completely defined by the set of edge lengths and the world coordinates of the vertices. The conjugate variables are the general nondegenerate matrices on the three-simplices which play the role of a general discrete connection. Our previous result on some representation of the Regge calculus action in terms of the local Euclidean (Minkowsky) frame vectors and orthogonal connection matrices as independent variables is somewhat modified for the considered case of the general linear group GL(4, R) of the connection matrices. As a result, we have some action invariant w.r.t. arbitrary change of coordinates of the vertices (and related GL(4, R) transformations in the four-simplices). Excluding GL(4, R) connection from this action via the equations of motion we have exactly the Regge action for the considered spacetime.

  13. Rapid and Reliable Binding Affinity Prediction of Bromodomain Inhibitors: A Computational Study

    PubMed Central

    2016-01-01

    Binding free energies of bromodomain inhibitors are calculated with recently formulated approaches, namely ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) and TIES (thermodynamic integration with enhanced sampling). A set of compounds is provided by GlaxoSmithKline, which represents a range of chemical functionality and binding affinities. The predicted binding free energies exhibit a good Spearman correlation of 0.78 with the experimental data from the 3-trajectory ESMACS, and an excellent correlation of 0.92 from the TIES approach where applicable. Given access to suitable high end computing resources and a high degree of automation, we can compute individual binding affinities in a few hours with precisions no greater than 0.2 kcal/mol for TIES, and no larger than 0.34 and 1.71 kcal/mol for the 1- and 3-trajectory ESMACS approaches. PMID:28005370

  14. Structure of Greyhound hemoglobin: origin of high oxygen affinity.

    PubMed

    Bhatt, Veer S; Zaldívar-López, Sara; Harris, David R; Couto, C Guillermo; Wang, Peng G; Palmer, Andre F

    2011-05-01

    This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Å resolution. GrHb was found to crystallize with an α₁β₁ dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

  15. Single-cell measurement of red blood cell oxygen affinity.

    PubMed

    Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M; Schonbrun, Ethan

    2015-08-11

    Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen-Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2-3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability.

  16. Single-cell measurement of red blood cell oxygen affinity

    PubMed Central

    Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

    2015-01-01

    Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen–Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2–3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability. PMID:26216973

  17. Label-Enhanced Surface Plasmon Resonance: A New Concept for Improved Performance in Optical Biosensor Analysis

    PubMed Central

    Granqvist, Niko; Hanning, Anders; Eng, Lars; Tuppurainen, Jussi; Viitala, Tapani

    2013-01-01

    Surface plasmon resonance (SPR) is a well-established optical biosensor technology with many proven applications in the study of molecular interactions as well as in surface and material science. SPR is usually applied in the label-free mode which may be advantageous in cases where the presence of a label may potentially interfere with the studied interactions per se. However, the fundamental challenges of label-free SPR in terms of limited sensitivity and specificity are well known. Here we present a new concept called label-enhanced SPR, which is based on utilizing strongly absorbing dye molecules in combination with the evaluation of the full shape of the SPR curve, whereby the sensitivity as well as the specificity of SPR is significantly improved. The performance of the new label-enhanced SPR method was demonstrated by two simple model assays: a small molecule assay and a DNA hybridization assay. The small molecule assay was used to demonstrate the sensitivity enhancement of the method, and how competitive assays can be used for relative affinity determination. The DNA assay was used to demonstrate the selectivity of the assay, and the capabilities in eliminating noise from bulk liquid composition variations. PMID:24217357

  18. US Consumers' Understanding of Nutrition Labels in 2013: The Importance of Health Literacy.

    PubMed

    Persoskie, Alexander; Hennessy, Erin; Nelson, Wendy L

    2017-09-28

    We examined US adults' understanding of a Nutrition Facts panel (NFP), which requires health literacy (ie, prose, document, and quantitative literacy skills), and the association between label understanding and dietary behavior. Data were from the Health Information National Trends Survey, a nationally representative survey of health information seeking among US adults (N = 3,185) conducted from September 6, 2013, through December 30, 2013. Participants viewed an ice cream nutrition label and answered 4 questions that tested their ability to apply basic arithmetic and understanding of percentages to interpret the label. Participants reported their intake of sugar-sweetened soda, fruits, and vegetables. Regression analyses tested associations among label understanding, demographic characteristics, and self-reported dietary behaviors. Approximately 24% of people could not determine the calorie content of the full ice-cream container, 21% could not estimate the number of servings equal to 60 g of carbohydrates, 42% could not estimate the effect on daily calorie intake of foregoing 1 serving, and 41% could not calculate the percentage daily value of calories in a single serving. Higher scores for label understanding were associated with consuming more vegetables and less sugar-sweetened soda, although only the association with soda consumption remained significant after adjusting for demographic factors. Many consumers have difficulty interpreting nutrition labels, and label understanding correlates with self-reported dietary behaviors. The 2016 revised NFP labels may address some deficits in consumer understanding by eliminating the need to perform certain calculations (eg, total calories per package). However, some tasks still require the ability to perform calculations (eg, percentage daily value of calories). Schools have a role in teaching skills, such as mathematics, needed for nutrition label understanding.

  19. Complex networks with large numbers of labelable attractors

    NASA Astrophysics Data System (ADS)

    Mi, Yuanyuan; Zhang, Lisheng; Huang, Xiaodong; Qian, Yu; Hu, Gang; Liao, Xuhong

    2011-09-01

    Information storage in many functional subsystems of the brain is regarded by theoretical neuroscientists to be related to attractors of neural networks. The number of attractors is large and each attractor can be temporarily represented or suppressed easily by corresponding external stimulus. In this letter, we discover that complex networks consisting of excitable nodes have similar fascinating properties of coexistence of large numbers of oscillatory attractors, most of which can be labeled with a few nodes. According to a simple labeling rule, different attractors can be identified and the number of labelable attractors can be predicted from the analysis of network topology. With the cues of the labeling association, these attractors can be conveniently retrieved or suppressed on purpose.

  20. Femtomolar Ln(III) affinity in peptide-based ligands containing unnatural chelating amino acids.

    PubMed

    Niedźwiecka, Agnieszka; Cisnetti, Federico; Lebrun, Colette; Delangle, Pascale

    2012-05-07

    The incorporation of unnatural chelating amino acids in short peptide sequences leads to lanthanide-binding peptides with a higher stability than sequences built exclusively from natural residues. In particular, the hexadentate peptide P(22), which incorporates two unnatural amino acids Ada(2) with aminodiacetate chelating arms, showed picomolar affinity for Tb(3+). To design peptides with higher denticity, expected to show higher affinity for Ln(3+), we synthesized the novel unnatural amino acid Ed3a(2) which carries an ethylenediamine triacetate side-chain and affords a pentadentate coordination site. The synthesis of the derivative Fmoc-Ed3a(2)(tBu)(3)-OH, with appropriate protecting groups for direct use in the solid phase peptide synthesis (Fmoc strategy), is described. The two high denticity peptides P(HD2) (Ac-Trp-Ed3a(2)-Pro-Gly-Ada(2)-Gly-NH(2)) and P(HD5) (Ac-Trp-Ada(2)-Pro-Gly-Ed3a(2)-Gly-NH(2)) led to octadentate Tb(3+) complexes with femtomolar stability in water. The position of the high denticity amino acid Ed3a(2) in the hexapeptide sequence appears to be critical for the control of the metal complex speciation. Whereas P(HD5) promotes the formation of polymetallic species in excess of Ln(3+), P(HD2) forms exclusively the mononuclear complex. The octadentate coordination of Tb(3+) by both P(HD) leads to total dehydration of the metal ion in the mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the novel peptide P(HD2) forms a unique octadentate Tb(3+) complex with femtomolar stability in water and an improvement of the luminescence properties with respect to the trisaquo TbP(22) complex by a factor of 4.

  1. Classical affine W-algebras associated to Lie superalgebras

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Suh, Uhi Rinn, E-mail: uhrisu1@math.snu.ac.kr

    2016-02-15

    In this paper, we prove classical affine W-algebras associated to Lie superalgebras (W-superalgebras), which can be constructed in two different ways: via affine classical Hamiltonian reductions and via taking quasi-classical limits of quantum affine W-superalgebras. Also, we show that a classical finite W-superalgebra can be obtained by a Zhu algebra of a classical affine W-superalgebra. Using the definition by Hamiltonian reductions, we find free generators of a classical W-superalgebra associated to a minimal nilpotent. Moreover, we compute generators of the classical W-algebra associated to spo(2|3) and its principal nilpotent. In the last part of this paper, we introduce a generalizationmore » of classical affine W-superalgebras called classical affine fractional W-superalgebras. We show these have Poisson vertex algebra structures and find generators of a fractional W-superalgebra associated to a minimal nilpotent.« less

  2. FRET-based quantum dot immunoassay for rapid and sensitive detection of Aspergillus amstelodami.

    PubMed

    Kattke, Michele D; Gao, Elizabeth J; Sapsford, Kim E; Stephenson, Larry D; Kumar, Ashok

    2011-01-01

    In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 10(3) spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 10(5) CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis.

  3. FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami

    PubMed Central

    Kattke, Michele D.; Gao, Elizabeth J.; Sapsford, Kim E.; Stephenson, Larry D.; Kumar, Ashok

    2011-01-01

    In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 103 spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 105 CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis. PMID:22163961

  4. Evaluating Kinase ATP Uptake and Tyrosine Phosphorylation using Multiplexed Quantification of Chemically Labeled and Post-Translationally Modified Peptides

    PubMed Central

    Fang, Bin; Hoffman, Melissa A.; Mirza, Abu-Sayeef; Mishall, Katie M.; Li, Jiannong; Peterman, Scott M.; Smalley, Keiran S. M.; Shain, Kenneth H.; Weinberger, Paul M.; Wu, Jie; Rix, Uwe; Haura, Eric B.; Koomen, John M.

    2015-01-01

    Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease. PMID:25782629

  5. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors.

