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Sample records for amplicon peptide display

  1. Peptide Amyloid Surface Display

    PubMed Central

    2015-01-01

    Homomeric self-assembly of peptides into amyloid fibers is a feature of many diseases. A central role has been suggested for the lateral fiber surface affecting gains of toxic function. To investigate this, a protein scaffold that presents a discrete, parallel β-sheet surface for amyloid subdomains up to eight residues in length has been designed. Scaffolds that present the fiber surface of islet amyloid polypeptide (IAPP) were prepared. The designs show sequence-specific surface effects apparent in that they gain the capacity to attenuate rates of IAPP self-assembly in solution and affect IAPP-induced toxicity in insulin-secreting cells. PMID:25541905

  2. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    NASA Astrophysics Data System (ADS)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  3. [Peptide phage display in biotechnology and biomedicine].

    PubMed

    Kuzmicheva, G A; Belyavskaya, V A

    2016-07-01

    To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

  4. Recognition of epoxy with phage displayed peptides.

    PubMed

    Swaminathan, Swathi; Cui, Yue

    2013-07-01

    The development of a general approach for non-destructive chemical and biological functionalization of epoxy could expand opportunities for both fundamental studies and creating various device platforms. Epoxy shows unique electrical, mechanical, chemical and biological compatibility and has been widely used for fabricating a variety of devices. Phage display has emerged as a powerful method for selecting peptides that possess enhanced selectivity and binding affinity toward a variety of targets. In this letter, we demonstrate for the first time a powerful yet benign approach for identifying binding motifs to epoxy via comprehensively screened phage displayed peptides. Our results show that the epoxy can be selectively recognized with peptide-displaying phages. Further, along with the development of epoxy-based microstructures; recognition of the epoxy with phage displayed peptides can be specifically localized in these microstructures. We anticipate that these results could open up exciting opportunities in the use of peptide-recognized epoxy in fundamental biochemical recognition studies, as well as in applications ranging from analytical devices, hybrid materials, surface and interface, to cell biology.

  5. Strategies for Vaccine Design Using Phage Display-Derived Peptides.

    PubMed

    Goulart, Luiz R; Santos, Paula de S

    2016-01-01

    Development of peptide vaccines through the phage display technology is a powerful strategy that relies on short peptides expressed in the phage capsid surface to induce highly targeted immune responses. Phage display-derived immunogenic peptides can be used directly as a phage-fused peptide reagent or as a synthetic peptide with specific modifications, according to target molecule and disease pathogen/parasite. Peptides' selection (mimotopes) can be performed against monoclonal or polyclonal antibodies to disclose determinant regions (epitopes) that can induce a neutralizing response. Validations of mimotopes are performed in vitro and in vivo, based on cell culture and animal models, to demonstrate its immunogenic potential for final vaccine formulations with an appropriate adjuvant. Here we present specific methods for the discovery of novel immunogenic peptides based on phage display.

  6. Combinatorial discovery of tumor targeting peptides using phage display.

    PubMed

    Landon, Linda A; Deutscher, Susan L

    2003-10-15

    Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents. Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available. Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting. Novel tumor-avid peptides can be efficiently identified via affinity selections using complex random peptide libraries containing millions of peptides that are displayed on bacteriophage. In vitro and in situ affinity selections may be used to identify peptides with high affinity for the target antigen in vitro. Unfortunately, it has been found that peptides selected in vitro or in situ may not effectively target tumors in vivo due to poor peptide stability and other problems. To improve in vivo targeting, methodological combinatorial chemistry innovations allow selections to be conducted in the environment of the whole animal. Thus, new targeting peptides with optimal in vivo properties can be selected in vivo in tumor-bearing animals. In vivo selections have been proven successful in identifying peptides that target the vasculature of specific organs. In addition, in vivo selections have identified peptides that bind specifically to the surface of or are internalized into tumor cells. In the future, direct selection of peptides for cancer imaging may be expedited using genetically engineered bacteriophage libraries that encode peptides with intrinsic radiometal-chelation or fluorescent sequences.

  7. Selection of peptide entry motifs by bacterial surface display.

    PubMed Central

    Taschner, Sabine; Meinke, Andreas; von Gabain, Alexander; Boyd, Aoife P

    2002-01-01

    Surface display technologies have been established previously to select peptides and polypeptides that interact with purified immobilized ligands. In the present study, we designed and implemented a surface display-based technique to identify novel peptide motifs that mediate entry into eukaryotic cells. An Escherichia coli library expressing surface-displayed peptides was combined with eukaryotic cells and the gentamicin protection assay was performed to select recombinant E. coli, which were internalized into eukaryotic cells by virtue of the displayed peptides. To establish the proof of principle of this approach, the fibronectin-binding motifs of the fibronectin-binding protein A of Staphylococcus aureus were inserted into the E. coli FhuA protein. Surface expression of the fusion proteins was demonstrated by functional assays and by FACS analysis. The fibronectin-binding motifs were shown to mediate entry of the bacteria into non-phagocytic eukaryotic cells and brought about the preferential selection of these bacteria over E. coli expressing parental FhuA, with an enrichment of 100000-fold. Four entry sequences were selected and identified using an S. aureus library of peptides displayed in the FhuA protein on the surface of E. coli. These sequences included novel entry motifs as well as integrin-binding Arg-Gly-Asp (RGD) motifs and promoted a high degree of bacterial entry. Bacterial surface display is thus a powerful tool to effectively select and identify entry peptide motifs. PMID:12144529

  8. Chemical posttranslational modification of phage-displayed peptides.

    PubMed

    Ng, Simon; Tjhung, Katrina F; Paschal, Beth M; Noren, Christopher J; Derda, Ratmir

    2015-01-01

    Phage-displayed peptide library has fueled the discovery of novel ligands for diverse targets. A new type of phage libraries that displays not only linear and disulfide-constrained cyclic peptides but moieties that cannot be encoded genetically or incorporated easily by bacterial genetic machinery has emerged recently. Chemical posttranslational modification of phage library is one of the simplest approaches to encode nonnatural moieties. It confers the library with new functionality and makes it possible to select and evolve molecules with properties not found in the peptides, for instance, glycopeptides recognized by carbohydrate-binding protein and peptides with photoswitching capability. To this end, we describe the newly emerging techniques to chemically modify the phage library and quantify the efficiency of the reaction with a biotin-capture assay. Finally, we provide the methods to construct N-terminal Ser peptide library that allows site-selective modification of phage.

  9. Peptide inhibitors of botulinum neurotoxin by mRNA display

    SciTech Connect

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H. . E-mail: yangdc@georgetown.edu

    2005-10-07

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs.

  10. Identification of gliadin-binding peptides by phage display

    PubMed Central

    2011-01-01

    Background Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products. Results Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin. Conclusions We believe that several of the isolated and

  11. Phage display screen for peptides that bind Bcl-2 protein.

    PubMed

    Park, Hye-Yeon; Kim, Joungmok; Cho, June-Haeng; Moon, Ji Young; Lee, Su-Jae; Yoon, Moon-Young

    2011-01-01

    Bcl-2 family proteins are key regulators of apoptosis associated with human disease, including cancer. Bcl-2 protein has been found to be overexpressed in many cancer cells. Therefore, Bcl-2 protein is a potential diagnostic target for cancer detection. In the present study, the authors have identified several Bcl-2 binding peptides with high affinity (picomolar range) from a 5-round M13 phage display library screening. These peptides can be used to develop novel diagnostic probes or potent inhibitors with diverse polyvalencies.

  12. Development of bacterial display peptides for use in biosensing applications

    NASA Astrophysics Data System (ADS)

    Stratis-Cullum, Dimitra N.; Kogot, Joshua M.; Sellers, Michael S.; Hurley, Margaret M.; Sarkes, Deborah A.; Pennington, Joseph M.; Val-Addo, Irene; Adams, Bryn L.; Warner, Candice R.; Carney, James P.; Brown, Rebecca L.; Pellegrino, Paul M.

    2012-06-01

    Recent advances in synthetic library engineering continue to show promise for the rapid production of reagent technology in response to biological threats. A synthetic library of peptide mutants built off a bacterial host offers a convenient means to link the peptide sequence, (i.e., identity of individual library members) with the desired molecular recognition traits, but also allows for a relatively simple protocol, amenable to automation. An improved understanding of the mechanisms of recognition and control of synthetic reagent isolation and evolution remain critical to success. In this paper, we describe our approach to development of peptide affinity reagents based on peptide bacterial display technology with improved control of binding interactions for stringent evolution of reagent candidates, and tailored performance capabilities. There are four key elements to the peptide affinity reagent program including: (1) the diverse bacterial library technology, (2) advanced reagent screening amenable to laboratory automation and control, (3) iterative characterization and feedback on both affinity and specificity of the molecular interactions, and (3) integrated multiscale computational prescreening of candidate peptide ligands including in silico prediction of improved binding performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be presented. Recent highlights of on cell vs. off-cell affinity behavior and correlation of the results with advanced docking simulations on the protein-peptide system(s) are included. The potential of this technology and approach to enable rapid development of a new affinity reagent with unprecedented speed (less than one week) would allow for rapid response to new and constantly emerging threats.

  13. Identification of a novel skin penetration enhancement peptide by phage display peptide library screening.

    PubMed

    Kumar, Sunny; Sahdev, Preety; Perumal, Omathanu; Tummala, Hemachand

    2012-05-07

    Skin is an important site for local or systemic application of drugs. However, a majority of drugs have poor permeability through the skin's topmost layer, stratum corneum (SC). The aim of this study was to identify safe and smaller peptides that could enhance the skin penetration of drug molecules. By screening phage display peptide library, we have identified a T2 peptide (LVGVFH), which enhanced the penetration of bacteriophages (~800 nm long bacterial viruses) across porcine and mouse skin. Pretreating the skin with synthetic T2 peptide at pH 4.5 resulted in significant penetration enhancement of hydrophilic drug 5-fluorouracil (5-FU) across skin. FTIR spectroscopy showed that the T2 peptide interacted with skin lipids to enhance the skin penetration. Pretreating the skin with T2 peptide enhanced the partitioning of small molecules with different lipophilicities (5-FU, fluorescein isothiocyanate, and rhodamine 123 hydrochloride) into skin. Fluorescence studies showed that T2 peptide enhanced the diffusion of these molecules into intercellular lipids of SC and thus enhanced the penetration into the skin. Histidine at the c-terminus of T2 peptide was identified to be critical for the skin penetration enhancement. T2 peptide interacted with skin lipids to cause skin penetration enhancement. The study identified a novel, safe, and noninvasive peptide to improve the skin penetration of drugs without chemical conjugation.

  14. Advancement and applications of peptide phage display technology in biomedical science.

    PubMed

    Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

    2016-01-19

    Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

  15. Phage-displayed peptide library screening for preferred human substrate peptide sequences for transglutaminase 7.

    PubMed

    Kuramoto, Katsuma; Yamasaki, Risa; Shimizu, Yoshitaka; Tatsukawa, Hideki; Hitomi, Kiyotaka

    2013-09-01

    Transglutaminases are a family of enzymes that catalyze cross-linking reactions between proteins. Among the members, there is currently no information regarding the substrate preferences of transglutaminase 7 (TG7), that would clarify its physiological significance. We previously obtained several highly reactive substrate peptide sequences of transglutaminases from a random peptide library. In this study, we screened for preferred substrate sequences for TG7 from a phage-displayed 12-mer peptide library. The most preferred sequence was selected based on reactivity and isozyme specificity. We firstly exhibited the tendency for the preference of substrate sequence for TG7. Then, using the most efficient peptide, Z3S, we established an in vitro assay system to assess enzymatic activity of TG7.

  16. Screening of a specific peptide binding to esophageal squamous carcinoma cells from phage displayed peptide library.

    PubMed

    Ma, Caixia; Li, Chunyan; Jiang, Dongliang; Gao, Xiaojie; Han, Juanjuan; Xu, Nan; Wu, Qiong; Nie, Guochao; Chen, Wei; Lin, Fenghuei; Hou, Yingchun

    2015-06-01

    To select a specifically binding peptide for imaging detection of human esophageal squamous cell carcinoma (ESCC), a phage-displayed 12-mer peptide library was used to screen the peptide that bind to ESCC cells specifically. After four rounds of bio-panning, the phage recovery rate gradually increased, and specific phage clones were effectively enriched. The 60 randomly selected phage clones were tested using cellular enzyme-linked immunosorbent assay (ELISA), and 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. From the sequencing results of the positive clones, 14 peptide sequences were obtained and ESCP9 consensus sequence was identified as the peptide with best affinity to ESCC cells via competitive inhibition, fluorescence microscopy, and flow cytometry. The results indicate that the peptide ESCP9 can bind to ESCC cells specifically and sensitively, and it is a potential candidate to be developed as an useful molecule to the imaging detection and targeting therapy for ESCC.