    PubMed

    García-Garayoa, E; Allemann-Tannahill, L; Bläuenstein, P; Willmann, M; Carrel-Rémy, N; Tourwé, D; Iterbeke, K; Conrath, P; Schubiger, P A

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [99mTc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

  6. Selection of imprinted nanoparticles by affinity chromatography.

    PubMed

    Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

    2009-04-15

    Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

  7. Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

    PubMed Central

    Verastegui, Y.; Cheng, J.; Engel, K.; Kolczynski, D.; Mortimer, S.; Lavigne, J.; Montalibet, J.; Romantsov, T.; Hall, M.; McConkey, B. J.; Rose, D. R.; Tomashek, J. J.; Scott, B. R.

    2014-01-01

    ABSTRACT Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. PMID:25028422

  8. The influence of need for cognition and principal display panel factors on over-the-counter drug facts label comprehension.

    PubMed

    Catlin, Jesse R; Pechmann, Cornelia; Brass, Eric P

    2012-01-01

    Nearly all work aimed at optimizing the ability of labeling to communicate over-the-counter (OTC) drug information has focused on back-of-the-package characteristics, such as the Drug Facts label. The effects of front of the package, or principal display panel (PDP) factors, have largely been neglected by researchers. Similarly, heterogeneity in consumers' approach to new information has received scant attention in the context of OTC drugs. This preliminary study tested the hypothesis that display of a drug's brand name on the PDP and individuals' need for cognition influence comprehension of Drug Facts label information. University students (n = 212) that had experienced heartburn but not used the drug class being studied constituted the primary analysis cohort. Students were randomly assigned to review one of two PDPs (brand name or generic), followed by a Drug Facts label and a series of questions related to selection and usage of the drug. Participants with low need for cognition were influenced by the brand name PDP, as those exposed to a PDP featuring a brand (vs. generic) spent less time reading the Drug Facts label and demonstrated lower comprehension of the label information on proper drug selection. These findings suggest that further research is needed to understand the impact of PDP contents and cognitive characteristics of consumers on the communication of OTC drug information. Health care providers should consider communication strategies that account for the challenges patients face in using OTC drugs properly.

  9. Expanding RNA binding specificity and affinity of engineered PUF domains.

    PubMed

    Zhao, Yang-Yang; Mao, Miao-Wei; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

    2018-05-18

    Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way.

  10. Expanding RNA binding specificity and affinity of engineered PUF domains

    PubMed Central

    Zhao, Yang-Yang; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

    2018-01-01

    Abstract Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way. PMID:29490074

  11. Anatomical, chemical, and ecological factors affecting tree species choice in dendrochemistry studies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cutter, B.E.; Guyette, R.P.

    1993-07-01

    Recently, element concentrations in tree rings have been used to monitor metal contamination, fertilization, and the effects of acid precipitation on soils. This has stimulated interest in which tree species may be suitable for use in studies of long-term trends in environmental chemistry. Potential radial translocation of elements across living boundaries can be a confounding factor in assessing environmental change. The selection of species which minimizes radial translocation of elements can be critical to the success of dendrochemical research. Criteria for selection of species with characteristics favorable for dendrochemical analysis are categorized into (1) habitat-based factors, (2) xylem-based factors, andmore » (3) element-based factors. A wide geographic range and ecological amplitude provide an advantage in calibration and better controls on the effects of soil chemistry. The most important xylem-based criteria are heartwood moisture content, permeability, and the nature of the sapwood-heartwood transition. The element of interest is important in determining suitable tree species because all elements are not equally mobile or detectable in the xylem. Ideally, the tree species selected for dendrochemical study will be long-lived, grow on a wide range of sites over a large geographic distribution, have a distinct heartwood with a low number of rings in the sapwood, a low heartwood moisture content, and have low radial permeability. Recommended temperate zone North American species include white oak (Quercus alba L.), post oak (Q. stellate Wangenh.), eastern redcedar (funiperus virginiana L.), old-growth Douglas-fir [Pseudoaugu menziesii (Mirb.) Franco] and big sagebrush (Artemisia tridentata Nutt.). In addition, species such as bristlecone pine (Pinus aristata Engelm. syn. longaeva), old-growth redwood [Sequoia sempervirens (D. Don) Endl.], and giant sequoia [S. gigantea (Lindl.) Deene] may be suitable for local purposes. 118 refs., 2 tabs.« less

  12. Side-by-Side Comparison of Commonly Used Biomolecules That Differ in Size and Affinity on Tumor Uptake and Internalization

    PubMed Central

    Leelawattanachai, Jeerapond; Kwon, Keon-Woo; Michael, Praveesuda; Ting, Richard; Kim, Ju-Young; Jin, Moonsoo M.

    2015-01-01

    The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization. PMID:25901755

  13. Na+/substrate Coupling in the Multidrug Antiporter NorM Probed with a Spin-labeled Substrate

    PubMed Central

    Steed, P. Ryan; Stein, Richard A.; Mishra, Smriti; Goodman, Michael C.; Mchaourab, Hassane S.

    2013-01-01

    NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na+ gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal to characterize mechanistically relevant binding interactions with NorM and to directly address the coupling of ion and drug binding. Fluorescence and EPR experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na+. Using double electron-electron resonance (DEER) spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na+ did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na+ does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na+ gradient across the native membrane, govern Na+/drug coupling during antiport. PMID:23902581

  14. Preparation and high-resolution microscopy of gold cluster labeled nucleic acid conjugates and nanodevices

    PubMed Central

    Powell, Richard D.; Hainfeld, James F.

    2013-01-01

    Nanogold and undecagold are covalently linked gold cluster labels which enable the identification and localization of biological components with molecular precision and resolution. They can be prepared with different reactivities, which means they can be conjugated to a wide variety of molecules, including nucleic acids, at specific, unique sites. The location of these sites can be synthetically programmed in order to preserve the binding affinity of the conjugate and impart novel characteristics and useful functionality. Methods for the conjugation of undecagold and Nanogold to DNA and RNA are discussed, and applications of labeled conjugates to the high-resolution microscopic identification of binding sites and characterization of biological macromolecular assemblies are described. In addition to providing insights into their molecular structure and function, high-resolution microscopic methods also show how Nanogold and undecagold conjugates can be synthetically assembled, or self-assemble, into supramolecular materials to which the gold cluster labels impart useful functionality. PMID:20869258

  15. A microfluidic laser scattering sensor for label-free detection of waterborne pathogens

    NASA Astrophysics Data System (ADS)

    Wei, Huang; Yang, Limei; Li, Feng

    2016-10-01

    A microfluidic-based multi-angle laser scattering (MALS) sensor capable of acquiring scattering pattern of single particle is demonstrated. The size and relative refractive index (RI) of polystyrene (PS) microspheres were deduced with accuracies of 60 nm and 0.001 by analyzing the scattering patterns. We measured scattering patterns of waterborne parasites i.e., cryptosporidium parvum (c.parvum) and giardia lamblia (g.lamblia), and some other representative species in 1 L water within 1 hour, and the waterborne parasites were identified with accuracy better than 96% by classification of distinctive scattering patterns with a support-vector-machine (SVM) algorithm. The system provides a promising tool for label-free and rapid detection of waterborne parasites.

  16. Coagulase-negative Staphylococcus species in bulk milk: Prevalence, distribution, and associated subgroup- and species-specific risk factors.

    PubMed

    De Visscher, A; Piepers, S; Haesebrouck, F; Supré, K; De Vliegher, S

    2017-01-01

    Coagulase-negative staphylococci (CNS) have become the main pathogens causing bovine mastitis in recent years. A huge variation in species distribution among herds has been observed in several studies, emphasizing the need to identify subgroup- and species-specific herd-level factors to improve our understanding of the differences in ecological and epidemiological nature between species. The use of bulk milk samples enables the inclusion of a large(r) number of herds needed to identify herd-level risk factors and increases the likelihood of recovering enough isolates per species needed for conducting subgroup- and, eventually, species-specific analyses at the same time. This study aimed to describe the prevalence and distribution of CNS species in bulk milk samples and to identify associated subgroup- and species-specific herd-level factors. Ninety percent of all bulk milk samples yielded CNS. Staphylococcus equorum was the predominant species, followed by Staphylococcus haemolyticus and Staphylococcus epidermidis. A seasonal effect was observed for several CNS species. Bulk milk samples from herds with a loose-pack or a tiestall housing system were more likely to yield CNS species compared with herds with a freestall barn, except for S. epidermidis, Staphylococcus simulans, and Staphylococcus cohnii. In September, herds in which udders were clipped had lower odds of yielding Staphylococcus chromogenes, S. simulans, and Staphylococcus xylosus, the CNS species assumed to be most relevant for udder health, in their bulk milk than herds in which udder clipping was not practiced. Bulk milk of herds participating in a monthly veterinary udder health-monitoring program was more likely to yield these 3 CNS species. Herds always receiving their milk quality premium or predisinfecting teats before attachment of the milking cluster had lower odds of having S. equorum in their bulk milk. Herds not using a single dry cotton or paper towel for each cow during premilking udder

  17. In situ assessment of the velocity of carbon transfer by tracing 13 C in trunk CO2 efflux after pulse labelling: variations among tree species and seasons.

    PubMed

    Dannoura, Masako; Maillard, Pascale; Fresneau, Chantal; Plain, Caroline; Berveiller, Daniel; Gerant, Dominique; Chipeaux, Christophe; Bosc, Alexandre; Ngao, Jérôme; Damesin, Claire; Loustau, Denis; Epron, Daniel

    2011-04-01

    Phloem is the main pathway for transferring photosynthates belowground. In situ(13) C pulse labelling of trees 8-10 m tall was conducted in the field on 10 beech (Fagus sylvatica) trees, six sessile oak (Quercus petraea) trees and 10 maritime pine (Pinus pinaster) trees throughout the growing season. Respired (13) CO2 from trunks was tracked at different heights using tunable diode laser absorption spectrometry to determine time lags and the velocity of carbon transfer (V). The isotope composition of phloem extracts was measured on several occasions after labelling and used to estimate the rate constant of phloem sap outflux (kP ). Pulse labelling together with high-frequency measurement of the isotope composition of trunk CO2 efflux is a promising tool for studying phloem transport in the field. Seasonal variability in V was predicted in pine and oak by bivariate linear regressions with air temperature and soil water content. V differed among the three species consistently with known differences in phloem anatomy between broadleaf and coniferous trees. V increased with tree diameter in oak and beech, reflecting a nonlinear increase in volumetric flow with increasing bark cross-sectional area, which suggests changes in allocation pattern with tree diameter in broadleaf species. Discrepancies between V and kP indicate vertical changes in functional phloem properties. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  18. Determinants of Nutrition Label Use among College Students

    ERIC Educational Resources Information Center

    Rasberry, Catherine N.; Chaney, Beth H.; Housman, Jeff M.; Misra, Ranjita; Miller, Paula J.

    2007-01-01

    The purpose of this research was to assess the frequency of nutrition label use among college students and its relationship to nutrition and label knowledge, attitudes, and beliefs regarding diet-disease relationships, and to determine factors predictive of frequent or infrequent label use. This study utilized a cross-sectional design. Volunteer…

  19. Vaccine Efficacy and Affinity Maturation

    NASA Astrophysics Data System (ADS)

    Lee, Hayoun; Deem, Michael W.

    2002-03-01

    We propose macroscopic equations to describe variable vaccine efficacy between repeated vaccinee and first time vaccinee. The main ingredients are antigenic distance between epidemic strain and vaccne strain, and affinity maturation dynamics which differs in primary and second response. Increase of affinity by repeated vaccine leads to localization in immune space. This localization decreases the ability of the immune system to response to distant, but related epidemic strains.

  20. On the Presence of Affine Fibril and Fiber Kinematics in the Mitral Valve Anterior Leaflet

    PubMed Central

    Lee, Chung-Hao; Zhang, Will; Liao, Jun; Carruthers, Christopher A.; Sacks, Jacob I.; Sacks, Michael S.

    2015-01-01

    In this study, we evaluated the hypothesis that the constituent fibers follow an affine deformation kinematic model for planar collagenous tissues. Results from two experimental datasets were utilized, taken at two scales (nanometer and micrometer), using mitral valve anterior leaflet (MVAL) tissues as the representative tissue. We simulated MVAL collagen fiber network as an ensemble of undulated fibers under a generalized two-dimensional deformation state, by representing the collagen fibrils based on a planar sinusoidally shaped geometric model. The proposed approach accounted for collagen fibril amplitude, crimp period, and rotation with applied macroscopic tissue-level deformation. When compared to the small angle x-ray scattering measurements, the model fit the data well, with an r2 = 0.976. This important finding suggests that, at the homogenized tissue-level scale of ∼1 mm, the collagen fiber network in the MVAL deforms according to an affine kinematics model. Moreover, with respect to understanding its function, affine kinematics suggests that the constituent fibers are largely noninteracting and deform in accordance with the bulk tissue. It also suggests that the collagen fibrils are tightly bounded and deform as a single fiber-level unit. This greatly simplifies the modeling efforts at the tissue and organ levels, because affine kinematics allows a straightforward connection between the macroscopic and local fiber strains. It also suggests that the collagen and elastin fiber networks act independently of each other, with the collagen and elastin forming long fiber networks that allow for free rotations. Such freedom of rotation can greatly facilitate the observed high degree of mechanical anisotropy in the MVAL and other heart valves, which is essential to heart valve function. These apparently novel findings support modeling efforts directed toward improving our fundamental understanding of tissue biomechanics in healthy and diseased conditions

  1. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    PubMed Central

    Abdolalizadeh, Jalal; Majidi Zolbanin, Jafar; Nouri, Mohammad; Baradaran, Behzad; Movassaghpour, AliAkbar; Farajnia, Safar; Omidi, Yadollah

    2013-01-01

    Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods:In this study, we examined the potential of our produced anti-TNF-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications. PMID:24312807

  2. Naming, labeling, and packaging of pharmaceuticals.

    PubMed

    Kenagy, J W; Stein, G C

    2001-11-01

    The problem of medical errors associated with the naming, labeling, and packaging of pharmaceuticals is discussed. Sound-alike and look-alike drug names and packages can lead pharmacists and nurses to unintended interchanges of drugs that can result in patient injury or death. The existing medication-use system is flawed because its safety depends on human perfection. Simplicity, standardization, differentiation, lack of duplication, and unambiguous communication are human factors concepts that are relevant to the medication-use process. These principles have often been ignored in drug naming, labeling, and packaging. Instead, current methods are based on long-standing commercial considerations and bureaucratic procedures. The process for naming a marketable drug is lengthy and complex and involves submission of a new chemical entity and patent application, generic naming, brand naming, FDA review, and final approval. Drug companies seek the fastest possible approval and may believe that the incremental benefit of human factors evaluation is small. "Trade dress" is the concept that underlies labeling and packaging issues for the drug industry. Drug companies are resistant to changing trade dress and brand names. Although a variety of private-sector organizations have called for reforms in drug naming, labeling, and packaging standards have been proposed, the problem remains. Drug names, labels, and packages are not selected and designed in accordance with human factors principles. FDA standards do not require application of these principles, the drug industry has struggled with change, and private-sector initiatives have had only limited success.

  3. Fluorescent Quantum Dots for Biological Labeling

    NASA Technical Reports Server (NTRS)

    McDonald, Gene; Nadeau, Jay; Nealson, Kenneth; Storrie-Lomardi, Michael; Bhartia, Rohit

    2003-01-01

    Fluorescent semiconductor quantum dots that can serve as "on/off" labels for bacteria and other living cells are undergoing development. The "on/off" characterization of these quantum dots refers to the fact that, when properly designed and manufactured, they do not fluoresce until and unless they come into contact with viable cells of biological species that one seeks to detect. In comparison with prior fluorescence-based means of detecting biological species, fluorescent quantum dots show promise for greater speed, less complexity, greater sensitivity, and greater selectivity for species of interest. There are numerous potential applications in medicine, environmental monitoring, and detection of bioterrorism.

  4. In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling*

    PubMed Central

    Boersema, Paul J.; Foong, Leong Yan; Ding, Vanessa M. Y.; Lemeer, Simone; van Breukelen, Bas; Philp, Robin; Boekhorst, Jos; Snel, Berend; den Hertog, Jeroen; Choo, Andre B. H.; Heck, Albert J. R.

    2010-01-01

    Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative

  5. Prediction of kinase-inhibitor binding affinity using energetic parameters

    PubMed Central

    Usha, Singaravelu; Selvaraj, Samuel

    2016-01-01

    The combination of physicochemical properties and energetic parameters derived from protein-ligand complexes play a vital role in determining the biological activity of a molecule. In the present work, protein-ligand interaction energy along with logP values was used to predict the experimental log (IC50) values of 25 different kinase-inhibitors using multiple regressions which gave a correlation coefficient of 0.93. The regression equation obtained was tested on 93 kinase-inhibitor complexes and an average deviation of 0.92 from the experimental log IC50 values was shown. The same set of descriptors was used to predict binding affinities for a test set of five individual kinase families, with correlation values > 0.9. We show that the protein-ligand interaction energies and partition coefficient values form the major deterministic factors for binding affinity of the ligand for its receptor. PMID:28149052

  6. Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Preston, D.A.; Wu, C.Y.; Blaszczak, L.C.

    Radiolabeled penicillin G is widely used as the imaging agent in penicillin-binding protein (PBP) assays. The disadvantages of most forms of labeled penicillin G are instability on storage and the long exposure times usually required for autoradiography or fluorography of electrophoretic gels. We investigated the utility of radioiodinated penicillin V as an alternative reagent. Radioiodination of p-(trimethylstannyl)penicillin V with ({sup 125}I)Na, using a modification of the chloramine-T method, is simple, high yielding, and site specific. We demonstrated the general equivalence of commercially obtained ({sup 3}H)penicillin G and locally synthesized ({sup 125}I)penicillin V (IPV) in their recognition of bacterial PBPs. Profilesmore » of PBPs in membranes from Bacteroides fragilis, Escherichia coli, Providencia rettgeri, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, and Enterococcus faecium labeled with IPV or (3H)penicillin G were virtually identical. Use of IPV as the imaging agent in competition experiments for determination of the affinities of various beta-lactam antibiotics for the PBPs of E. coli yielded results similar to those obtained in experiments with ({sup 3}H)penicillin G. Dried electrophoretic gels from typical PBP experiments, using IPV at 37.3 Ci/mmol and 30 micrograms/ml, exposed X-ray film in 8 to 24 h. The stability of IPV on storage at 4{degrees}C was inversely proportional to specific activity. At 37.3 Ci/mmol and 60 micrograms/ml, IPV retained useful activity for at least 60 days at 4{degrees}C. IPV represents a practical and stable reagent for rapid PBP assays.« less

  7. Semi-Supervised Projective Non-Negative Matrix Factorization for Cancer Classification.

    PubMed

    Zhang, Xiang; Guan, Naiyang; Jia, Zhilong; Qiu, Xiaogang; Luo, Zhigang

    2015-01-01

    Advances in DNA microarray technologies have made gene expression profiles a significant candidate in identifying different types of cancers. Traditional learning-based cancer identification methods utilize labeled samples to train a classifier, but they are inconvenient for practical application because labels are quite expensive in the clinical cancer research community. This paper proposes a semi-supervised projective non-negative matrix factorization method (Semi-PNMF) to learn an effective classifier from both labeled and unlabeled samples, thus boosting subsequent cancer classification performance. In particular, Semi-PNMF jointly learns a non-negative subspace from concatenated labeled and unlabeled samples and indicates classes by the positions of the maximum entries of their coefficients. Because Semi-PNMF incorporates statistical information from the large volume of unlabeled samples in the learned subspace, it can learn more representative subspaces and boost classification performance. We developed a multiplicative update rule (MUR) to optimize Semi-PNMF and proved its convergence. The experimental results of cancer classification for two multiclass cancer gene expression profile datasets show that Semi-PNMF outperforms the representative methods.

  8. Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein

    NASA Technical Reports Server (NTRS)

    Kutner, A.; Link, R. P.; Schnoes, H. K.; DeLuca, H. F.