  17. Trastuzumab-binding peptide display by Tobacco mosaic virus

    SciTech Connect

    Frolova, Olga Y.; Petrunia, Igor V.; Komarova, Tatiana V.; Kosorukov, Vyacheslav S.; Sheval, Eugene V.; Gleba, Yuri Y.; Dorokhov, Yuri L.

    2010-11-10

    Human epidermal growth factor receptor-2 (HER2/neu) is a target for the humanized monoclonal antibody trastuzumab. Recently, trastuzumab-binding peptides (TBP) of HER2/neu that inhibit proliferation of breast cancer cells were identified. We have now studied conditions of efficient assembly in vivo of Tobacco mosaic virus (TMV)-based particles displaying TBP on its surface. The system is based on an Agrobacterium-mediated co-delivery of binary vectors encoding TMV RNA and coat protein (CP) with TBP in its C-terminal extension into plant leaves. We show how the fusion of amino acid substituted TBP (sTBP) to CP via a flexible peptide linker can improve the manufacturability of recombinant TMV (rTMV). We also reveal that rTMV particles with exposed sTBP retained trastuzumab-binding capacity but lost an anti-HER2/neu immunogenic scaffold function. Mouse antibodies against rTMV did not recognize HER2/neu on surface of human SK-BR-3 cells.

  18. Genetically engineered peptides for inorganics: study of an unconstrained bacterial display technology and bulk aluminum alloy.

    PubMed

    Adams, Bryn L; Finch, Amethist S; Hurley, Margaret M; Sarkes, Deborah A; Stratis-Cullum, Dimitra N

    2013-09-06

    The first-ever peptide biomaterial discovery using an unconstrained engineered bacterial display technology is reported. Using this approach, we have developed genetically engineered peptide binders for a bulk aluminum alloy and use molecular dynamics simulation of peptide conformational fluctuations to demonstrate sequence-dependent, structure-function relationships for metal and metal oxide interactions.

  19. Isolation and characterization of anti-SEB peptides using magnetic sorting and bacterial peptide display library technology

    NASA Astrophysics Data System (ADS)

    Pennington, Joseph M.; Kogot, Joshua M.; Sarkes, Deborah A.; Pellegrino, Paul M.; Stratis-Cullum, Dimitra N.

    2012-06-01

    Peptide display libraries offer an alternative method to existing antibody development methods enabling rapid isolation of highly stable reagents for detection of new and emerging biological threats. Bacterial display libraries are used to isolate new peptide reagents within 1 week, which is simpler and timelier than using competing display library technology based on phage or yeast. Using magnetic sorting methods, we have isolated peptide reagents with high affinity and specificity to staphylococcal enterotoxin B (SEB), a suspected food pathogen. Flow cytometry methods were used for on-cell characterization and the binding affinity (Kd) of this new peptide reagent was determined to be 56 nm with minimal cross-reactivity to other proteins. These results demonstrated that magnetic sorting for new reagents using bacterial display libraries is a rapid and effective method and has the potential for current and new and emerging food pathogen targets.

  20. Isolation and Characterization of Anti-SEB Peptides Using Magnetic Sorting and Bacterial Peptide Display Library Technology

    DTIC Science & Technology

    2012-09-01

    than using competing display library technology based on phage or yeast. Using magnetic sorting methods, we have isolated peptide reagents with high...characterization and the binding affinity (Kd) of this new peptide reagent was determined to be 56 nm with minimal cross- reactivity to other proteins . These...library technology based on phage or yeast. Using magnetic sorting methods, we have isolated peptide reagents with high affinity and specificity to

  1. Attaching the phage display-selected GLA peptide to liposomes: factors influencing target binding.

    PubMed

    van Rooy, Inge; Hennink, Wim E; Storm, Gert; Schiffelers, Raymond M; Mastrobattista, Enrico

    2012-02-14

    In our previous study, phage display selections were performed by in situ perfusion of a random peptide library through a mouse brain. This yielded two peptides (GLA and GYR) that showed significant binding to human brain endothelial cells (hCMEC/D3) when displayed on phage particles, but not to human umbilical vein endothelial cells (HUVECs). In the present study, these peptides were produced synthetically and coupled to liposomes to investigate the capacity of the peptides to act as ligands for targeting to hCMEC/D3 cells. Flow cytometry studies showed that these peptides when coupled to liposomes showed weak binding to the target brain endothelial cells. We hypothesized that the weak endothelial cell binding of the selected peptides when coupled to liposomes as compared to the binding of the peptides displayed on phage particles may be ascribed to: change of vehicle shape, change of peptide density, or change of peptide conformation. Peptide density on the liposomes influenced binding of the liposomes to the cells, however, this effect was minor. To study the influence of the peptide conformation, the GLA peptide was recombinantly produced fused to the N1-N2 domains of the phage p3 minor coat protein (p3-GLA) to mimic its conformation when displayed on phage particles. Binding of liposomes modified with either the GLA peptide or the p3-GLA protein to hCMEC/D3 cells was studied, and the p3-GLA-liposomes showed a higher binding to the cells compared to the GLA-liposomes. The experiments demonstrate that bringing the GLA peptide into the original phage protein environment restores and improves the peptide binding capacity and suggest that the GLA peptide, with some modifications, may be used as a brain-targeting ligand in the future.

  2. Phage displayed peptide recognizing porcine aminopeptidase N is a potent small molecule inhibitor of PEDV entry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by po...

  3. Analysis of protective antigen peptide binding motifs using bacterial display technology

    NASA Astrophysics Data System (ADS)

    Sarkes, Deborah A.; Dorsey, Brandi L.; Stratis-Cullum, Dimitra N.

    2015-05-01

    In today's fast-paced world, a new biological threat could emerge at any time, necessitating a prompt, reliable, inexpensive detection reagent in each case. Combined with magnetic-activated cell sorting (MACS), bacterial display technology makes it possible to isolate selective, high affinity peptide reagents in days to weeks. Utilizing the eCPX display scaffold is also a rapid way to screen potential peptide reagents. Peptide affinity reagents for protective antigen (PA) of the biothreat Bacillus anthracis were previously discovered using bacterial display. Bioinformatics analysis resulted in the consensus sequence WXCFTC. Additionally, we have discovered PA binding peptides with a WW motif, one of which, YGLHPWWKNAPIGQR, can pull down PA from 1% human serum. The strength of these two motifs combined, to obtain a WWCFTC consensus, is assessed here using Fluorescence Activated Cell Sorting (FACS). While monitoring binding to PA, overall expression of the display scaffold was assessed using the YPet Mona expression control tag (YPet), and specificity was assessed by binding to Streptavidin R-Phycoerythrin (SAPE). The importance of high YPet binding is highlighted as many of the peptides in one of the three replicate experiments fell below our 80% binding threshold. We demonstrate that it is preferable to discard this experiment, due to questionable expression of the peptide itself, than to try to normalize for relative expression. The peptides containing the WWCFTC consensus were of higher affinity and greater specificity than the peptides containing the WW consensus alone, validating further investigation to optimize known PA binders.

  4. Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening.

    PubMed

    Lee, Kyoung Jin; Lee, Jae Hee; Chung, Hye Kyung; Choi, Jinhyang; Park, Jaesook; Park, Seok Soon; Ju, Eun Jin; Park, Jin; Shin, Seol Hwa; Park, Hye Ji; Ko, Eun Jung; Suh, Nayoung; Kim, InKi; Hwang, Jung Jin; Song, Si Yeol; Jeong, Seong-Yun; Choi, Eun Kyung

    2015-02-01

    Discovery of the cancer-specific peptidic ligands have been emphasized for active targeting drug delivery system and non-invasive imaging. For the discovery of useful and applicable peptidic ligands, in vivo peptide-displayed phage screening has been performed in this study using a xenograft mouse model as a mimic microenvironment to tumor. To seek human lung cancer-specific peptides, M13 phage library displaying 2.9 × 10(9) random peptides was intravenously injected into mouse model bearing A549-derived xenograft tumor through the tail vein. Then the phages emerged from a course of four rounds of biopanning in the xenograft tumor tissue. Novel peptides were categorized into four groups according to a sequence-homology phylogenicity, and in vivo tumor-targeting capacity of these peptides was validated by whole body imaging with Cy5.5-labeled phages in various cancer types. The result revealed that novel peptides accumulated only in adenocarcinoma lung cancer cell-derived xenograft tissue. For further confirmation of the specific targeting ability, in vitro cell-binding assay and immunohistochemistry in vivo tumor tissue were performed with a selected peptide. The peptide was found to bind intensely to lung cancer cells both in vitro and in vivo, which was efficiently compromised with unlabeled phages in an in vitro competition assay. In conclusion, the peptides specifically targeting human lung cancer were discovered in this study, which is warranted to provide substantive feasibilities for drug delivery and imaging in terms of a novel targeted therapeutics and diagnostics.

  5. Is phage display technology on target for developing peptide-based cancer drugs?

    PubMed

    Landon, Linda A; Zou, Jun; Deutscher, Susan L

    2004-06-01

    New tumor targeting agents are required to advance cancer diagnosis and treatment. Bacteriophage (phage) display technology, a molecular genetic means of combinatorial drug discovery, is an emerging approach to identify and improve peptide molecules as pharmaceuticals. Peptides are thought to have clinically desirable benefits over currently used biomolecules, such as antibodies, because of their rapid blood clearance, increased diffusion and tissue penetration, non-immunogenic nature and ease of synthesis. Using phage display, one can rapidly and simultaneously survey billion-clone peptide libraries, resulting in large numbers of "hits". However, only a few lead compounds resulting from the hits historically reach the drug market. Hence determining which peptide may best translate into a useful drug is of particular importance. Examination of successfully marketed drugs has highlighted key features of a winning agent, including low molecular weight, high affinity, stability, solubility, lipophilicity and conformational rigidity. Although peptide modulators of tumor cell function and cancer targeting agents have been developed, the majority of peptide-based drugs reported thus far are immune and cardiac regulators. In this review, we will highlight how phage display has been employed to isolate peptides that target key steps in cancer progression--from tumor growth to metastasis--and how phage display technology can be harnessed to select a priori peptides with inherent features essential for anti-cancer drug efficacy. In 2003, phage display provided us with several novel peptides not only in clinical trials but approved by the FDA for use as therapeutics in a variety of diseases--suggesting that the future looks bright for phage display in anti-cancer drug development.

  6. Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries.

    PubMed

    Flachbartova, Z; Pulzova, L; Bencurova, E; Potocnakova, L; Comor, L; Bednarikova, Z; Bhide, M

    2016-01-01

    The aim of the study was to isolate and characterize novel antimicrobial peptides from peptide phage library with antimicrobial activity against multidrug resistant Listeria monocytogenes. Combinatorial phage-display library was used to affinity select peptides binding to the cell surface of multidrug resistant L. monocytogenes. After several rounds of affinity selection followed by sequencing, three peptides were revealed as the most promising candidates. Peptide L2 exhibited features common to antimicrobial peptides (AMPs), and was rich in Asp, His and Lys residues. Peptide L3 (NSWIQAPDTKSI), like peptide L2, inhibited bacterial growth in vitro, without any hemolytic or cytotoxic effects on eukaryotic cells. L1 peptide showed no inhibitory effect on Listeria. Structurally, peptides L2 and L3 formed random coils composed of α-helix and β-sheet units. Peptides L2 and L3 exhibited antimicrobial activity against multidrug resistant isolates of L. monocytogenes with no haemolytic or toxic effects. Both peptides identified in this study have the potential to be beneficial in human and veterinary medicine.

  7. Selection of phage-displayed peptides that bind to a particular ligand-bound antibody.

    PubMed

    Tanaka, Fujie; Hu, Yunfeng; Sutton, Jori; Asawapornmongkol, Lily; Fuller, Roberta; Olson, Arthur J; Barbas, Carlos F; Lerner, Richard A

    2008-06-01

    Phage-displayed peptides that selectively bind to aldolase catalytic antibody 93F3 when bound to a particular 1,3-diketone hapten derivative have been developed using designed selection strategies with libraries containing 7-12 randomized amino acid residues. These phage-displayed peptides discriminated the particular 93F3-diketone complex from ligand-free 93F3 and from 93F3 bound to other 1,3-diketone hapten derivatives. By altering the selection procedures, phage-displayed peptides that bind to antibody 93F3 in the absence of 1,3-diketone hapten derivatives have also been developed. With using these phage-displayed peptides, ligand-bound states of the antibody were distinguished from each other. A docking model of one of the peptides bound to the antibody 93F3-diketone complex was created using a sequential divide-and-conquer peptide docking strategy; the model suggests that the peptide interacts with both the antibody and the ligand through a delicate hydrogen bonding network.