    1986-01-01

    3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

  9. Complete inactivation of HIV-1 using photo-labeled non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Rios, Adan; Quesada, Jorge; Anderson, Dallas; Goldstein, Allan; Fossum, Theresa; Colby-Germinario, Susan; Wainberg, Mark A

    2011-01-01

    We demonstrate that a photo-labeled derivative of the non-nucleoside reverse transcriptase inhibitor (NNRTI) dapivirine termed DAPY, when used together with exposure to ultraviolet light, was able to completely and irreversibly inactivate both HIV-1 RT activity as well as infectiousness in each of a T cell line and peripheral blood mononuclear cells. Control experiments using various concentrations of DAPY revealed that a combination of exposure to ultraviolet light together with use of the specific, high affinity photo-labeled compound was necessary for complete inactivation to occur. This method of HIV RT inactivation may have applicability toward preservation of an intact viral structure and warrants further investigation in regard to the potential of this approach to elicit a durable, broad protective immune response. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Recombinant spider silk genetically functionalized with affinity domains.

    PubMed

    Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

    2014-05-12

    Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

  11. Description of two new species of the Exocelina broschii-group from Papua New Guinea, with revision and key to all representatives of this species group (Coleoptera, Dytiscidae, Copelatinae)

    PubMed Central

    Shaverdo, Helena; Sagata, Katayo; Balke, Michael

    2016-01-01

    Abstract Two new species of Exocelina Broun, 1886 from Papua New Guinea are described herein: Exocelina mondmillensis sp. n. and Exocelina pseudomarinae sp. n. They are placed into the Exocelina broschii-group based on the shovel/fork-like ventral sclerites of their median lobe. While the former has rather distinct combination of the morphological characters (inconspicuous dorsal punctation, thin apex of the median lobe and ventral sclerite of the median lobe with two tips of different length), the latter is very similar to already described species Exocelina marinae (Shaverdo, Sagata & Balke, 2005). All described species of the group are revised and a key to their identification is provided. Important diagnostic characters (habitus, color, protarsomeres 4–5, median lobes, and parameres) are illustrated. Data on the distribution of all species of the group are given showing that its representatives occur only in Papua New Guinea and most of them are widely distributed in it central part. PMID:27110191

  12. Advantages and application of label-free detection assays in drug screening.

    PubMed

    Cunningham, Brian T; Laing, Lance G

    2008-08-01

    Adoption is accelerating for a new family of label-free optical biosensors incorporated into standard format microplates owing to their ability to enable highly sensitive detection of small molecules, proteins and cells for high-throughput drug discovery applications. Label-free approaches are displacing other detection technologies owing to their ability to provide simple assay procedures for hit finding/validation, accessing difficult target classes, screening the interaction of cells with drugs and analyzing the affinity of small molecule inhibitors to target proteins. This review describes several new drug discovery applications that are under development for microplate-based photonic crystal optical biosensors and the key issues that will drive adoption of the technology. Microplate-based optical biosensors are enabling a variety of cell-based assays, inhibition assays, protein-protein binding assays and protein-small molecule binding assays to be performed with high-throughput and high sensitivity.

  13. What is prescription labeling communicating to doctors about hepatotoxic drugs? A study of FDA approved product labeling.

    PubMed

    Willy, Mary E; Li, Zili

    2004-04-01

    The objective of this study was to evaluate the informativeness and consistency of product labeling of hepatotoxic drugs marketed in the United States. We searched the Physicians' Desk Reference-2000 for prescription drugs with hepatic failure and/or hepatic necrosis listed in the labeling. We used a six-item checklist to evaluate the 'informativeness' and consistency of the labeling content. An informativeness score equaled the proportion of checklist items present in each drug's labeling. Ninety-five prescription drugs were included in the study. Eleven (12%) of the drugs had information related to hepatic failure in a Black Boxed Warning, 52 (54%) in the Warnings section and 32 (34%) in the Adverse Reactions section of the label. The mean informativeness score was 35%; the score was significantly higher, 61%, when the risk was perceived to be high. The informativeness of labeling was not affected by the time of the labeling, but differed across the Center for Drug Evaluation and Research (CDER) Review Division responsible for the labeling. The information provided in labeling is variable and affected by many factors, including the perceived level of risk and review division strategy. Product labeling may benefit from current FDA initiatives to improve the consistency of risk-related labeling.

  14. Cyclic versus Noncyclic Chelating Scaffold for 89Zr-Labeled ZEGFR:2377 Affibody Bioconjugates Targeting Epidermal Growth Factor Receptor Overexpression

    PubMed Central

    2017-01-01

    Zirconium-89 is an emerging radionuclide for positron emission tomography (PET) especially for biomolecules with slow pharmacokinetics as due to its longer half-life, in comparison to fluorine-18 and gallium-68, imaging at late time points is feasible. Desferrioxamine B (DFO), a linear bifunctional chelator (BFC) is mostly used for this radionuclide so far but shows limitations regarding stability. Our group recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study was designed to compare FSC and DFO head-to-head as bifunctional chelators for 89Zr-radiolabeled EGFR-targeting ZEGFR:2377 affibody bioconjugates. FSC-ZEGFR:2377 and DFO-ZEGFR:2377 were evaluated regarding radiolabeling, in vitro stability, specificity, cell uptake, receptor affinity, biodistribution, and microPET-CT imaging. Both conjugates were efficiently labeled with zirconium-89 at room temperature but radiochemical yields increased substantially at elevated temperature, 85 °C. Both 89Zr-FSC-ZEGFR:2377 and 89Zr-DFO-ZEGFR:2377 revealed remarkable specificity, affinity and slow cell-line dependent internalization. Radiolabeling at 85 °C showed comparable results in A431 tumor xenografted mice with minor differences regarding blood clearance, tumor and liver uptake. In comparison 89Zr-DFO-ZEGFR:2377, radiolabeled at room temperature, showed a significant difference regarding tumor-to-organ ratios. MicroPET-CT imaging studies of 89Zr-FSC-ZEGFR:2377 as well as 89Zr-DFO-ZEGFR:2377 confirmed these findings. In summary we were able to show that FSC is a suitable alternative to DFO for radiolabeling of biomolecules with zirconium-89. Furthermore, our findings indicate that 89Zr-radiolabeling of DFO conjugates at higher temperature reduces off-chelate binding leading to significantly improved tumor-to-organ ratios and therefore enhancing image contrast. PMID:29160082

  15. Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

    PubMed Central

    Berger, Christian; Ho, Jenny T.C.; Kimura, Tomohiro; Hess, Sonja; Gawrisch, Klaus; Yeliseev, Alexei

    2010-01-01

    We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and 15N2-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2 per liter of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two 15N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of 15N chemical shifts strongly suggests that the 15N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation. PMID:20044006

  16. Cholera Toxin Inhibitors Studied with High-Performance Liquid Affinity Chromatography: A Robust Method to Evaluate Receptor–Ligand Interactions

    PubMed Central

    Bergström, Maria; Liu, Shuang; Kiick, Kristi L.; Ohlson, Sten

    2009-01-01

    Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The KD values of galactose and meta-nitrophenyl α-D-galactoside were determined with weak affinity chromatography to be 52 and 1 mM, respectively, which agree well with IC50 values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but KD values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct KD values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis. PMID:19152642

  17. Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

    PubMed

    Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

    2015-10-21

    Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.

  18. Measuring the electron affinity of organic solids: an indispensable new tool for organic electronics.

    PubMed

    Yoshida, Hiroyuki

    2014-04-01

    Electron affinity is a fundamental energy parameter of materials. In organic semiconductors, the electron affinity is closely related to electron conduction. It is not only important to understand fundamental electronic processes in organic solids, but it is also indispensable for research and development of organic semiconductor devices such as organic light-emitting diodes and organic photovoltaic cells. However, there has been no experimental technique for examining the electron affinity of organic materials that meets the requirements of such research. Recently, a new method, called low-energy inverse-photoemission spectroscopy, has been developed. A beam of low-energy electrons is focused onto the sample surface, and photons emitted owing to the radiative transition to unoccupied states are then detected. From the onset of the spectral intensity, the electron affinity is determined within an uncertainty of 0.1 eV. Unlike in conventional inverse-photoemission spectroscopy, sample damage is negligible and the resolution is improved by a factor of 2. The principle of the method and several applications are reported.

  19. A 45-Amino-Acid Scaffold Mined from the PDB for High-Affinity Ligand Engineering.

    PubMed

    Kruziki, Max A; Bhatnagar, Sumit; Woldring, Daniel R; Duong, Vandon T; Hackel, Benjamin J

    2015-07-23

    Small protein ligands can provide superior physiological distribution compared with antibodies, and improved stability, production, and specific conjugation. Systematic evaluation of the PDB identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α helix opposite a β sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 10(8) mutants and directed evolution toward four targets yielded target-specific binders with affinities as strong as 200 ± 100 pM, Tms from 65 °C ± 3 °C to 80°C ± 1 °C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ± 8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

    PubMed Central

    Nampally, Malathi; Moerschbacher, Bruno Maria

    2012-01-01

    Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining. PMID:22367086

  1. Electron Detachment Dissociation (EDD) of Fluorescently Labeled Sialylated Oligosaccharides

    PubMed Central

    Zhou, Wen; Håkansson, Kristina

    2012-01-01

    We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared to IRMPD. Neutral losses and satellite ions such as C – 2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared to 2-AA labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. PMID:22120881

  2. Development of High-Performance Chemical Isotope Labeling LC-MS for Profiling the Carbonyl Submetabolome.

    PubMed

    Zhao, Shuang; Dawe, Margot; Guo, Kevin; Li, Liang

    2017-06-20

    Metabolites containing a carbonyl group represent several important classes of molecules including various forms of ketones and aldehydes such as steroids and sugars. We report a high-performance chemical isotope labeling (CIL) LC-MS method for profiling the carbonyl submetabolome with high coverage and high accuracy and precision of relative quantification. This method is based on the use of dansylhydrazine (DnsHz) labeling of carbonyl metabolites to change their chemical and physical properties to such an extent that the labeled metabolites can be efficiently separated by reversed phase LC and ionized by electrospray ionization MS. In the analysis of six standards representing different carbonyl classes, acetaldehyde could be ionized only after labeling and MS signals were significantly increased for other 5 standards with an enhancement factor ranging from ∼15-fold for androsterone to ∼940-fold for 2-butanone. Differential 12 C- and 13 C-DnsHz labeling was developed for quantifying metabolic differences in comparative samples where individual samples were separately labeled with 12 C-labeling and spiked with a 13 C-labeled pooled sample, followed by LC-MS analysis, peak pair picking, and peak intensity ratio measurement. In the replicate analysis of a 1:1 12 C-/ 13 C-labeled human urine mixture (n = 6), an average of 2030 ± 39 pairs per run were detected with 1737 pairs in common, indicating the possibility of detecting a large number of carbonyl metabolites as well as high reproducibility of peak pair detection. The average RSD of the peak pair ratios was 7.6%, and 95.6% of the pairs had a RSD value of less than 20%, demonstrating high precision for peak ratio measurement. In addition, the ratios of most peak pairs were close to the expected value of 1.0 (e.g., 95.5% of them had ratios of between 0.67 and 1.5), showing the high accuracy of the method. For metabolite identification, a library of DnsHz-labeled standards was constructed, including 78 carbonyl

  3. A novel strategy using MASCOT Distiller for analysis of cleavable isotope-coded affinity tag data to quantify protein changes in plasma.