  8. Peptide phage display as a tool for drug discovery: targeting membrane receptors.

    PubMed

    Molek, Peter; Strukelj, Borut; Bratkovic, Tomaz

    2011-01-21

    Ligands selected from phage-displayed random peptide libraries tend to be directed to biologically relevant sites on the surface of the target protein. Consequently, peptides derived from library screenings often modulate the target protein's activity in vitro and in vivo and can be used as lead compounds in drug design and as alternatives to antibodies for target validation in both genomics and drug discovery. This review discusses the use of phage display to identify membrane receptor modulators with agonistic or antagonistic activities. Because isolating or producing recombinant membrane proteins for use as target molecules in library screening is often impossible, innovative selection strategies such as panning against whole cells or tissues, recombinant receptor ectodomains, or neutralizing antibodies to endogenous binding partners were devised. Prominent examples from a two-decade history of peptide phage display will be presented, focusing on the design of affinity selection experiments, methods for improving the initial hits, and applications of the identified peptides.

  9. Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

    PubMed Central

    Toledo-Machado, Christina Monerat; Machado de Avila, Ricardo Andrez; NGuyen, Christophe; Granier, Claude; Bueno, Lilian Lacerda; Carneiro, Claudia Martins; Menezes-Souza, Daniel; Carneiro, Rubens Antonio; Chávez-Olórtegui, Carlos; Fujiwara, Ricardo Toshio

    2015-01-01

    ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL. PMID:25710003

  10. Phage display as a technology delivering on the promise of peptide drug discovery.

    PubMed

    Hamzeh-Mivehroud, Maryam; Alizadeh, Ali Akbar; Morris, Michael B; Church, W Bret; Dastmalchi, Siavoush

    2013-12-01

    Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries can be displayed on the phage surface. The phage library can be incubated with a target of interest and the phage which bind can be isolated and sequenced to reveal the displayed peptides' primary structure. In this review, we focus on the 'mechanics' of the phage display process, whilst highlighting many diverse and subtle ways it has been used to further the drug-development process, including the potential for the phage particle itself to be used as a drug carrier targeted to a particular pathogen or cell type in the body.

  11. Construction of a high efficiency copper adsorption bacterial system via peptide display and its application on copper dye polluted wastewater.

    PubMed

    Maruthamuthu, Murali Kannan; Nadarajan, Saravanan Prabhu; Ganesh, Irisappan; Ravikumar, Sambandam; Yun, Hyungdon; Yoo, Ik-Keun; Hong, Soon Ho

    2015-11-01

    For the construction of an efficient copper waste treatment system, a cell surface display strategy was employed. The copper adsorption ability of recombinant bacterial strains displaying three different copper binding peptides were evaluated in LB Luria-Bertani medium (LB), artificial wastewater, and copper phthalocyanine containing textile dye industry wastewater samples. Structural characteristics of the three peptides were also analyzed by similarity-based structure modeling. The best binding peptide was chosen for the construction of a dimeric peptide display and the adsorption ability of the monomeric and dimeric peptide displayed strains were compared. The dimeric peptide displayed strain showed superior copper adsorption in all three tested conditions (LB, artificial wastewater, and textile dye industry wastewater). When the strains were exposed to copper phthalocyanine dye polluted wastewater, the dimeric peptide display [543.27 µmol/g DCW dry cell weight (DCW)] showed higher adsorption of copper when compared with the monomeric strains (243.53 µmol/g DCW).

  12. From phage display to nanoparticle delivery: functionalizing liposomes with multivalent peptides improves targeting to a cancer biomarker.

    PubMed

    Gray, Bethany Powell; Li, Shunzi; Brown, Kathlynn C

    2013-01-16

    Phage display is commonly used to isolate peptides that bind to a desired cell type. While chemical synthesis of selected peptides often results in ligands with low affinity, a multivalent tetrameric presentation of the peptides dramatically improves affinity. One of the primary uses of these peptides is conjugation to nanoparticle-based therapeutics for specific delivery to target cell types. We set out to optimize the path from phage display peptide selection to peptide presentation on a nanoparticle surface for targeted delivery. Here, we examine the effects of peptide valency, density, and affinity on nanoparticle delivery and therapeutic efficacy, using the α(v)β(6)-specific H2009.1 peptide as a model phage-selected peptide and liposomal doxorubicin as a model therapeutic nanoparticle. Liposomes displaying the higher affinity multivalent H2009.1 tetrameric peptide demonstrate 5-10-fold higher drug delivery than liposomes displaying the lower affinity monomeric H2009.1 peptide, even when the same number of peptide subunits are displayed on the liposome. Importantly, a 6-fold greater toxicity is observed toward α(v)β(6)-expressing cells for liposomes displaying tetrameric verses monomeric H2009.1 peptides. Additionally, liposomal targeting and toxicity increase with increasing concentrations of H2009.1 tetrameric peptide on the liposome surface. Thus, both the multivalent peptide and the multivalent liposome scaffold work together to increase targeting to α(v)β(6)-expressing cells. This multilayered approach to developing high affinity targeted nanoparticles may improve the utility of moderate affinity peptides. As tetramerization is known to increase affinity for a variety of phage-selected peptides, it is anticipated that the tetrameric scaffold may act as a general method for taking peptides from phage display to nanoparticle display.

  13. An in vitro polysome display system for identifying ligands from very large peptide libraries.

    PubMed Central

    Mattheakis, L C; Bhatt, R R; Dower, W J

    1994-01-01

    We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system. Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems. Images PMID:7522328

  14. In vitro affinity screening of protein and peptide binders by megavalent bead surface display.

    PubMed

    Diamante, Letizia; Gatti-Lafranconi, Pietro; Schaerli, Yolanda; Hollfelder, Florian

    2013-10-01

    The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead K(d) measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

  15. Exploiting bacterial peptide display technology to engineer biomaterials for neural stem cell culture.

    PubMed

    Little, Lauren E; Dane, Karen Y; Daugherty, Patrick S; Healy, Kevin E; Schaffer, David V

    2011-02-01

    Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering.

  16. MULTIVALENT DISPLAY OF PENDANT PRO-APOPTOTIC PEPTIDES INCREASES CYTOTOXIC ACTIVITY

    PubMed Central

    Chu, David S.H.; Bocek, Michael J.; Shi, Julie; Ta, Anh; Ngambenjawong, Chayanon; Rostomily, Robert C.; Pun, Suzie H.

    2015-01-01

    Several cationic antimicrobial peptides have been investigated as potential anti-cancer drugs due to their demonstrated selective toxicity towards cancer cells relative to normal cells. For example, intracellular delivery of KLA, a pro-apoptotic peptide, results in toxicity against a variety of cancer cell lines; however, the relatively low activity and small size leads to rapid renal excretion when applied in vivo, limiting its therapeutic potential. In this work, apoptotic peptide-polymer hybrid materials were developed to increase apoptotic peptide activity via multivalent display. Multivalent peptide materials were prepared with comb-like structure by RAFT copolymerization of peptide macromonomers with N-(2-hydroxypropyl) methacrylamide (HPMA). Polymers displayed a GKRK peptide sequence for targeting p32, a protein often overexpressed on the surface of cancer cells, either fused with or as a comonomer to a KLA macromonomer. In three tested cancer cell lines, apoptotic polymers were significantly more cytotoxic than free peptides as evidenced by an order of magnitude decrease in IC50 values for the polymers compared to free peptide. The uptake efficiency and intracellular trafficking of one polymer construct was determined by radiolabeling and subcellular fractionation. Despite their more potent cytotoxic profile, polymeric KLA constructs have poor cellular uptake efficiency (<1%). A significant fraction (20%) of internalized constructs localize with intact mitochondrial fractions. In an effort to increase cellular uptake, polymer amines were converted to guanidines by reaction with O-methylisourea. Guanidinylated polymers disrupted function of isolated mitochondria more than their lysine-based analogs, but overall toxicity was decreased, likely due to inefficient mitochondrial trafficking. Thus, while multivalent KLA polymers are more potent than KLA peptides, these materials can be substantially improved by designing next generation materials with improved

  17. Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library

    PubMed Central

    Wu, Yan; Li, Cai-Yun

    2015-01-01

    Objective Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy. Methods The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides. Results After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with Kaff (affinity constant) of 16.4±8.6 µg/mL (n=3), 9.2±2.1 µg/mL (n=3), and 174.8±31.1 µg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed. Conclusion These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer. PMID:26197772

  18. Screening specific polypeptides of breast cancer stem cells from a phage display random peptide library

    PubMed Central

    Liu, Fei; Qi, Chun-Ling; Kong, Mian; Liu, Ting-Ting; Li, Lei; Li, Bao-Jiang

    2016-01-01

    The present study aimed to identify polypeptides that specifically bond to breast cancer stem cells from a phage display random 12 peptide library, in addition to the affinity and specificity of polypeptides. A phage display random 12 peptide library was screened using breast cancer stem cells as targets isolated from the MDA-MB-231 cell line using the serum-free culture technique with hs578bst and MDA-MB-231 cells as subtract-screening cells. Positive and specific binding clones were amplified and sent for sequencing. The affinity and specificity of the positive clones were subsequently identified by ELISA and 3,3′-diaminobenzidine staining. The results demonstrated that phages were gathered ~500 times following three rounds of biopanning. ELISA identified that the affinity to breast cancer stem cells of the no. 6 phage was 6.14 times higher than that in the control group. In addition, immunohistochemistry observed that the no. 6 phage exhibited high-specificity bonding to breast cancer stem cells, and the peptide sequence of the positive phage was GYSASRSTIPGK following DNA sequencing and translation. Thus, the present study isolated a specific peptide that bonds to breast cancer stem cells from a phage display random peptide library, which may facilitate further studies regarding the stem cell-targeted therapy of breast cancer. PMID:28105180

  19. Labeling and distribution of linear peptides identified using in vivo phage display selection for tumors.

    PubMed

    Kennel, S J; Mirzadeh, S; Hurst, G B; Foote, L J; Lankford, T K; Glowienka, K A; Chappell, L L; Kelso, J R; Davern, S M; Safavy, A; Brechbiel, M W

    2000-11-01

    To develop targeting molecules to be used for vascular targeting of short half-lived alpha-emitters for radioimmunotherapy, linear peptide phage display libraries were selected in vivo for binding to IC-12 rat tracheal tumors growing in severe combined immune deficient mice. After three rounds of selection, 15 phage clones were analyzed for DNA sequence, and the deduced translation products of cDNA inserts were compared. Three consensus sequences were chosen from three separate experimental selection series and peptides of these sequences with added -gly-gly-tyr were obtained. Peptides were radiolabeled on tyrosine with (125)I and the biodistribution in tumor-bearing mice was determined. The radioiodinated peptides were stable in vitro and when injected in tumor-bearing mice approximately 3.0 %ID/g accumulated in the tumor; however, much of the (125)I was found in the gastrointestinal tract and thyroid, indicative of dehalogenation of the labeled peptide. Radiolabeling peptide 2 with N-succinimidyl-3-(125)I-iodobenzoate resulted in faster excretion, which in turn resulted in lower levels in tumor and other organs, especially thyroid and gastrointestinal tract. Peptide 2 was derivatized with the bifunctional isothiocyanates of cyclohexyl-B diethylenetriaminepentaacetic acid (DTPA) or CHX-A" DTPA by direct conjugation or with a hydroxylamine derivative of 1B4M-DTPA (2-(p-[O-(carboxamylmethyl)hydroxylamine]benzyl)-6-methyl-diethylenetriamine-N,N,N',N",N"-pentaacetic acid ) coupled at the N-terminus. The primary molecular species in the conjugated products were shown by mass spectrometry to have one DTPA per peptide. Peptide chelate conjugates were radiolabeled with (213)Bi and the products tested for biodistribution in tumor-bearing mice. The data show that chelation of (213)Bi to peptides was accomplished by both the direct method of DTPA attachment and by the method using the linker at the N-terminus. Only small amounts of peptide accumulated at tumor sites. We

  20. Engineering RNA phage MS2 virus-like particles for peptide display

    NASA Astrophysics Data System (ADS)

    Jordan, Sheldon Keith

    Phage display is a powerful and versatile technology that enables the selection of novel binding functions from large populations of randomly generated peptide sequences. Random sequences are genetically fused to a viral structural protein to produce complex peptide libraries. From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified by infection of bacteria. For certain applications however, existing phage display platforms have limitations. One such area is in the field of vaccine development, where the goal is to identify relevant epitopes by affinity-selection against an antibody target, and then to utilize them as immunogens to elicit a desired antibody response. Today, affinity selection is usually conducted using display on filamentous phages like M13. This technology provides an efficient means for epitope identification, but, because filamentous phages do not display peptides in the high-density, multivalent arrays the immune system prefers to recognize, they generally make poor immunogens and are typically useless as vaccines. This makes it necessary to confer immunogenicity by conjugating synthetic versions of the peptides to more immunogenic carriers. Unfortunately, when introduced into these new structural environments, the epitopes often fail to elicit relevant antibody responses. Thus, it would be advantageous to combine the epitope selection and immunogen functions into a single platform where the structural constraints present during affinity selection can be preserved during immunization. This dissertation describes efforts to develop a peptide display system based on the virus-like particles (VLPs) of bacteriophage MS2. Phage display technologies rely on (1) the identification of a site in a viral structural protein that is

  1. Screening and identification of a peptide specifically targeted to NCI-H1299 from a phage display peptide library.

    PubMed

    Zang, Linquan; Shi, Lei; Guo, Jiao; Pan, Qin; Wu, Wei; Pan, Xuediao; Wang, Junye

    2009-08-18

    In this study, a NCI-H1299 (Non-Small Cell Lung Cancer, NSCLC) and a normal lung cell line (Small Airway Epithelial Cells, SAEC) were used for the subtractive screening in vitro with a phage display-12 peptide library. After three rounds of panning, there was an obvious enrichment for the phages specifically binding to the NCI-H1299 cells, and the output/input ratio of phages increased about 875-fold (from 0.4x10(4) to 3.5x10(6)). A group of peptides being capable of binding specifically to the NCI-H1299 cells were obtained, and the affinity of these peptides to bind to the targeted cells and tissues was studied. Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, a M13 phage isolated and identified from the above screenings, and a synthetic peptide ZS-1 (sequence EHMALTYPFRPP) corresponded to the sequence of the surface protein of the M13 phage were demonstrated to be capable of binding to the tumor cell surfaces of NCI-H1299 and A549 cell lines and biopsy specimens, but not to normal lungs tissue samples, other different cancer cells, or nontumor surrounding lung tissues. In conclusion, the peptide ZS-1 may be a potential candidate of biomarker ligands used for targeted drug delivery in therapy of lung cancer.

  2. Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

    PubMed Central

    Rudolph, Stephan; Klein, Antonia Nicole; Tusche, Markus; Schlosser, Christine; Elfgen, Anne; Brener, Oleksandr; Teunissen, Charlotte; Gremer, Lothar; Funke, Susanne Aileen; Kutzsche, Janine; Willbold, Dieter

    2016-01-01

    Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ1–42 species, reduced Aβ1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein. PMID:26840229

  3. Screening and identification of a peptide specifically targeted to NCI-H1299 cells from a phage display peptide library.

    PubMed

    Tu, Xiangan; Zang, Linquan; Lan, Daiyan; Liang, Weican

    2009-01-01

    Ligands that are capable of binding to tumor cell surface biomarkers specifically used in the early diagnosis of cancer and targeted drug delivery in cancer chemotherapy have been extensively investigated. Phage display technology has been demonstrated to be a powerful tool in this field. In this study, the non-small cell lung cancer NCI-H1299 and the normal lung small airway epithelial cell lines were used for subtractive screening in vitro with a phage display 12-peptide library. After three rounds of panning, there was an obvious enrichment in the phages specifically binding to the NCI-H1299 cells, and the output/input ratio of phages increased approximately 875-fold (from 0.4x104 to 3.5x106). A group of peptides capable of binding specifically to the NCI-H1299 cells was obtained, and the affinity of these peptides to bind to the targeted cells and tissues was studied. Through cell-based ELISA, immunocytochemical staining, immunohistochemical staining and immunofluorescence, an M13 phage was isolated and identified from the above screenings, and a synthetic peptide, ZT-1 (sequence QQMHLMSYAPGP), corresponding to the sequence of the surface protein of the M13 phage, was demonstrated to be capable of binding to the tumor cell surfaces of NCI-H1299 and A549 cells and biopsy specimens, but not to normal lung tissue samples, other cancer cells, or non-tumor adjacent lung tissues. In conclusion, the peptide ZT-1 may be a potential candidate biomarker ligand that can be used for targeted drug delivery in lung cancer therapy.

  4. Multidrug-resistance drug-binding peptides generated by using a phage display library.

    PubMed

    Popkov, M; Lussier, I; Medvedkine, V; Estève, P O; Alakhov, V; Mandeville, R

    1998-01-15

    A phage display library of random decapeptides was used to generate peptide ligands that can bind multidrug-resistance (MDR) drugs mimicking, in this respect, the drug-binding activity of P-glycoprotein. Seven peptide sequences were identified that specifically bound doxorubicin. Five of these sequences expressed the core consensus motif WXXW. The displacement assay showed that the phages expressing these peptides bound MDR type drugs (vinblastine, doxorubicin, verapamil, and genistein) with the same selectivity as P-glycoprotein and did not interact with non-MDR type drugs, such as arabinosylcytosine (Ara-C) and melphalan. One of the selected peptides that showed a highest capacity for the binding (VCDWWGWGIC) was synthesized and displayed competition with the phage for doxorubicin binding. The structure modeling suggested that all the selected sequences contained a hydrophobic envelope in which MDR drugs could be docked with substantial energy minimization. Western blot analysis showed that monospecific antibody obtained against the phage expressing VCDWWGWGIC peptide could specifically recognize P-glycoprotein in the membrane fraction of MDR phenotype MCF-7ADR cells. The MDR drug-binding sequences generated during this work could provide an important tool for design and screening of new chemotherapeutic agents.

  5. Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob.

    PubMed

    Miura, Y; Yoshida, K; Nishimoto, T; Hatanaka, K; Ohnami, S; Asaka, M; Douglas, J T; Curiel, D T; Yoshida, T; Aoki, K

    2007-10-01

    Targeting of gene transfer at the level of cell entry is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success because proper targeting ligand-receptor systems on the cells of interest are generally unknown. Systematic approaches to generate adenovirus vectors targeting any given cell type need to be developed to achieve this goal. Here, we constructed an adenovirus library that was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob. As proof of concept, we screened the adenovirus display library on a glioma cell line and observed selection of several particular peptide sequences. The targeted vector carrying the most frequently isolated peptide significantly enhanced gene transduction in the glioma cell line but not in many other cell lines. Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. This vector-screening system can facilitate the development of a targeted adenovirus vector for a variety of applications in medicine.

  6. Identification of peptide sequences that target to the brain using in vivo phage display.

    PubMed

    Li, Jingwei; Zhang, Qizhi; Pang, Zhiqing; Wang, Yuchen; Liu, Qingfeng; Guo, Liangran; Jiang, Xinguo

    2012-06-01

    Phage display technology could provide a rapid means for the discovery of novel peptides. To find peptide ligands specific for the brain vascular receptors, we performed a modified phage display method. Phages were recovered from mice brain parenchyma after administrated with a random 7-mer peptide library intravenously. A longer circulation time was arranged according to the biodistributive brain/blood ratios of phage particles. Following sequential rounds of isolation, a number of phages were sequenced and a peptide sequence (CTSTSAPYC, denoted as PepC7) was identified. Clone 7-1, which encodes PepC7, exhibited translocation efficiency about 41-fold higher than the random library phage. Immunofluorescence analysis revealed that Clone 7-1 had a significant superiority on transport efficiency into the brain compared with native M13 phage. Clone 7-1 was inhibited from homing to the brain in a dose-dependent fashion when cyclic peptides of the same sequence were present in a competition assay. Interestingly, the linear peptide (ATSTSAPYA, Pep7) and a scrambled control peptide PepSC7 (CSPATSYTC) did not compete with the phage at the same tested concentration (0.2-200 pg). Labeled by Cy5.5, PepC7 exhibited significant brain-targeting capability in in vivo optical imaging analysis. The cyclic conformation of PepC7 formed by disulfide bond, and the correct structure itself play a critical role in maintaining the selectivity and affinity for the brain. In conclusion, PepC7 is a promising brain-target motif never been reported before and it could be applied to targeted drug delivery into the brain.

  7. Display of Peptides and Proteins on the Surface of Bacteriophage λ

    NASA Astrophysics Data System (ADS)

    Sternberg, Nat; Hoess, Ronald H.

    1995-02-01

    The display of peptides or proteins on the surface of viruses is an important technology for studying peptides or proteins and their interaction with other molecules. Here we describe a display vehicle based on bacteriophage λ that incorporates a number of features distinct from other currently used display systems. Fusions of peptides or protein domains have been made to the amino terminus of the 11-kDa D protein of the λ capsid. These fusions assemble onto the viral capsid and appear to be accessible to ligand interactions, based on the ability of a monoclonal antibody to recognize an epitope fused to the D protein on phage heads. To produce large D fusion display libraries and yet avoid the cumbersome task of cloning many fragments into λ DNA, we have used the Cre-loxP site-specific recombination system in vivo to incorporate plasmids encoding the D fusions into the phage genome. Finally, we show that D fusion proteins can be added in vitro to phage lacking D protein and be assembled onto the viral capsid.

  8. Phage Display against Corneal Epithelial Cells Produced Bioactive Peptides That Inhibit Aspergillus Adhesion to the Corneas

    PubMed Central

    Zhao, Ge; Li, Siyuan; Zhao, Wei; He, Kun; Xi, Haijie; Li, Weihua; Zhou, Qingjun; Wang, Yiqiang

    2012-01-01

    Dissection of host-pathogen interactions is important for both understanding the pathogenesis of infectious diseases and developing therapeutics for the infectious diseases like various infectious keratitis. To enhance the knowledge about pathogenesis infectious keratitis, a random 12-mer peptide phage display library was screened against cultured human corneal epithelial cells (HCEC). Fourteen sequences were obtained and BLASTp analysis showed that most of their homologue counterparts in GenBank were for defined or putative proteins in various pathogens. Based on known or predicted functions of the homologue proteins, ten synthetic peptides (Pc-A to Pc-J) were measured for their affinity to bind cells and their potential efficacy to interfere with pathogen adhesion to the cells. Besides binding to HCEC, most of them also bound to human corneal stromal cells and umbilical endothelial cells to different extents. When added to HCEC culture, the peptides induced expression of MyD88 and IL-17 in HCEC, and the stimulated cell culture medium showed fungicidal potency to various extents. While peptides Pc-C and Pc-E inhibited Aspergillus fumigatus (A.f) adhesion to HCEC in a dose-dependent manner, the similar inhibition ability of peptides Pc-A and Pc-B required presence of their homologue ligand Alb1p on A.f. When utilized in an eyeball organ culture model and an in vivo A.f keratitis model established in mouse, Pc-C and Pc-E inhibited fungal adhesion to corneas, hence decreased corneal disruption caused by inflammatory infiltration. Affinity pull-down of HCEC membrane proteins with peptide Pc-C revealed several molecules as potential receptors for this peptide. In conclusion, besides proving that phage display-selected peptides could be utilized to interfere with adhesion of pathogens to host cells, hence could be exploited for managing infectious diseases including infectious keratitis, we also proposed that the phage display technique and the resultant peptides could

  9. Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library.

    PubMed

    Wang, Hongjun; Gao, Yaping; Gong, Yumei; Chen, Xiaoling; Liu, Chuan; Zhou, Xuemei; Blackall, P J; Zhang, Peijun; Yang, Hanchun

    2007-01-31

    Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

  10. Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library.

    PubMed

    Smith, Richard G; Missailidis, Sotiris; Price, Michael R

    2002-01-05

    A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.

  11. Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display.

    PubMed

    Hacker, David E; Hoinka, Jan; Iqbal, Emil S; Przytycka, Teresa M; Hartman, Matthew C T

    2017-03-17

    Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nanomolar affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.

  12. Generating high accuracy peptide binding data in high throughput with yeast surface display and SORTCERY

    PubMed Central

    Reich, Lothar “Luther”; Dutta, Sanjib; Keating, Amy E.