    PubMed

    Leung, Kit-Yi; Lescuyer, Pierre; Campbell, James; Byers, Helen L; Allard, Laure; Sanchez, Jean-Charles; Ward, Malcolm A

    2005-08-01

    A novel strategy consisting of cleavable Isotope-Coded Affinity Tag (cICAT) combined with MASCOT Distiller was evaluated as a tool for the quantification of proteins in "abnormal" patient plasma, prepared by pooling samples from patients with acute stroke. Quantification of all light and heavy cICAT-labelled peptide ion pairs was obtained using MASCOT Distiller combined with a proprietary software. Peptides displaying differences were selected for identification by MS. These preliminary results show the promise of our approach to identify potential biomarkers.

  4. Immunolocalization of ciliary neurotrophic factor receptor alpha (CNTFRalpha) in mammalian photoreceptor cells.

    PubMed

    Beltran, William A; Rohrer, Hermann; Aguirre, Gustavo D

    2005-04-01

    To characterize the site of expression of the alpha subunit of the receptor for ciliary neurotrophic factor (CNTFRalpha) in the retina of a variety of mammalian species, and determine whether CNTFRalpha is localized to photoreceptor cells. The cellular distribution of CNTFRalpha(protein) was examined by immunocytochemistry in the adult retinas of several mammalian species that included mouse, rat, dog, cat, sheep, pig, horse, monkey, and human. Developing retinas from 3-day-old and 6-day-old rats were also included in this study. The molecular weight of CNTFRalpha in rat, dog, cat, pig, and human retinas was determined by immunoblotting. CNTFRalpha immunolabeling was present in the retina of all species. A common pattern was observed in all species, and represented labeling of the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL). CNTFRalpha did not immunolocalize to photoreceptor cells in both adult and developing rodent retinas, but was consistently observed in both rods and cones of non-rodent species. The molecular weight of CNTFRalpha in mammalian retinas was approximately 61-64 kDa. These findings highlight a significant difference in the expression of CNTFRalpha in the retina of rodent and non-rodent mammalian species. The expression of CNTFRalpha by rods and cones in non-rodent species may suggest a direct mechanism of action if CNTF administration results in photoreceptor rescue.

  5. Characterization of the translation elongation factor 1-α gene in a wide range of pathogenic Aspergillus species.

    PubMed

    Nouripour-Sisakht, Sadegh; Ahmadi, Bahram; Makimura, Koichi; Hoog, Sybren de; Umeda, Yoshiko; Alshahni, Mohamed Mahdi; Mirhendi, Hossein

    2017-04-01

    We aimed to evaluate the resolving power of the translation elongation factor (TEF)-1α gene for phylogenetic analysis of Aspergillus species. Sequences of 526 bp representing the coding region of the TEF-1α gene were used for the assessment of levels of intra- and inter-specific nucleotide polymorphism in 33 species of Aspergillus, including 57 reference, clinical and environmental strains. Analysis of TEF-1α sequences indicated a mean similarity of 92.6 % between the species, with inter-species diversity ranging from 0 to 70 nucleotides. The species with the closest resemblance were A. candidus/A. carneus, and A. flavus/A. oryzae/A. ochraceus, with 100 and 99.8 % identification, respectively. These species are phylogenetically very close and the TEF-1α gene appears not to have sufficient discriminatory power to differentiate them. Meanwhile, intra-species differences were found within strains of A. clavatus, A. clavatonanicus, A. candidus, A. fumigatus, A. terreus, A. alliaceus, A. flavus, Eurotium amstelodami and E. chevalieri. The tree topology with strongly supported clades (≥70 % bootstrap values) was almost compatible with the phylogeny inferred from analysis of the DNA sequences of the beta tubulin gene (BT2). However, the backbone of the tree exhibited low bootstrap values, and inter-species correlations were not obvious in some clades; for example, tree topologies based on BT2 and TEF-1α genes were incompatible for some species, such as A. deflectus, A. janus and A. penicillioides. The gene was not phylogenetically more informative than other known molecular markers. It will be necessary to test other genes or larger genomic regions to better understand the taxonomy of this important group of fungi.

  6. Environmental factors-ecological species group relationships in the Surash lowland-mountain forests in northern Iran

    Treesearch

    Fatemeh Bazdid Vahdati; Shahryar Saeidi Mehrvarz; Daniel C. Dey; Alireza Naqinezhad

    2016-01-01

    Identification of the primary factors that influence the ecological distribution of species groups is important to managers of lowland-mountain forests in northern Iran. The aim of this study was to identify main ecological species groups, describe the site conditions associated with these species groups and the relationships between environmental factors and the...

  7. NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vourtsis, Dionysios J.; Chasapis, Christos T.; Pairas, George

    2014-07-18

    Highlights: • A polypeptide, N-ALF{sub 233}, was overexpressed in E. coli and successfully isolated. • We produced {sup 2}H/{sup 15}N/{sup 13}C labeled protein samples. • Amino acid selective approaches were applied. • We acquired several heteronuclear NMR spectra, to complete the backbone assignment. • Prediction of the secondary structure was performed. - Abstract: NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successfulmore » overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.« less

  8. College student reactions to health warning labels: Sociodemographic and psychosocial factors related to perceived effectiveness of different approaches

    PubMed Central

    Berg, Carla J.; Thrasher, James F.; Westmaas, J. Lee; Buchanan, Taneisha; Pinsker, Erika A.; Ahluwalia, Jasjit S.

    2011-01-01

    Objective To examine factors associated with perceiving different types of pictorial cigarette health warning labels as most effective in motivating smokers to quit or preventing smoking initiation among college students. Method We administered an online survey to 24,055 students attending six Southeast colleges in Fall, 2010. We obtained complete data for the current analyses from 2,600. Results Current smoking prevalence was 23.5%. The largest majority (78.6%) consistently rated gruesome images as most effective, 19.5% rated testimonial images as most effective, and only a small proportion rated either standard (1.6%) or human suffering images (0.3%) as most effective. Subsequent analyses focused on differences between those endorsing gruesome images or testimonials as most effective. Factors related to ranking testimonials versus gruesome images as most effective included being female (p<0.01), White (p<0.01), and nonsmokers (p=0.04), lower perceived smoking prevalence (p<0.01), and greater receptivity to laws/restrictions around smoking (p<0.01) and tobacco marketing (p=0.01). Among smokers, factors related to ranking testimonials as most effective versus gruesome images included being female (p=0.03), being White (p=0.03), higher autonomous motivation (p=0.03), and greater extrinsic self-efficacy (p=0.02). Conclusions Understanding factors related to perceived effectiveness of different pictorial warnings among subpopulations should inform health warning labels released by the FDA. PMID:21945706

  9. A Mixtures-of-Trees Framework for Multi-Label Classification

    PubMed Central

    Hong, Charmgil; Batal, Iyad; Hauskrecht, Milos

    2015-01-01

    We propose a new probabilistic approach for multi-label classification that aims to represent the class posterior distribution P(Y|X). Our approach uses a mixture of tree-structured Bayesian networks, which can leverage the computational advantages of conditional tree-structured models and the abilities of mixtures to compensate for tree-structured restrictions. We develop algorithms for learning the model from data and for performing multi-label predictions using the learned model. Experiments on multiple datasets demonstrate that our approach outperforms several state-of-the-art multi-label classification methods. PMID:25927011

  10. Proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface.

    PubMed

    Kastritis, Panagiotis L; Rodrigues, João P G L M; Folkers, Gert E; Boelens, Rolf; Bonvin, Alexandre M J J

    2014-07-15

    Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions. Copyright © 2014. Published by Elsevier Ltd.

  11. Identification of cellular MMP substrates using quantitative proteomics: isotope-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification (iTRAQ).

    PubMed

    Butler, Georgina S; Dean, Richard A; Morrison, Charlotte J; Overall, Christopher M

    2010-01-01

    Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cellular context. Here we describe the use of two protein labeling techniques, Isotope-Coded Affinity Tags (ICAT and Isobaric Tags for Relative and Absolute Quantification (iTRAQ), which we have used successfully to identify novel matrix metalloproteinase (MMP) substrates in cell culture systems (1-4). ICAT and iTRAQ can label proteins and protease cleavage products of secreted proteins, protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium, or cell surface proteins in membrane preparations; isotopically distinct labels are used for control cells. Tryptic digestion and tandem mass spectrometry of the generated fragments enable sequencing of differentially labeled but otherwise identical pooled peptides. The isotopic tag, which is unique for each label, identifies the peptides originating from each sample, for instance, protease-transfected or control cells, and comparison of the peak areas enables relative quantification of the peptide in each sample. Thus proteins present in altered amounts between protease-expressing and null cells are implicated as protease substrates and can be further validated as such.

  12. Teat apex colonization with coagulase-negative Staphylococcus species before parturition: Distribution and species-specific risk factors.