    2016-01-01

    Library methods are widely used to study protein-protein interactions, and high-throughput screening or selection followed by sequencing can identify a large number of peptide ligands for a protein target. In this chapter we describe a procedure called "SORTCERY" that can rank the affinities of library members for a target with high accuracy. SORTCERY follows a three-step protocol. First, fluorescence activated cell sorting (FACS) is used to sort a library of yeast displayed peptide ligands according to their affinities for a target. Second, all sorted pools are deep sequenced. Third, the resulting data are analyzed to create a ranking. We demonstrate an application of SORTCERY to the problem of ranking peptide ligands for the anti-apoptotic regulator Bcl-xL. PMID:27094295

  13. Differential Bacterial Surface Display of Peptides by the Transmembrane Domain of OmpA

    PubMed Central

    Verhoeven, Gertjan S.; Alexeeva, Svetlana; Dogterom, Marileen; den Blaauwen, Tanneke

    2009-01-01

    Peptide libraries or antigenic determinants can be displayed on the surface of bacteria through insertion in a suitable outer membrane scaffold protein. Here, we inserted the well-known antibody epitopes 3xFLAG and 2xmyc in exterior loops of the transmembrane (TM) domain of OmpA. Although these highly charged epitopes were successfully displayed on the cell surface, their levels were 10-fold reduced due to degradation. We verified that the degradation was not caused by the absence of the C-terminal domain of OmpA. In contrast, a peptide that was only moderately charged (SA-1) appeared to be stably incorporated in the outer membrane at normal protein levels. Together, these results suggest that the display efficiency is sensitive to the charge of the inserted epitopes. In addition, the high-level expression of OmpA variants with surface-displayed epitopes adversely affected growth in a strain dependent, transient manner. In a MC4100 derived strain growth was affected, whereas in MC1061 derived strains growth was unaffected. Finally, results obtained using a gel-shift assay to monitor β-barrel folding in vivo show that the insertion of small epitopes can change the heat modifiability of the OmpA TM domain from ‘aberrant’ to normal, and predict that some β-barrels will not display any significant heat-modifiability at all. PMID:19707582

  14. Programmable Multivalent Display of Receptor Ligands using Peptide Nucleic Acid Nanoscaffolds

    PubMed Central

    Englund, Ethan A.; Wang, Deyun; Fujigaki, Hidetsugu; Sakai, Hiroyasu; Micklitsch, Christopher M.; Ghirlando, Rodolfo; Martin-Manso, Gema; Pendrak, Michael L.; Roberts, David D.; Durell, Stewart R.; Appella, Daniel H.

    2012-01-01

    Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to study multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1 to 45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models. PMID:22233624

  15. Identification of nose-to-brain homing peptide through phage display.

    PubMed

    Wan, Xiao-Mei; Chen, Yong-Ping; Xu, Wen-Rui; Yang, Wen-Jun; Wen, Long-Ping

    2009-02-01

    Brain delivery of drug molecules through the nasal passage represents a viable approach for bypassing the blood-brain barrier (BBB) but remains a major challenge due to lack of efficient homing carriers. To screen for potential peptides with the ability to transport into the brain via the nasal passage, we applied a C7C phage peptide display library (Ph.D.-C7C) intra-nasally to anesthetized rats and recovered phage from the brain tissue 45 min after phage administration. After three rounds of panning, 10 positive phage clones were selected and sequenced. Clone7, which exhibited highest translocation efficiency, was chosen for further studies. After nasal administration, Clone7 entered the brain within 30 min and exhibited translocation efficiency about 50-fold higher than a random phage. A 11-amino acid synthetic peptide derived from the displayed sequence of Clone7 (ACTTPHAWLCG) efficiently inhibited the nasal-brain translocation of Clone7. Both phage recovery results and fluorescent microscopy images revealed the presence of many more Clone7 phage in the brain than in the liver, kidney and other internal organs after the nasal administration, suggesting that Clone7 bypassed the BBB and entered brain directly. Furthermore, both Clone7 and the ACTTPHAWLCG peptide were found to be heavily distributed along the olfactory nerve after the nasal administration, further suggesting a direct passage route into the brain via the olfactory region. These results demonstrated the feasibility of using the in vivo phage display approach for selecting peptides with the nose-to-brain homing capability and may have implications for the development of novel targeting carriers useful for brain delivery.

  16. Screening of cell-penetrating peptides using mRNA display.

    PubMed

    Lee, Jae-Hun; Song, Hyun Seok; Park, Tai Hyun; Park, Tae Hyun; Lee, Sun-Gu; Kim, Byung-Gee

    2012-03-01

    Cell-penetrating peptides (CPPs) are attractive vectors for in vivo and in vitro cellular uptake. Their use is, however, limited by insufficient understanding of their preference for a target cell. Here, a new CPP screening method is presented that uses mRNA display. After incubating the target cell lines, such as human embryonic kidney 293 (HEK 293) and HeLa cells, with an mRNA display library for 3 h at 37°C, the CPP-mRNA nucleotide conjugates were harvested. These were amplified with PCR and subsequently sequenced. The screened CPPs for each cell line were identified after four rounds of selection. Among them, two peptides, MAMPGEPRRANVMAHKLEPASLQLR NSCA (CPPK) and MAPQRDTVGGRTTPPSWGPAKAQLRNSCA (CPPL) were selected, and the FITC-labeled peptides were evaluated for their ability to penetrate cells. The screened CPPs were superior to polyarginine (R(11) ), which is widely used as a standard peptide and shows good cell penetration efficiency. Our method can be applied to other target cells for which CPPs have not yet been elucidated.

  17. Baculoviral capsid display of His-tagged ZnO inorganic binding peptide

    PubMed Central

    Song, Lei; Liu, Yingying

    2010-01-01

    Virus-templated fabrication of compound structures can be made through incorporating the specifically inorganic-binding peptide into the viral scaffold, widely used is phage display system. Compared to prokaryotic phages, insect cell-based baculovirus has some strengths such as the adaptability to the proteins’ posttranslational modification and non-replication in mammalian cells. As an attempt to explore the baculovirus-mediated bioconjugates, we show in this study that a genetically engineered baculovirus, with a hexahistidine (His6) tagged ZnO binding peptide fused to the N-terminus of the viral capsid protein vp39 of AcNPV, was constructed. It maintains both the viral infectivity and the fusion protein’s activity. The presence of the fusion protein on the baculovirus particle was demonstrated by western blot analysis of purified budded virus. Its display on the virus capsid was revealed by virus fractionation analysis. The binding of nanosized ZnO powders to the virus capsid was visualized by transmission electron microscopy (TEM). This is the first report of the display of the inorganic-binding peptide on the capsid of eukaryotic baculovirus. Aimed at the nanomaterials’ application in the biological field, this research could find useful in the biotracking of the baculovirus transduction process and the preparation of novel functional nanodevices. PMID:20407822

  18. Adhesive peptides selected by phage display: characterization, applications and similarities with fibrinogen.

    PubMed

    Gebhardt, K; Lauvrak, V; Babaie, E; Eijsink, V; Lindqvist, B H

    1996-01-01

    Phase clones with affinity for polystyrene/polyurethane magnetic particles were isolated from a 10-men peptide display library. Sequence analysis revealed that 40 out of 80 clones contained the consensus WXXWXXXW. Some of the selected phages showed high surface activity and adsorbed to plastic surfaces even in the presence of blocking agents or surfactants. Covalent attachment of a synthetic peptide (KG), carrying one of the selected sequences to alkaline phosphatase (AP) or bovine serum albumin (BSA) enhanced binding of AP to a wide range of materials and improved the ability of BSA to prevent binding of antibodies and phages to polystyrene. Interestingly, the WXXW/XXXW motif occurs in the beta- and gamma-chains of the natural "adhesive" protein fibrinogen, and a synthetic peptide carrying the gamma-chain 369-376 sequence turned out to have essentially the same binding properties as the KG peptide. Furthermore, adsorption in different types of polystyrene was similar for AP carrying either the KG or gamma-chain peptide intact fibrinogen and plasmin-generated fragment D1. The latter fragment contains two copies of the WXXWXXXW motif but lacks the alpha-chain: protuberances previously implicated in fibrinogen adsorption. Thus, our study may have revealed a hitherto unknown structural determinant for fibrinogen's adsorptivity, located in the 13-kDa C terminal region of the gamma-chain.

  19. Selective inhibition of miR-21 by phage display screened peptide

    PubMed Central

    Bose, Debojit; Nahar, Smita; Rai, Manish Kumar; Ray, Arjun; Chakraborty, Kausik; Maiti, Souvik

    2015-01-01

    miRNAs are nodal regulators of gene expression and deregulation of miRNAs is causally associated with different diseases, including cancer. Modulation of miRNA expression is thus of therapeutic importance. Small molecules are currently being explored for their potential to downregulate miRNAs. Peptides have shown to have better potency and selectivity toward their targets but their potential in targeting and modulating miRNAs remain unexplored. Herein, using phage display we found a very selective peptide against pre-miR-21. Interestingly, the peptide has the potential to downregulate miR-21, by binding to pre-miR-21 and hindering Dicer processing. It is selective towards miR-21 inside the cell. By antagonising miR-21 function, the peptide is able to increase the expression of its target proteins and thereby increase apoptosis and suppress cell proliferation, invasion and migration. This peptide can further be explored for its anti-cancer activity in vivo and may be even extended to clinical studies. PMID:25824952

  20. Immunogenic peptides from phage display libraries with potential of protecting mice against the Pseudorabies virus.

    PubMed

    Gazarian, Karlen; Gazarian, Tatiana; Betancourt, José Iván Sánchez; Alonso Morales, Rogelio A

    2011-12-29

    Phage display selection of combinatorial peptide libraries has demonstrated its almost unlimited potential in identifying binding ligands for many targets. The method shows promise for selection of immunogenic peptides against pathogens by antibodies. We have undertaken a study designed to select such mimics for one of the representatives of Herpesviridae, the Pseudorabies virus (PrV), infecting pigs and causing severe neurological complications known as Aujeszky's disease. By screening a 12mer linear and a 7mer cysteine-constrained libraries with immunoglobulins of a rabbit immunized with the virus, a family of 10 antigenic and immunogenic peptides was derived sharing a sequence motif K(L/P/V)GDP(R/K/L). Groups of six C57BL/6 mice were immunized with bacteriophages expressing peptides with this motif sequences. Some of the mice were found to be positive in seroneutralization assay; in a challenge setting, all but two immunized mice survived, albeit presenting some disease symptoms. We discuss the perspectives and limits of generating peptide leads by library screening with immune polyclonal antiserum for designing pure epitope-based vaccines to PrV in the future.

  1. Blocking peptides against HBV: PreS1 protein selected from a phage display library

    SciTech Connect

    Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui; Xiao, Gengfu

    2011-09-09

    Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

  2. Inorganic binding peptides designed by phage display techniques for biotechnology applications

    NASA Astrophysics Data System (ADS)

    Liao, Chih-Wei

    Biomacromolecules play an important role in the control of hard tissue structure and function via specific molecular recognition interactions between proteins of the matrix and inorganic species of the biomineral phase. During the construction of the tissue, biomacromolecules are usually folded into a certain comformation, analogous to a "lock" for fitting with other proteins or smaller molecules as a "key". Currently, the rational design of molecular recognition in biomacro-molecules is still hard to accomplish because the protein conformation is too complex to precisely predict based on the existing conformational information of proteins found in biological systems. In the past two decades, the combinatorial approach (e.g. phage display techniques) has been used to select short binding peptides with molecular recognition to an inorganic target material without a prior knowledge of the amino acid sequence required for the specific binding. The technique has been referred to as "biopanning" because bacteriophages are used to "screen" for peptides that exhibit strong binding to a target material of interest. In this study, two diverse applications were chosen to demonstrate the utility of the biopanning approach. In one project, phage display techniques were used to pan for Indium Zinc Oxide (InZnO) binding peptides to serve as linkers between transducer devices and biosensing elements for demonstration of the feasibility of reversibly electro-activated biosensors. The amorphous InZnO, with its homogeneous surface, led to three consensus peptide sequences, AGFPNSTHSSNL, SHAPDSTWFALF, and TNSSSQFVVAIP. In addition, it was demonstrated that some selected phage clones of the InZnO binding peptides were able to be released from the InZnO surface after applying a voltage of 1400 mV on an electro-activated releasing device. In the second project, phage display techniques were used to select phage clones that bind specifically to francolite mineral in order to achieve

  3. Phage Displayed Peptides to Avian H5N1 Virus Distinguished the Virus from Other Viruses

    PubMed Central

    Qin, Chengfeng; Ren, Xiaofeng

    2011-01-01

    The purpose of the current study was to identify potential ligands and develop a novel diagnostic test to highly pathogenic avian influenza A virus (HPAI), subtype H5N1 viruses using phage display technology. The H5N1 viruses were used as an immobilized target in a biopanning process using a 12-mer phage display random peptide library. After five rounds of panning, three phages expressing peptides HAWDPIPARDPF, AAWHLIVALAPN or ATSHLHVRLPSK had a specific binding activity to H5N1 viruses were isolated. Putative binding motifs to H5N1 viruses were identified by DNA sequencing. In terms of the minimum quantity of viruses, the phage-based ELISA was better than antiserum-based ELISA and a manual, semi-quantitative endpoint RT-PCR for detecting H5N1 viruses. More importantly, the selected phages bearing the specific peptides to H5N1 viruses were capable of differentiating this virus from other avian viruses in enzyme-linked immunosorbent assays. PMID:21887228

  4. Strategies for the construction and use of peptide and antibody libraries displayed on phages.

    PubMed

    Pini, Alessandro; Giuliani, Andrea; Ricci, Claudia; Runci, Ylenia; Bracci, Luisa

    2004-12-01

    Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.

  5. Phage-displayed peptides selected for binding to Campylobacter jejuni are antimicrobial.

    PubMed

    Bishop-Hurley, Sharon L; Rea, Philippa J; McSweeney, Christopher S

    2010-10-01

    In developed countries, Campylobacter jejuni is a leading cause of zoonotic bacterial gastroenteritis in humans with chicken meat implicated as a source of infection. Campylobacter jejuni colonises the lower gastrointestinal tract of poultry and during processing is spread from the gastrointestinal tract onto the surface of dressed carcasses. Controlling or eliminating C.jejuni on-farm is considered to be one of the best strategies for reducing human infection. Molecules on the cell surface of C.jejuni interact with the host to facilitate its colonisation and persistence in the gastrointestinal tract of poultry. We used a subtractive phage-display protocol to affinity select for peptides binding to the cell surface of a poultry isolate of C.jejuni with the aim of finding peptides that could be used to control this microorganism in chickens. In total, 27 phage peptides, representing 11 unique clones, were found to inhibit the growth of C.jejuni by up to 99.9% in vitro. One clone was bactericidal, reducing the viability of C.jejuni by 87% in vitro. The phage peptides were highly specific. They completely inhibited the growth of two of the four poultry isolates of C.jejuni tested with no activity detected towards other Gram-negative and Gram-positive bacteria.

  6. K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

    PubMed

    Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya; Yaguchi, Masahiro; Niida, Ayumu; Sasaki, Shigekazu; Miwa, Masanori; Ohkubo, Shoichi; Sakamoto, Jun-Ichi; Kamaura, Masahiro; Cho, Nobuo; Tani, Akiyoshi

    2017-03-11

    Amino-acid mutations of Gly(12) (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs.

  7. Enhanced pulmonary absorption of a macromolecule through coupling to a sequence-specific phage display-derived peptide.

    PubMed

    Morris, Christopher J; Smith, Mathew W; Griffiths, Peter C; McKeown, Neil B; Gumbleton, Mark

    2011-04-10

    With the aim of identifying a peptide sequence that promotes pulmonary epithelial transport of macromolecule cargo we used a stringent peptide-phage display library screening protocol against rat lung alveolar epithelial primary cell cultures. We identified a peptide-phage clone (LTP-1) displaying the disulphide-constrained 7-mer peptide sequence, C-TSGTHPR-C, that showed significant pulmonary epithelial translocation across highly restrictive polarised cell monolayers. Cell biological data supported a differential alveolar epithelial cell interaction of the LTP-1 peptide-phage clone and the corresponding free synthetic LTP-1 peptide. Delivering select phage-clones to the intact pulmonary barrier of an isolated perfused rat lung (IPRL) resulted in 8.7% of lung deposited LTP-1 peptide-phage clone transported from the IPRL airways to the vasculature compared (p<0.05) to the cumulative transport of less than 0.004% for control phage-clone groups. To characterise phage-independent activity of LTP-1 peptide, the LTP-1 peptide was conjugated to a 53kDa anionic PAMAM dendrimer. Compared to respective peptide-dendrimer control conjugates, the LTP-1-PAMAM conjugate displayed a two-fold (bioavailability up to 31%) greater extent of absorption in the IPRL. The LTP-1 peptide-mediated enhancement of transport, when LTP-1 was either attached to the phage clone or conjugated to dendrimer, was sequence-dependent and could be competitively inhibited by co-instillation of excess synthetic free LTP-1 peptide. The specific nature of the target receptor or mechanism involved in LTP-1 lung transport remains unclear although the enhanced transport is enabled through a mechanism that is non-disruptive with respect to the pulmonary transport of hydrophilic permeability probes. This study shows proof-of principle that array technologies can be effectively exploited to identify peptides mediating enhanced transmucosal delivery of macromolecule therapeutics across an intact organ.

  8. Application of phage peptide display technology for the study of food allergen epitopes.

    PubMed

    Chen, Xueni; Dreskin, Stephen C

    2016-12-20

    Phage peptide display technology has been used to identify IgE-binding mimotopes (mimics of natural epitopes) that mimic conformational epitopes. This approach is effective in the characterization of those epitopes that are important for eliciting IgE-mediated allergic responses by food allergens and those that are responsible for cross-reactivity among allergenic food proteins. Application of this technology will increase our understanding of the mechanisms whereby food allergens elicit allergic reactions, will facilitate the discovery of diagnostic reagents and may lead to mimotope-based immunotherapy.

  9. PEGylation enables the specific tumor accumulation of a peptide identified by phage display.

    PubMed

    Mier, Walter; Krämer, Susanne; Zitzmann, Sabine; Altmann, Annette; Leotta, Karin; Schierbaum, Ursula; Schnölzer, Martina; Eisenhut, Michael; Haberkorn, Uwe

    2013-04-28

    Peptides are excellent alternatives to small molecules and proteinaceous drugs. Their high medicinal potential for diagnostic and therapeutic applications has prompted the development of tumor targeting peptides. Despite its excellent tumor binding capacity, FROP-DOTA (H-Glu-Asn-Tyr-Glu-Leu-Met-Asp-Leu-Leu-Ala-Tyr-Leu-Lys(DOTA)-NH2), a peptide that we had identified in phage display libraries, revealed slow binding kinetics. Consequently, biodistribution studies showed that its excretion forestalled a significant tumor accumulation. The aim of this study was to investigate whether the conjugation of PEG to FROP-DOTA resulted in a derivative with a prolonged residence time in the blood. A synthetic method for the PEGylation of the tumor specific peptide FROP-DOTA was developed. Thereafter, binding studies were done in vitro and a biodistribution was performed in tumor bearing animals. These were compared to the data obtained with FROP-DOTA. The binding kinetics of the PEGylated FROP-DOTA was even slower than that of FROP-DOTA. Biodistribution studies of the labeled conjugate in mice bearing human FRO82-2 tumors showed a time dependent increased uptake of the PEGylated peptide with a high retention (at 24 h p.i. 76% of the maximal activity concentration persisted in the tumor). The highest uptake values were determined at 120 min p.i. reaching 2.3%ID/g tumor as compared to 0.06%ID/g observed for the non-PEGylated derivative at 135 min p.i. Apparently, PEGylation provides a substantially improved stabilization in the circulation which allowed a stable tumor accumulation.

  10. Selection of antitumor displayed peptides for the specific delivery of the anticancer drug lactaptin

    PubMed Central

    Nemudraya, Anna Andreevna; Kuligina, Elena Vladimirovna; Ilyichev, Alexandr Alexeevich; Fomin, Alexandr Sergeevich; Stepanov, Grigory Alexandrovich; Savelyeva, Anna Valentinovna; Koval, Olga Alexandrovna; Richter, Vladimir Alexandrovich

    2016-01-01

    It has been previously demonstrated that lactaptin, the proteolytic fragment of human milk protein κ-casein, induces the death of various cultured cancer cells. The recombinant analog of lactaptin, RL2, effectively induces the apoptosis of mouse hepatocarcinoma-1 (HA-1) tumor cells in vitro and suppress the growth of HA-1 tumors and metastases in vivo. The antitumor drug Lactaptin developed on the basis of RL2 has been successful in preclinical trials. Lactaptin shows its efficiency in relation to mouse and human cancer cells and tumors. However, Lactaptin, as with the majority of protein-based therapeutic drugs, is distributed evenly throughout the organism, which reduces its antitumor efficacy. To develop the targeted delivery of lactaptin, the present study selected tumor-specific peptides by screening a phage display peptide library in vivo on A/Sn strain mice with subcutaneously transplanted HA-1 cells. Two genetic constructs were made for the production of recombinant fusion proteins composed of RL2 and the selected tumor-targeting peptide. In vitro experiments involving HA-1, MDA-MB-231 and MCF-7 cells cultures demonstrated that the fusion proteins induce apoptotic death in mouse and human tumor cells, as with RL2. The in vivo experiments involving the mouse HA-1 tumor model demonstrated that the tumor fluorescence intensity of the Cy5-fusion protein conjugates is higher than that of RL2-Cy5. As conjugation of the tumor-specific peptides to RL2 provided retention of RL2 in the tumor tissues, fusion proteins composed of lactaptin and peptides specific for human tumors are deemed promising to improve the antitumor efficiency of lactaptin. PMID:28105163

  11. Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

    NASA Astrophysics Data System (ADS)

    Essler, Markus; Ruoslahti, Erkki

    2002-02-01

    In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

  12. Phage-displayed peptide targeting on the Puumala hantavirus neutralization site.

    PubMed Central

    Heiskanen, T; Lundkvist, A; Vaheri, A; Lankinen, H

    1997-01-01

    We have selected ligands for Puumala hantavirus, the causative agent of nephropathia epidemica, from a seven-amino-acid peptide library flanked by cysteines and displayed on a filamentous phage. To direct the selection to areas on the virus particle which are essential for infection, phages were competitively eluted with neutralizing monoclonal antibodies specific for the viral glycoproteins. The selected phage populations were specific for the same sites as the antibodies and mimicked their functions. The peptide insert, CHWMFSPWC, when displayed on the phages, completely inhibited Puumala virus infection in cell culture at the same effective concentration as the eluting antibody specific for envelope glycoprotein G2. The binding of the phage clones to the virus and inhibition of infection were not necessarily coincident; Pro-6 was critical for virus inhibition, while consensus residues Trp-2 and Phe-4 were essential for binding. The strategy described can be applied to any virus for production of molecules mimicking the effect of neutralizing antibodies. PMID:9094664

  13. Phage display of ScFv peptides recognizing the thymidine(6–4)thymidine photoproduct

    PubMed Central

    Zavala, Anamaria G.; Lancaster, Thaddeus; Groopman, John D.; Strickland, Paul T.; Chandrasegaran, Srinivasan

    2000-01-01

    Solar ultraviolet (UV) radiation induces DNA photoproducts in skin cells and is the predominant cause of human skin cancers. To understand human susceptibility to skin cancer and to facilitate the development of prevention measures, highly specific reagents to detect and quantitate UV-induced DNA adducts in human skin will be needed. One approach towards this end is the use of monoclonal antibody-based molecular dosimetry methods. To facilitate the development of photoproduct-specific antibody reagents we have: (i) cloned and sequenced a single chain variable fragment (ScFv) gene coding for one such high affinity monoclonal antibody, αUVssDNA-1 (mAb C3B6), recognizing the thymidine(6–4)thymidine photoproduct; (ii) expressed and displayed the cloned ScFv gene on the surface of phage; (iii) selected functional recombinant phage by panning; (iv) purified the ScFv peptide; (v) shown that the purified ScFv peptide binds to UV-irradiated polythymidylic acid but not unirradiated polythymidylic acid. This is the first demonstration of the use of phage display to select a ScFv recognizing DNA damage. In addition, this is the initial step towards immortalizing the antibody gene for genetic manipulation, structure–function studies and application to human investigations. PMID:10710441

  14. A New Peptide Ligand for Targeting Human Carbonic Anhydrase IX, Identified through the Phage Display Technology

    PubMed Central

    Askoxylakis, Vasileios; Garcia-Boy, Regine; Rana, Shoaib; Krämer, Susanne; Hebling, Ulrike; Mier, Walter; Altmann, Annette; Markert, Annette; Debus, Jürgen; Haberkorn, Uwe

    2010-01-01

    Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. Methods Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. Results In vitro binding experiments of 125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. Conclusions These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX. PMID:21209841

  15. Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

    SciTech Connect

    Rodi, D. J.; Soares, A. S.; Makowski, L.; Biosciences Division; BNL

    2002-01-01

    Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.

  16. Discovery of peptidic miR-21 processing inhibitor by mirror image phage display: A novel method to generate RNA binding D-peptides.