    PubMed

    De Visscher, A; Piepers, S; Haesebrouck, F; De Vliegher, S

    2016-02-01

    Coagulase-negative staphylococci (CNS) are the main cause of bovine intramammary infections and are also abundantly present in extramammary habitats such as teat apices. Teat apex colonization (TAC) with CNS has already been explored in lactating dairy cows at the species level, whereas this is not true for dry cows and end-term heifers. Therefore, the aim of this observational study was to describe CNS TAC in nonlactating dairy cows and end-term heifers in Flemish dairy herds and to identify associated risk factors at the herd, cow, and quarter level. All CNS were molecularly identified to the species level using transfer RNA intergenic spacer PCR (tDNA-PCR) and sequencing of the 16S rRNA gene, allowing for species-specific statistical analyses using multivariable, multilevel logistic regression. Staphylococcus devriesei, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus equorum were the most frequently isolated species. Staphylococcus chromogenes was the sole species colonizing teat apices of cows and heifers in all herds, whereas large between-herd differences were observed for the other species. Teat apices of red and white Holstein Friesians, of quarters dried off without an internal teat sealer, and swabbed in months with lower precipitation and higher ambient temperature were significantly more likely to be colonized by S. devriesei. Slightly dirty teat apices and teat apices swabbed in months with lower precipitation had higher odds of being colonized by S. chromogenes, whereas teat apices sampled in months with lower precipitation and higher ambient temperature were more likely to be colonized by S. haemolyticus. Dirty teat apices and teat apices swabbed in months with lower ambient temperature in combination with low precipitation had higher odds of being colonized by S. equorum. Diverse factors explaining CNS TAC, yet mostly related to humidity, ambient temperature, and hygiene, substantiate differences in epidemiological

  13. The internalization of fluorescence-labeled PLA nanoparticles by macrophages.

    PubMed

    Li, Fengjuan; Zhu, Aiping; Song, Xiaoli; Ji, Lijun; Wang, Juan

    2013-09-10

    Rhodamine B (RhB)-labeled PLA nanoparticles were prepared through surface grafting copolymerization of glycidyl methacrylate (GMA) onto PLA nanoparticles during the emulsion/evaporation process. RhB firstly interacts with sodium dodecyl sulfate (SDS) through electrostatic interaction to form hydrophobic complex (SDS-RhB). Due to the high-affinity of SDS-RhB with GMA, hydrophilic RhB can be successfully combined into PLA nanoparticles. The internalization of RhB-labeled PLA nanoparticles by macrophages was investigated with fluorescence microscope technology. The effects of the PLA nanoparticle surface nature and size on the internalization were investigated. The results indicate that the PLA particles smaller than 200 nm can avoid the uptake of phagocytosis. The bigger PLA particles (300 nm) with polyethylene glycol (PEG) surface showed less internalization by macrophage compared with those with poly(ethylene oxide-propylene oxide) copolymer (F127) or poly(vinyl alcohol) (PVA) surface. The "stealth" function of PEG on the PLA nanoparticles from internalization of macrophages due to the low protein adsorption is revealed by electrochemical impedance technology. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Progressive multi-atlas label fusion by dictionary evolution.

    PubMed

    Song, Yantao; Wu, Guorong; Bahrami, Khosro; Sun, Quansen; Shen, Dinggang

    2017-02-01

    Accurate segmentation of anatomical structures in medical images is important in recent imaging based studies. In the past years, multi-atlas patch-based label fusion methods have achieved a great success in medical image segmentation. In these methods, the appearance of each input image patch is first represented by an atlas patch dictionary (in the image domain), and then the latent label of the input image patch is predicted by applying the estimated representation coefficients to the corresponding anatomical labels of the atlas patches in the atlas label dictionary (in the label domain). However, due to the generally large gap between the patch appearance in the image domain and the patch structure in the label domain, the estimated (patch) representation coefficients from the image domain may not be optimal for the final label fusion, thus reducing the labeling accuracy. To address this issue, we propose a novel label fusion framework to seek for the suitable label fusion weights by progressively constructing a dynamic dictionary in a layer-by-layer manner, where the intermediate dictionaries act as a sequence of guidance to steer the transition of (patch) representation coefficients from the image domain to the label domain. Our proposed multi-layer label fusion framework is flexible enough to be applied to the existing labeling methods for improving their label fusion performance, i.e., by extending their single-layer static dictionary to the multi-layer dynamic dictionary. The experimental results show that our proposed progressive label fusion method achieves more accurate hippocampal segmentation results for the ADNI dataset, compared to the counterpart methods using only the single-layer static dictionary. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. 16 CFR 306.12 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... biodiesel, biomass-based diesel, biodiesel blends, and biomass-based diesel blends. The label is 3 inches (7... biodiesel blends containing more than 5 percent and no greater than 20 percent biodiesel by volume. (i) The...” followed immediately by the numerical value representing the volume percentage of biodiesel in the fuel (e...

  16. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hall, Justin; Brault, Amy; Vincent, Fabien

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a highmore » affinity (K D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.« less

  17. Relations of Environmental Factors with Mussel-Species Richness in the Neversink River, New York

    USGS Publications Warehouse

    Baldigo, Barry P.; Ernst, Anne G.; Schuler, George E.; Apse, Colin D.

    2007-01-01

    INTRODUCTION Declines in the distribution, abundance, and diversity of freshwater-mussel species (family Unionidae1) have been reported worldwide (Bogan, 1993; Strayer and Jirka, 1997). The principal causes of the observed declines are difficult to confirm, however, because only a few of the many factors that affect mussel-species populations have been identified (Strayer and Ralley, 1993; Strayer, 1999; Baldigo and others, 2003; Strayer and others, 2006). The Neversink River, which drains the Catskill Mountains in southeastern New York (fig. 1), contains seven species of mussels (Strayer and Ralley, 1991; Strayer and Jirka, 1997). Populations of the endangered dwarf wedgemussel (Alasmidonta heterodon) and the threatened swollen wedgemussel (Alasmidonta varicosa) coexist with other unionid mussels in the Neversink River (Strayer and Ralley, 1991, 1993; Baldigo and others, 2003). Dwarf wedgemussel populations had previously been found only downstream from the site of an abandoned dam in the lower part of the river at Cuddebackville (fig. 1), and swollen wedgemussels were only found in the lower and middle reaches of the river. The limited distribution of these two species suggests that they may be susceptible to local extinctions. The distribution of mussel populations can be limited by impoundments. Mussel larvae develop in species-specific host fish; thus, impoundments that restrict passage of these host fish also restrict the extent of mussels. The Neversink River is impounded by the Neversink Reservoir [241 square kilometers (km2)], a major source of drinking water for the City of New York, and was also impounded 50 km downstream by the Cuddebackville Dam until 2004, when the latter was removed by The Nature Conservancy (TNC) and the U.S. Army Corps of Engineers to improve fish passage. The removal of this dam has provided previously unavailable habitat for diadromous and other fish species that act as hosts for rare mussel species. In addition, releases from

  18. Cabazitaxel is more active than first-generation taxanes in ABCB1(+) cell lines due to its reduced affinity for P-glycoprotein.

    PubMed

    Duran, George E; Derdau, Volker; Weitz, Dietmar; Philippe, Nicolas; Blankenstein, Jörg; Atzrodt, Jens; Sémiond, Dorothée; Gianolio, Diego A; Macé, Sandrine; Sikic, Branimir I

    2018-04-19

    The primary aim of this study was to determine cabazitaxel's affinity for the ABCB1/P-glycoprotein (P-gp) transporter compared to first-generation taxanes. We determined the kinetics of drug accumulation and retention using [ 14 C]-labeled taxanes in multidrug-resistant (MDR) cells. In addition, membrane-enriched fractions isolated from doxorubicin-selected MES-SA/Dx5 cells were used to determine sodium orthovanadate-sensitive ATPase stimulation after exposure to taxanes. Custom [ 3 H]-azido-taxane analogues were synthesized for the photoaffinity labeling of P-gp. The maximum intracellular drug concentration was achieved faster with [ 14 C]-cabazitaxel (5 min) than [ 14 C]-docetaxel (15-30 min). MDR cells accumulated twice as much cabazitaxel than docetaxel, and these levels could be restored to parental levels in the presence of the P-gp inhibitor PSC-833 (valspodar). Efflux in drug-free medium confirmed that MDR cells retained twice as much cabazitaxel than docetaxel. There was a strong association (r 2  = 0.91) between the degree of taxane resistance conferred by P-gp expression and the accumulation differences observed with the two taxanes. One cell model expressing low levels of P-gp was not cross-resistant to cabazitaxel while demonstrating modest resistance to docetaxel. Furthermore, there was a 1.9 × reduction in sodium orthovanadate-sensitive ATPase stimulation resulting from treatment with cabazitaxel compared to docetaxel. We calculated a dissociation constant (Kd) value of 1.7 µM for [ 3 H]-azido-docetaxel and ~ 7.5 µM for [ 3 H]-azido-cabazitaxel resulting in a 4.4 × difference in P-gp labeling, and cold docetaxel was a more effective competitor than cabazitaxel. Our studies confirm that cabazitaxel is more active in ABCB1(+) cell models due to its reduced affinity for P-gp compared to docetaxel.

  19. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  20. Methylsorb: a simple method for quantifying DNA methylation using DNA-gold affinity interactions.

    PubMed

    Sina, Abu Ali Ibn; Carrascosa, Laura G; Palanisamy, Ramkumar; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

    2014-10-21

    The analysis of DNA methylation is becoming increasingly important both in the clinic and also as a research tool to unravel key epigenetic molecular mechanisms in biology. Current methodologies for the quantification of regional DNA methylation (i.e., the average methylation over a region of DNA in the genome) are largely affected by comprehensive DNA sequencing methodologies which tend to be expensive, tedious, and time-consuming for many applications. Herein, we report an alternative DNA methylation detection method referred to as "Methylsorb", which is based on the inherent affinity of DNA bases to the gold surface (i.e., the trend of the affinity interactions is adenine > cytosine ≥ guanine > thymine).1 Since the degree of gold-DNA affinity interaction is highly sequence dependent, it provides a new capability to detect DNA methylation by simply monitoring the relative adsorption of bisulfite treated DNA sequences onto a gold chip. Because the selective physical adsorption of DNA fragments to gold enable a direct read-out of regional DNA methylation, the current requirement for DNA sequencing is obviated. To demonstrate the utility of this method, we present data on the regional methylation status of two CpG clusters located in the EN1 and MIR200B genes in MCF7 and MDA-MB-231 cells. The methylation status of these regions was obtained from the change in relative mass on gold surface with respect to relative adsorption of an unmethylated DNA source and this was detected using surface plasmon resonance (SPR) in a label-free and real-time manner. We anticipate that the simplicity of this method, combined with the high level of accuracy for identifying the methylation status of cytosines in DNA, could find broad application in biology and diagnostics.