    PubMed

    Sakamoto, Kotaro; Otake, Kentaro; Umemoto, Tadashi

    2017-02-15

    A novel method to generate RNA binding D-peptide has been developed. To achieve the screening method, phage display was applied to "Mirrored" RNA (L-enantiomer of RNA). We have selected pre-miR21 as an initial screening target to demonstrate the method. The mirrored pre-miR-21 binding peptide sequences were successfully obtained, and were chemically synthesized using D-amino acids. D-peptide is expected to have favorable properties as a drug candidate such as protease resistance and low immunogenicity. As a result of binding evaluation of the D-peptide to pre-miR-21, the EC50 value was 440nM. In addition, the D-peptide possessed inhibition activity to miR-21 processing.

  17. Targeting prostate cancer cells with genetically engineered polypeptide-based micelles displaying gastrin-releasing peptide.

    PubMed

    Zhang, Wei; Garg, Sanjay; Eldi, Preethi; Zhou, Fiona Huan-Huan; Johnson, Ian R D; Brooks, Doug A; Lam, Frankie; Rychkov, Grigori; Hayball, John; Albrecht, Hugo

    2016-11-20

    In recent years G protein-coupled receptors (GPCRs) have emerged as crucial tumorigenic factors that drive aberrant cancer growth, metastasis and angiogenesis. Consequently, a number of GPCRs are strongly expressed in cancer derived cell lines and tissue samples. Therefore a rational anti-cancer strategy is the design of nano-medicines that specifically target GPCRs to bind and internalise cytotoxic drugs into cancer cells. Herein, we report the genetic engineering of a self-assembling nanoparticle based on elastin-like polypeptide (ELP), which has been fused with gastrin releasing peptide (GRP). These nanoparticles increased intracellular calcium concentrations when added to GRP receptor positive PC-3 prostate cancer cells, demonstrating specific receptor activation. Moreover, GRP-displaying fluorescent labelled nanoparticles showed specific cell-surface interaction with PC-3 prostate cancer cells and increased endocytic uptake. These nanoparticles therefore provide a targeted molecular carrier system for evaluating the delivery of cytotoxic drugs into cancer cells.

  18. Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display.

    PubMed

    Tjhung, Katrina F; Deiss, Frédérique; Tran, Jessica; Chou, Ying; Derda, Ratmir

    2015-01-01

    In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a negatively charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus, as well as positively charged sequences which suppress production of phage when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs) co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc.).

  19. Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

    SciTech Connect

    Nemoto, Naoto; Tsutsui, Chihiro; Yamaguchi, Junichi; Ueno, Shingo; Machida, Masayuki; Kobayashi, Toshikatsu; Sakai, Takafumi

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. Black-Right-Pointing-Pointer Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. Black-Right-Pointing-Pointer Inhibitory effect of peptide depends on number and pattern of its disulfide bonds. -- Abstract: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method 'cDNA display'. In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.

  20. Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides

    SciTech Connect

    Kotrba, P.; Ruml, T.; Doleckova, L.; Lorenzo, V. de

    1999-03-01

    Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd{sup 2+}-to-HP and Cd{sup 2+}-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane of E. coli. Isolated cell envelopes of E. coli bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd{sup 2+} binding capacity. The bioaccumulation of Cd{sup 2+}, Cu{sup 2+}, and Zn{sup 2+} by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd{sup 2+} from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu{sup 2+} and Zn{sup 2+}. However, Cu{sup 2+} ceased contribution of HP for Cd{sup 2+} accumulation, probably due to the strong binding of Cu{sup 2+} to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.

  1. Selection of phage-displayed peptides for the detection of imidacloprid in water and soil.

    PubMed

    Liu, Zhiping; Liu, Jianfeng; Wang, Kai; Li, Wenhui; Shelver, Weilin L; Li, Qing X; Li, Ji; Xu, Ting

    2015-09-15

    Imidacloprid is the most widely used neonicotinoid insecticide in the world and shows widespread environment and human exposures. A phage clone designated L7-1 that selectively binds to imidacloprid was selected from a commercial phage display library containing linear 7-mer randomized amino acid residues. Using the clone L7-1, a competitive enzyme-linked immunosorbent assay (ELISA) for imidacloprid was developed. The half-maximum signal inhibition concentration (IC50) and the limit of detection (LOD) of the phage ELISA for imidacloprid were 96 and 2.3 ng ml(-1), respectively. This phage ELISA showed relatively low cross-reactivity with all of the tested compounds structurally similar to imidacloprid, less than 2% with the exception of 6-chloronicotinic acid, a metabolite of imidacloprid that showed 11.5%. The average recoveries of the phage ELISA for imidacloprid in water and soil samples were in the ranges of 74.6 to 86.3% and 72.5 to 93.6%, respectively. The results of the competitive phage ELISA for imidacloprid in the fortified samples agreed well with those of a high-performance liquid chromatography (HPLC) method. The simple phage-displayed peptide technology has been proven to be a convenient and efficient method for the development of an alternative format of ELISA for small molecules.

  2. Peptide sequences mediating tropism to intact blood-brain barrier: an in vivo biodistribution study using phage display.

    PubMed

    Smith, Mathew W; Al-Jayyoussi, Ghaith; Gumbleton, Mark

    2012-11-01

    Peptide motifs that demonstrate tropism for the blood brain barrier (BBB) are of real translational value in developing innovative delivery strategies for biological brain targeted therapies. In vivo peptide-phage display affords peptide selection against the full complement of biological markers within the correct cellular macro- and micro-environments. Here a stringent in vivo biopanning protocol was employed in the rat aimed at identifying cyclic 7-mer peptide motifs that mediate tropism to brain microvasculature. Five rounds of biopanning identified 349 unique peptide motifs in the brain tissue gray matter compartment (microvasculature and parenchyma). While in general no consensus was evident linking peptide physico-chemical properties and brain tropism, peptides bearing c-SxTSSTx-c or c-xxxSSTx-c motifs were found to be present in high abundance. Based on amino acid frequency distribution of the 349 unique peptides sequences a theoretical 'idealized' peptide pattern, c-PP(S/P)SSST-c, could be derived. For the most abundant experimental peptide sequence found in brain tissue, c-SYTSSTM-c, an in vivo pharmacokinetic and whole body tissue biodistribution study was performed. Based upon tissue exposure data (i.e. tissue AUC((0-infinity))) the sequence c-SYTSSTM-c efficiently retargeted phage virions to the brain providing an approximate 5-fold greater (P<0.05) accumulation in brain over control phage; in all other organs no significant (P>0.05) difference in tissue tropism between c-SYTSSTM-c and control phages were evident. This peptide and more generally the peptide motifs, -SxTSSTx- or -xxxSSTx-, warrant further investigation as agents mediating sequence-dependent tropism to brain microvasculature potentially able to deliver biologic cargo to the CNS.

  3. PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

    PubMed Central

    Dasa, Siva Sai Krishna; Kelly, Kimberly A.

    2016-01-01

    Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

  4. RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic Efficacy of Attenuated Salmonella-mediated Cancer Therapy.

    PubMed

    Park, Seung-Hwan; Zheng, Jin Hai; Nguyen, Vu Hong; Jiang, Sheng-Nan; Kim, Dong-Yeon; Szardenings, Michael; Min, Jung Hyun; Hong, Yeongjin; Choy, Hyon E; Min, Jung-Joon

    2016-01-01

    Bacteria-based anticancer therapies aim to overcome the limitations of current cancer therapy by actively targeting and efficiently removing cancer. To achieve this goal, new approaches that target and maintain bacterial drugs at sufficient concentrations during the therapeutic window are essential. Here, we examined the tumor tropism of attenuated Salmonella typhimurium displaying the RGD peptide sequence (ACDCRGDCFCG) on the external loop of outer membrane protein A (OmpA). RGD-displaying Salmonella strongly bound to cancer cells overexpressing αvβ3, but weakly bound to αvβ3-negative cancer cells, suggesting the feasibility of displaying a preferential homing peptide on the bacterial surface. In vivo studies revealed that RGD-displaying Salmonellae showed strong targeting efficiency, resulting in the regression in αvβ3-overexpressing cancer xenografts, and prolonged survival of mouse models of human breast cancer (MDA-MB-231) and human melanoma (MDA-MB-435). Thus, surface engineering of Salmonellae to display RGD peptides increases both their targeting efficiency and therapeutic effect.

  5. Camptothecin binds to a synthetic peptide identified by a T7 phage display screen.

    PubMed

    Takakusagi, Yoichi; Kobayashi, Susumu; Sugawara, Fumio

    2005-11-01

    An analysis of non-biotinylated camptothecin (CPT) binding to the C-20-biotinylated CPT binding peptide NSSQSARR was carried out using two methods, quartz-crystal microbalance (QCM) and surface plasmon resonance (SPR). The peptide was immobilized peptide on a sensor chip and showed a dissociation constant (KD) of approximately 0.1 microM against CPT in QCM and SPR experiments.

  6. Induction of anti-EGFR immune response with mimotopes identified from a phage display peptide library by panitumumab

    PubMed Central

    Qu, Hong; Di, Jiabo; Wang, Zaozao; Xing, Jiadi; Wu, Fan; Wu, Wei; Wang, Xicheng; Shen, Lin; Jiang, Beihai; Su, Xiangqian

    2016-01-01

    The epidermal growth factor receptor (EGFR) is overexpressed in several epithelial tumors. Anti-EGFR humanized monoclonal antibodies, cetuximab and panitumumab, in combination with chemotherapy have improved the prognosis for patients with wild-type RAS tumors. To identify mimotopes of EGFR and develop mimotope-based EGFR vaccines, we screened a phage display peptide library with panitumumab. Two EGFR mimotopes P19 and P26, which could be recognized by panitumumab specifically, were isolated. To enhance the immune responses, we generated recombinant proteins of P19 or P26 fused to a heat-shock cognate protein 70 (Hsc70), and evaluated the efficacy of Hsc70-P19 and Hsc70-P26 as vaccines in vivo. Immunization with Hsc70-P19 or Hsc70-P26 fusion protein stimulated the immune system to produce specific antibodies against peptides as well as EGFR. Moreover, antibodies elicited against mimotopes could induce antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and inhibit the proliferation of EGFR-overexpressing A431 cells. Treatment with Hsc70-P19 and Hsc70-P26 significantly reduced tumor growth in BALB/c transplantable lung cancer models. Although there was no sequence homology between the phage-derived peptides and EGFR by alignments, both peptides mimic the conformational structure of EGFR binding to panitumumab. In conclusion, the mimotopes we identified from phage display peptide library could be promising candidate vaccines for active anti-EGFR immunotherapy against cancers. PMID:27659529

  7. Induction of anti-EGFR immune response with mimotopes identified from a phage display peptide library by panitumumab.

    PubMed

    Wang, Aidong; Cui, Ming; Qu, Hong; Di, Jiabo; Wang, Zaozao; Xing, Jiadi; Wu, Fan; Wu, Wei; Wang, Xicheng; Shen, Lin; Jiang, Beihai; Su, Xiangqian

    2016-11-15

    The epidermal growth factor receptor (EGFR) is overexpressed in several epithelial tumors. Anti-EGFR humanized monoclonal antibodies, cetuximab and panitumumab, in combination with chemotherapy have improved the prognosis for patients with wild-type RAS tumors. To identify mimotopes of EGFR and develop mimotope-based EGFR vaccines, we screened a phage display peptide library with panitumumab. Two EGFR mimotopes P19 and P26, which could be recognized by panitumumab specifically, were isolated. To enhance the immune responses, we generated recombinant proteins of P19 or P26 fused to a heat-shock cognate protein 70 (Hsc70), and evaluated the efficacy of Hsc70-P19 and Hsc70-P26 as vaccines in vivo. Immunization with Hsc70-P19 or Hsc70-P26 fusion protein stimulated the immune system to produce specific antibodies against peptides as well as EGFR. Moreover, antibodies elicited against mimotopes could induce antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and inhibit the proliferation of EGFR-overexpressing A431 cells. Treatment with Hsc70-P19 and Hsc70-P26 significantly reduced tumor growth in BALB/c transplantable lung cancer models. Although there was no sequence homology between the phage-derived peptides and EGFR by alignments, both peptides mimic the conformational structure of EGFR binding to panitumumab. In conclusion, the mimotopes we identified from phage display peptide library could be promising candidate vaccines for active anti-EGFR immunotherapy against cancers.

  8. "Blind" targeting in action: From phage display to breast cancer cell targeting with peptide-gold nanoconjugates.