  1. [Academic production on food labeling in Brazil].

    PubMed

    Câmara, Maria Clara Coelho; Marinho, Carmem Luisa Cabral; Guilam, Maria Cristina; Braga, Ana Maria Cheble Bahia

    2008-01-01

    To review and discuss academic production (theses and dissertations) on the topic of labeling of prepackaged foods in Brazil. A search of the database maintained by the Coordination for the Development of Higher Education Professionals (CAPES), one of the two Brazilian government research funding and support agencies, was conducted on the following keywords: "rotulagem" (labeling), "rotulagem nutricional" (food labeling) and "rótulo de alimentos" (food labels). The search covered the years 1987 (earliest year available) to 2004. We identified 49 studies on this topic. Content analysis identified three major themes: the extent to which food labels meet specific legal requirements (57.2%); the degree to which consumers understand the information on labels (22.4%); and the labeling of transgenic or genetically-modified foods (20.4%). Food labeling is a frequent topic and is adequately covered by the Brazilian academic production. In most of the studies, ineffective law enforcement appears to be the main factor in the lack of compliance with and disrespect for the food labeling rules and regulations in Brazil.

  2. High-affinity K+ uptake in pepper plants.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2005-06-01

    High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

  3. Modulating the DNA affinity of Elk-1 with computationally selected mutations.

    PubMed

    Park, Sheldon; Boder, Eric T; Saven, Jeffery G

    2005-04-22

    In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

  4. Capture and detection of Staphylococcus aureus with dual labeled aptamers to cell surface components.

    PubMed

    Ramlal, Shylaja; Mondal, Bhairab; Lavu, Padma Sudharani; N, Bhavanashri; Kingston, Joseph

    2018-01-16

    In the present study, a high throughput whole cell SELEX method has been applied successfully in selecting specific aptamers against whole cells of Staphylococcus aureus, a potent food poisoning bacterium. A total ten rounds of SELEX and three rounds of intermittent counter SELEX, was performed to obtain specific aptamers. Obtained oligonucleotide pool were cloned, sequenced and then grouped into different families based on their primary sequence homology and secondary structure similarity. FITC labeled sequences from different families were selected for further characterization via flow cytometry analysis. The dissociation constant (K d ) values of specific and higher binders ranged from 34 to 128nM. Binding assays to assess the selectivity of aptamer RAB10, RAB 20, RAB 28 and RAB 35 demonstrated high affinity against S. aureus and low binding affinity for other bacteria. To demonstrate the potential use of the aptamer a sensitive dual labeled sandwich detection system was developed using aptamer RAB10 and RAB 35 with a detection limit of 10 2 CFU/mL. Furthermore detection from mixed cell population and spiked sample emphasized the robustness as well as applicability of the developed method. Altogether, the established assay could be a reliable detection tool for the routine investigation of Staphylococcus aureus in samples from food and clinical sources. Copyright © 2017. Published by Elsevier B.V.

  5. Improved full analytical polygon-based method using Fourier analysis of the three-dimensional affine transformation.

    PubMed

    Pan, Yijie; Wang, Yongtian; Liu, Juan; Li, Xin; Jia, Jia

    2014-03-01

    Previous research [Appl. Opt.52, A290 (2013)] has revealed that Fourier analysis of three-dimensional affine transformation theory can be used to improve the computation speed of the traditional polygon-based method. In this paper, we continue our research and propose an improved full analytical polygon-based method developed upon this theory. Vertex vectors of primitive and arbitrary triangles and the pseudo-inverse matrix were used to obtain an affine transformation matrix representing the spatial relationship between the two triangles. With this relationship and the primitive spectrum, we analytically obtained the spectrum of the arbitrary triangle. This algorithm discards low-level angular dependent computations. In order to add diffusive reflection to each arbitrary surface, we also propose a whole matrix computation approach that takes advantage of the affine transformation matrix and uses matrix multiplication to calculate shifting parameters of similar sub-polygons. The proposed method improves hologram computation speed for the conventional full analytical approach. Optical experimental results are demonstrated which prove that the proposed method can effectively reconstruct three-dimensional scenes.

  6. Support for the elevational Rapoport's rule among seed plants in Nepal depends on biogeographical affinities and boundary effects.

    PubMed

    Feng, Jianmeng; Hu, Xiaokang; Wang, Jie; Wang, Yanmei

    2016-10-01

    As one of the most important hypotheses on biogeographical distribution, Rapoport's rule has attracted attention around the world. However, it is unclear whether the applicability of the elevational Rapoport's Rule differs between organisms from different biogeographical regions. We used Stevens' method, which uses species diversity and the averaged range sizes of all species within each (100 m) elevational band to explore diversity-elevation, range-elevation, and diversity-range relationships. We compared support for the elevational Rapoport's rule between tropical and temperate species of seed plants in Nepal. Neither tropical nor temperate species supported the predictions of the elevational Rapoport's rule along the elevation gradient of 100-6,000 m a.s.l. for any of the studied relationships. However, along the smaller 1,000-5,000 m a.s.l. gradient (4,300 m a.s.l. for range-elevation relationships) which is thought to be less influenced by boundary effects, we observed consistent support for the rule by tropical species, although temperate species did not show consistent support. The degree of support for the elevational Rapoport's rule may not only be influenced by hard boundary effects, but also by the biogeographical affinities of the focal taxa. With ongoing global warming and increasing variability of temperature in high-elevation regions, tropical taxa may shift upward into higher elevations and expand their elevational ranges, causing the loss of temperate taxa diversity. Relevant studies on the elevational Rapoport's rule with regard to biogeographical affinities may be a promising avenue to further our understanding of this rule.

  7. Electron affinity of liquid water

    DOE PAGES

    Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco; ...

    2018-01-16

    Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1-0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential ofmore » the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.« less

  8. Electron affinity of liquid water

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco

    Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1-0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential ofmore » the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.« less

  9. Thermus arciformis sp. nov., a thermophilic species from a geothermal area.

    PubMed

    Zhang, Xin-Qi; Ying, Yi; Ye, Ying; Xu, Xue-Wei; Zhu, Xu-Fen; Wu, Min

    2010-04-01

    Two aerobic, Gram-negative, non-motile, non-sporulating, yellow-pigmented bacteria, strains TH92(T) and TH91, were isolated from a hot spring located in Laibin, Guangxi, in the south-eastern geothermal area of China. The isolates grew at 40-77 degrees C (optimally at 70 degrees C) and at pH 6.0-9.5 (optimally at pH 7.5-8.0). Phylogenetic analysis of 16S rRNA gene sequences and levels of DNA-DNA relatedness together indicated that the new isolates represented a novel species of the genus Thermus with closest affinity to Thermus aquaticus, Thermus igniterrae and Thermus thermophilus. Compared with their closest relatives, strains TH92( T) and TH91 were able to assimilate a wider range of carbohydrates, amino acids and organic acids as sole carbon sources for growth, such as lactose and melibiose. The new isolates had lower combined levels of C(16 : 0 ) and iso-C(16 : 0) compared with their closest relatives. On the basis of polyphasic taxonomic characterization, strains TH92(T) and TH91 are considered to represent a single novel species of the genus Thermus, for which the name Thermus arciformis sp. nov. is proposed. The type strain is TH92(T) (=CGMCC 1.6992(T) =JCM 15153(T)).

  10. Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Feng; Jaitly, Navdeep; Jayachandran, Hemalatha

    2007-10-12

    To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative Phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation sitemore » and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.« less

  11. Determination of trace alkaline phosphatase by affinity adsorption solid substrate room temperature phosphorimetry based on wheat germ agglutinin labeled with 8-quinolineboronic acid phosphorescent molecular switch and prediction of diseases

    NASA Astrophysics Data System (ADS)

    Liu, Jia-Ming; Gao, Hui; Li, Fei-Ming; Shi, Xiu-Mei; Lin, Chang-Qing; Lin, Li-Ping; Wang, Xin-Xing; Li, Zhi-Ming

    2010-09-01

    The 8-quinolineboronic acid phosphorescent molecular switch (abbreviated as PMS-8-QBA. Thereinto, 8-QBA is 8-quinolineboronic acid, and PMS is phosphorescent molecular switch) was found for the first time. PMS-8-QBA, which was in the "off" state, could only emit weak room temperature phosphorescence (RTP) on the acetyl cellulose membrane (ACM). However, PMS-8-QBA turned "on" automatically for its changed structure, causing that the RTP of 8-QBA in the system increased, after PMS-8-QBA-WGA (WGA is wheat germ agglutinin) was formed by reaction between -OH of PMS-8-QBA and -COOH of WGA. More interesting is that the -NH 2 of PMS-8-QBA-WGA could react with the -COOH of alkaline phosphatase (AP) to form the affinity adsorption (AA) product WGA-AP-WGA-8-QBA-PMS (containing -NH-CO- bond), which caused RTP of the system to greatly increase. Thus, affinity adsorption solid substrate room temperature phosphorimetry using PMS-8-QBA as labelling reagent (PMS-8-QBA-AA-SSRTP) for the determination of trace AP was established. The method had many advantages, such as high sensitivity (the detection limit (LD) was 2.5 zg spot -1. For sample volume of 0.40 μl spot -1, corresponding concentration was 6.2 × 10 -18 g ml -1), good selectivity (the allowed concentration of coexisting material was higher, when the relative error was ±5%), high accuracy (applied to detection of AP content in serum samples, the result was coincided with those obtained by enzyme-linked immunoassay), which was suitable for the detection of trace AP content in serum samples and the forecast of human diseases. Meanwhile, the mechanism of PMS-8-QBA-AASSRTP was discussed. The new field of analytical application and clinic diagnosis technique of molecule switch are exploited, based on the phosphorescence characteristic of PMS-8-QBA, the AA reaction between WGA and AP, as well as the relation between AP content and human diseases. The research results promote the development and interpenetrate among molecule

  12. Pharmacological and gene regulation properties point to the SlHAK5 K+ transporter as a system for high-affinity Cs+ uptake in tomato plants.

    PubMed

    Ródenas, Reyes; Nieves-Cordones, Manuel; Rivero, Rosa M; Martinez, Vicente; Rubio, Francisco

    2018-04-01

    Potassium (K + ) and cesium (Cs + ) are chemically similar but while K + is an essential nutrient, Cs + can be toxic for living organisms, plants included. Two different situations could lead to problems derived from the presence of Cs + in agricultural systems: (1) presence of Cs + at high concentrations that could produce toxic effects on plants, (2) presence of micromolar concentrations of radiocesium, which can be accumulated in the plant and affect animal and human health through the food chain. While K + uptake has been well described in tomato plants, information on molecular mechanisms involved in Cs + accumulation in this species is absent. Here, we show that in tomato plants, high concentrations of Cs + produce deficiency of K + but do not induce high-affinity K + uptake or the gene encoding the high-affinity K + transporter SlHAK5. At these concentrations, Cs + uptake takes place through a Ca 2+ -sensitive pathway, probably a non-selective cation channel. At micromolar concentrations, Cs + is accumulated by a high-affinity uptake system upregulated in K + -starved plants. This high-affinity Cs + uptake shares features with high-affinity K + uptake. It is sensitive to NH 4 + and insensitive to Ba 2+ and Ca 2+ and its presence parallels the pattern of SlHAK5 expression. Moreover, blockers of reactive oxygen species and ethylene action repress SlHAK5 and negatively regulate both high-affinity K + and Cs + uptake. Thus, we propose that SlHAK5 contributes to Cs + uptake from micromolar concentrations in tomato plants and can constitute a pathway for radiocesium transfer from contaminated areas to the food chain. © 2017 Scandinavian Plant Physiology Society.

  13. Template CoMFA Generates Single 3D-QSAR Models that, for Twelve of Twelve Biological Targets, Predict All ChEMBL-Tabulated Affinities

    PubMed Central

    Cramer, Richard D.

    2015-01-01

    The possible applicability of the new template CoMFA methodology to the prediction of unknown biological affinities was explored. For twelve selected targets, all ChEMBL binding affinities were used as training and/or prediction sets, making these 3D-QSAR models the most structurally diverse and among the largest ever. For six of the targets, X-ray crystallographic structures provided the aligned templates required as input (BACE, cdk1, chk2, carbonic anhydrase-II, factor Xa, PTP1B). For all targets including the other six (hERG, cyp3A4 binding, endocrine receptor, COX2, D2, and GABAa), six modeling protocols applied to only three familiar ligands provided six alternate sets of aligned templates. The statistical qualities of the six or seven models thus resulting for each individual target were remarkably similar. Also, perhaps unexpectedly, the standard deviations of the errors of cross-validation predictions accompanying model derivations were indistinguishable from the standard deviations of the errors of truly prospective predictions. These standard deviations of prediction ranged from 0.70 to 1.14 log units and averaged 0.89 (8x in concentration units) over the twelve targets, representing an average reduction of almost 50% in uncertainty, compared to the null hypothesis of “predicting” an unknown affinity to be the average of known affinities. These errors of prediction are similar to those from Tanimoto coefficients of fragment occurrence frequencies, the predominant approach to side effect prediction, which template CoMFA can augment by identifying additional active structural classes, by improving Tanimoto-only predictions, by yielding quantitative predictions of potency, and by providing interpretable guidance for avoiding or enhancing any specific target response. PMID:26065424

  14. Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

    PubMed

    Venkataramani, Varun; Kardorff, Markus; Herrmannsdörfer, Frank; Wieneke, Ralph; Klein, Alina; Tampé, Robert; Heilemann, Mike; Kuner, Thomas

    2018-04-03

    With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

  15. H 2-saturation of high affinity H 2-oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups

    DOE PAGES

    Piché-Choquette, Sarah; Tremblay, Julien; Tringe, Susannah G.; ...

    2016-03-10

    Soil microbial communities are continuously exposed to H 2 diffusing into the soil from the atmosphere. N 2-fixing nodules represent a peculiar microniche in soil where H 2 can reach concentrations up to 20,000 fold higher than in the global atmosphere (0.530 ppmv). In this study, we investigated the impact of H 2 exposure on soil bacterial community structure using dynamic microcosm chambers simulating soil H 2 exposure from the atmosphere and N 2-fixing nodules. Biphasic kinetic parameters governing H 2 oxidation activity in soil changed drastically upon elevated H 2 exposure, corresponding to a slight but significant decay ofmore » high affinity H 2-oxidizing bacteria population, accompanied by an enrichment or activation of microorganisms displaying low-affinity for H 2. In contrast to previous studies that unveiled limited response by a few species, the relative abundance of 958 bacterial ribotypes distributed among various taxonomic groups, rather than a few distinct taxa, was influenced by H 2 exposure. Furthermore, correlation networks showed important alterations of ribotype covariation in response to H 2 exposure, suggesting that H 2 affects microbe-microbe interactions in soil. Taken together, our results demonstrate that H 2-rich environments exert a direct influence on soil H 2-oxidizing bacteria in addition to indirect effects on other members of the bacterial communities.« less

  16. H2-saturation of high affinity H2-oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups

    PubMed Central

    Piché-Choquette, Sarah; Tremblay, Julien; Tringe, Susannah G.

    2016-01-01

    Soil microbial communities are continuously exposed to H2 diffusing into the soil from the atmosphere. N2-fixing nodules represent a peculiar microniche in soil where H2 can reach concentrations up to 20,000 fold higher than in the global atmosphere (0.530 ppmv). In this study, we investigated the impact of H2 exposure on soil bacterial community structure using dynamic microcosm chambers simulating soil H2 exposure from the atmosphere and N2-fixing nodules. Biphasic kinetic parameters governing H2 oxidation activity in soil changed drastically upon elevated H2 exposure, corresponding to a slight but significant decay of high affinity H2-oxidizing bacteria population, accompanied by an enrichment or activation of microorganisms displaying low-affinity for H2. In contrast to previous studies that unveiled limited response by a few species, the relative abundance of 958 bacterial ribotypes distributed among various taxonomic groups, rather than a few distinct taxa, was influenced by H2 exposure. Furthermore, correlation networks showed important alterations of ribotype covariation in response to H2 exposure, suggesting that H2 affects microbe-microbe interactions in soil. Taken together, our results demonstrate that H2-rich environments exert a direct influence on soil H2-oxidizing bacteria in addition to indirect effects on other members of the bacterial communities. PMID:26989620

  17. Towards a tunable tautomeric switch in azobenzene biomimetics: implications for the binding affinity of 2-(4'-hydroxyphenylazo)benzoic acid to streptavidin.

    PubMed

    Farrera, Joan-Antoni; Canal, Ivan; Hidalgo-Fernández, Pedro; Pérez-García, M Lluïsa; Huertas, Oscar; Luque, F Javier

    2008-01-01

    The tautomeric equilibria of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) and 2-(3',5'-dimethyl-4'-hydroxyphenylazo)benzoic acid (3',5'-dimethyl-HABA) have been studied by a combination of spectroscopic and computational methods. For neutral HABA in solvents of different polarity (toluene, chloroform, DMSO, DMF, butanol, and ethanol) the azo tautomer (AT) is largely predominant. For monoanionic HABA, the hydrazone tautomer (HT) is the only detected species in apolar solvents such as toluene and chloroform, while the AT is the only detected species in water and a mixture of both tautomers is detected in ethanol. Comparison of the results obtained for HABA and its 3',5'-dimethylated derivative shows that dimethylation of the hydroxybenzene ring shifts the tautomeric preferences towards the hydrazone species. These findings have been used to examine the differences in binding affinity to streptavidin, as the lower affinity of HABA can be explained in terms of the larger energetic cost associated with the tautomeric shift to the bioactive hydrazone species. Overall, these results suggest that a balanced choice of chemical substituents, embedding environment, and pH can be valuable for exploitation of the azo-hydrazone tautomerism of HABA biomimetics in biotechnological applications.

  18. How Structure Defines Affinity in Protein-Protein Interactions

    PubMed Central

    Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

    2014-01-01

    Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

  19. Contextualizing Informal Labeling Effect on Adolescent Recidivism in South Korea.

    PubMed

    Lee, Jonathan

    2017-08-01

    Symbolic interactionism argues that the effect of informal labeling by general others, such as family and friends, on behavior depends on the social context under which labeling takes place. Despite abundant research on informal labeling, little effort has been made to contextualize its impact on adolescent reoffending. Also, compared with other theories, only a few studies have been conducted among youths in Asian population. Using three consecutive waves of self-reported survey data from a nationally representative sample of 2,406 Korean adolescents, this study examined an interactional model for the informal labeling effect. Findings suggest that informal labeling, as well as school commitment and delinquent peer association, has an independent effect on delinquency. Also supported is the symbolic interactionist hypothesis that adolescents with greater involvement in delinquent subcultures were less susceptible to informal labeling. Implications of the findings are discussed.

  20. Synthesis of Cryptophycin Affinity Labels and Tubulin Labeling

    DTIC Science & Technology

    2006-05-01

    Nostoc sp.), are a new and potent tumor-selective class of tubulin-binding antimitotic agents1 that show excellent activity against MDR cancer cell...lines and were exceptionally active against mammary derived tumors.2,3 Cryptophycin-1 (1, Fig. 1) is the major cytotoxin in Nostoc sp.4,5 and...arenastatin A), isolated from the Okinawan marine sponge Dysidea arenaria6 and later from Nostoc sp. strain GSV 224,7 is also a potent inhibitor of tubulin

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