    PubMed

    Galbiati, Elisabetta; Gambini, Luca; Civitarese, Viola; Bellini, Michela; Ambrosini, Dario; Allevi, Raffaele; Avvakumova, Svetlana; Romeo, Sergio; Prosperi, Davide

    2016-09-01

    Tumor homing peptides (THPs) specific for a representative breast cancer cell line (MCF-7) were carefully selected basing on a phage-displayed peptide library freely available on the web, namely the "TumorHoPe: A Database of Tumor Homing Peptides". The selected THPs were synthesized and evaluated in terms of their affinity toward MCF-7 cells. Out of 5 tested THPs, 3 best-performing peptide sequences and 1 scrambled sequence were separately conjugated to spherical gold nanoparticles yielding stable nanoconjugates. THP nanoconjugates were examined for their ability to actively target MCF-7 cells in comparison to noncancerous 3T3-L1 fibroblast cells. These THP-gold nanoconjugates exhibited good selectivity and binding affinity by flow cytometry, and low cytotoxicity as assayed by cell death experiments. The uptake of targeted nanoconjugates by the breast cancer cells was confirmed by transmission electron microscopy analysis. This work demonstrates that it is possible to exploit the conjugation of short peptides selected from phage-displayed libraries to develop nanomaterials reliably endowed with tumor targeting potential irrespective of a specific knowledge of the target cell biology.

  9. Humoral immune responses against gonadotropin releasing hormone elicited by immunization with phage-peptide constructs obtained via phage display.

    PubMed

    Samoylov, Alexandre; Cochran, Anna; Schemera, Bettina; Kutzler, Michelle; Donovan, Caitlin; Petrenko, Valery; Bartol, Frank; Samoylova, Tatiana

    2015-12-20

    Phage display is based on genetic engineering of phage coat proteins resulting in fusion peptides displayed on the surface of phage particles. The technology is widely used for generation of phages with novel characteristics for numerous applications in biomedicine and far beyond. The focus of this study was on development of phage-peptide constructs that stimulate production of antibodies against gonadotropin releasing hormone (GnRH). Phage-peptide constructs that elicit production of neutralizing GnRH antibodies can be used for anti-fertility and anti-cancer applications. Phage-GnRH constructs were generated via selection from a phage display library using several types of GnRH antibodies as selection targets. Such phage constructs were characterized for sequence similarities to GnRH peptide and frequency of their occurrence in the selection rounds. Five of the constructs with suitable characteristics were tested in mice as a single dose 5×10(11) virions (vir) vaccine and were found to be able to stimulate production of GnRH-specific antibodies, but not to suppress testosterone (indirect indicator of GnRH antibody neutralizing properties). Next, one of the constructs was tested at a higher dose of 2×10(12) vir per mouse in combination with a poly(lactide-co-glycolide) (PLGA)-based adjuvant. This resulted in multifold increase in GnRH antibody production and significant reduction of serum testosterone, indicating that antibodies produced in response to the phage-GnRH immunization possess neutralizing properties. To achieve optimal immune responses for desired applications, phage-GnRH constructs can be modified with respect to flanking sequences of GnRH-like peptides displayed on phage. Anticipated therapeutic effects also might be attained using optimized phage doses, a combination of several constructs in a single treatment, or application of adjuvants and advanced phage delivery systems.

  10. Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

    PubMed

    Bakhshinejad, Babak; Zade, Hesam Motaleb; Shekarabi, Hosna Sadat Zahed; Neman, Sara

    2016-12-01

    Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

  11. Chemical Synthesis and In Vitro Evaluation of a Phage Display-Derived Peptide Active against Infectious Salmon Anemia Virus

    PubMed Central

    Ojeda, Nicolás; Cárdenas, Constanza; Guzmán, Fanny

    2016-01-01

    ABSTRACT Infectious salmon anemia virus (ISAV) is the etiological agent of the disease by the same name and causes major losses in the salmon industry worldwide. Epizootic ISAV outbreaks have occurred in Norway and, to a lesser degree, in Canada. In 2007, an ISAV outbreak in Chile destroyed most of the seasonal production and endangered the entire Chilean salmon industry. None of the existing prophylactic approaches have demonstrated efficacy in providing absolute protection from or even a palliative effect on ISAV proliferation. Sanitary control measures for ISAV, based on molecular epidemiology data, have proven insufficient, mainly due to high salmon culture densities and a constant presence of a nonpathogenic strain of the virus. This report describes an alternative treatment approach based on interfering peptides selected from a phage display library. The screening of a phage display heptapeptide library resulted in the selection of a novel peptide with significant in vitro antiviral activity against ISAV. This peptide specifically interacted with the viral hemagglutinin-esterase protein, thereby impairing virus binding, with plaque reduction assays showing a significant reduction in viral yields. The identified peptide acts at micromolar concentrations against at least two different pathogenic strains of the virus, without detectable cytotoxic effects on the tested fish cells. Therefore, antiviral peptides represent a novel alternative for controlling ISAV and, potentially, other fish pathogens. IMPORTANCE Identifying novel methods for the efficient control of infectious diseases is imperative for the future of global aquaculture. The present study used a phage display heptapeptide library to identify a peptide with interfering activity against a key protein of the infectious salmon anemia virus (ISAV). A piscine orthomyxovirus, ISAV is a continuous threat to the commercial sustainability of cultured salmon production worldwide. The complex epidemiological

  12. Novel cell-surface peptides specific to human oral squamous cell carcinoma using an E. coli peptide display library.

    PubMed

    Kawai, Noriko; Asaumi, Junichi; Murakami, Jun; Wakasa, Toru; Kuroda, Masahiro; Hisatomi, Miki; Unetsubo, Teruhisa; Maki, Yuu; Matsuzaki, Hidenobu; Yanagi, Yoshinonu; Konouchi, Hironobu

    2007-04-01

    We attempted to find a specific antigen of oral squamous cell carcinoma (SCC) cells that could be safely applied to gene therapy in the conservative clinical treatment of oral cancer. We performed subtraction using normal human keratinocyte cells, followed by selection using four oral SCC cell lines. We isolated three clones from poorly differentiated SCC cells and four from well-differentiated SCC cells. These seven clones adsorbed to the oral SCC cells at rates 10-100 times those of normal human keratinocyte cells. The three clones from the poorly differentiated SCC cells showed the same peptide sequence (LAPRTHP). Of the four clones from the well-differentiated SCC cells, three showed the same peptide sequence (FGTLPGT) and the fourth showed a different one (VTPNSTP). Each peptide sequence may recognize the material that exists specifically on the oral SCC cell cortex. We can expect applications not only for tumor-targeting treatment using a gene therapy virus vector but also for diagnosis using, as a tumor marker, the peculiar SCC surface material that these peptides recognize.

  13. A new non-muscle-invasive bladder tumor-homing peptide identified by phage display in vivo

    PubMed Central

    YANG, XIAOFENG; ZHANG, FAN; LUO, JUNQIAN; PANG, JIANZHI; YAN, SANHUA; LUO, FANG; LIU, JIEHAO; WANG, WEI; CUI, YONGPING; SU, XIXI

    2016-01-01

    Bladder cancer is common and widespread, and its incidence is increasing. Many new diagnostic methods combined with state-of-the-art technology have been introduced in cystoscopy to collect real-time images of the bladder mucosa for diagnosis, but often miss inconspicuous early-stage tumors. Fluorophore-labeled peptides with high sensitivity and specificity for cancer would be a desirable tool for the detection and treatment of tiny or residual bladder tumors. Phage display and the human non-muscle-invasive bladder cancer cell line BIU-87 were used to identify a peptide. The isolated phage display peptide (CSSPIGRHC, named NYZL1) was tested in vitro for its binding specificity and affinity. Accumulation into xenograft tumors in a nude mouse model was analyzed with FITC-labeled NYZL1. NYZL1, with strong tumor-homing ability, was identified by in vivo phage library selection in the bladder cancer model. The NYZL1 phage and synthetic FITC-labeled NYZL1 peptides bound to tumor tissues and cells, but were hardly detected in normal control organs. Notably, accumulation of FITC-NYZL1 in bladder tumor cells was time-dependent. Biodistribution studies of xenografts of BIU-87 cells showed accumulation of injected FITC-NYZL1 in the tumors, and the bound peptide could not be removed by perfusion after 24 h. The mouse model of bladder tumor showed increased fluorescence intensity in the tumor-bearing bladder in comparison with normal bladder tissues after 4–6 h. In conclusion, NYZL1 may represent a lead peptide structure applicable in the development of optical molecular imaging. PMID:27221614

  14. Cu-free cycloaddition for identifying catalytic active adenylation domains of nonribosomal peptide synthetases by phage display.

    PubMed

    Zou, Yekui; Yin, Jun

    2008-10-15

    To engineer the substrate specificities of nonribosomal peptide synthetases (NRPS), we developed a method to display NRPS modules on M13 phages and select catalytically active adenylation (A) domains that would load azide functionalized substrate analogs to the neighboring peptidyl carrier protein (PCP) domains. Biotin conjugated difluorinated cyclooctyne was used for copper free cycloaddition with an azide substituted substrate attached to PCP. Biotin-labeled phages were selected by binding to streptavidin.

  15. Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

    PubMed

    Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

    2016-10-01

    Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium.

  16. Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

    DOEpatents

    Peabody, David S.; Chackerian, Bryce; Ashley, Carlee; Carnes, Eric; Negrete, Oscar

    2017-01-24

    The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referred to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.

  17. Peptides Displayed as High Density Brush Polymers Resist Proteolysis and Retain Bioactivity

    PubMed Central

    2015-01-01

    We describe a strategy for rendering peptides resistant to proteolysis by formulating them as high-density brush polymers. The utility of this approach is demonstrated by polymerizing well-established cell-penetrating peptides (CPPs) and showing that the resulting polymers are not only resistant to proteolysis but also maintain their ability to enter cells. The scope of this design concept is explored by studying the proteolytic resistance of brush polymers composed of peptides that are substrates for either thrombin or a metalloprotease. Finally, we demonstrate that the proteolytic susceptibility of peptide brush polymers can be tuned by adjusting the density of the polymer brush and offer in silico models to rationalize this finding. We contend that this strategy offers a plausible method of preparing peptides for in vivo use, where rapid digestion by proteases has traditionally restricted their utility. PMID:25314576

  18. In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display

    PubMed Central

    Ueno, Shingo; Yoshida, Sayaka; Mondal, Anupom; Nishina, Kazuya; Koyama, Makoto; Sakata, Ichiro; Miura, Kenju; Hayashi, Yujiro; Nemoto, Naoto; Nishigaki, Koichi; Sakai, Takafumi

    2012-01-01

    G protein-coupled receptors (GPCRs) are major drug targets, and their ligands are currently being explored and developed by many pharmaceutical companies and independent researchers. Class A (rhodopsin-like) GPCRs compose a predominant GPCR family; therefore, class A GPCR ligands are in demand. Growth hormone secretagogue receptor (GHS-R) is a class A GPCR that stimulates food intake by binding to its peptide ligand, ghrelin. Therefore, antagonists of GHS-R are expected to exert antiobesity function. In this article, we describe the use of cDNA display to screen for successfully and identify an antagonistic peptide of GHS-R. The antagonistic peptide inhibited the ghrelin-induced increase in intracellular Ca2+ in vitro (IC50 = approximately 10 μM) and repressed the contraction of isolated animal stomach in response to ghrelin. Furthermore, peripheral administration of the peptide inhibited the food intake of mice. This work provides new insight into the development of antiobesity drugs and describes a method for the discovery of unique peptide ligands for class A GPCRs. PMID:22723348

  19. Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from Lactobacillus fermentum BR11

    PubMed Central

    Turner, Mark S.; Hafner, Louise M.; Walsh, Terry; Giffard, Philip M.

    2003-01-01

    A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His6). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His6 and CFTR peptides or His6 peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors. PMID:14532035

  20. Efficient identification of murine M2 macrophage peptide targeting ligands by phage display and next-generation sequencing

    PubMed Central

    Liu, Gary W.; Livesay, Brynn R.; Kacherovsky, Nataly A.; Cieslewicz, Maryelise; Lutz, Emi; Waalkes, Adam; Jensen, Michael C.; Salipante, Stephen J.; Pun, Suzie H.

    2015-01-01

    Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (< 0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by non-specific, preferentially amplifying “parasitic sequences” and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively-activated (M2) macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences. PMID:26161996

  1. Identification of a novel peptide ligand targeting visceral adipose tissue via transdermal route by in vivo phage display.

    PubMed

    Lee, Nam Kyung; Kim, Hong Shin; Kim, Kyung Hyun; Kim, Eun-Bae; Cho, Chong Su; Kang, Sang Kee; Choi, Yun Jaie

    2011-11-01

    To find novel peptide ligands targeting visceral adipose tissue (visceral fat) via transdermal route, in vivo phage display screening was conducted by dermal administration of a phage-peptide library to rats and a peptide sequence, CGLHPAFQC (designated as TDA1), was identified as a targeting ligand to visceral adipose tissue through the consecutive transdermal biopannings. Adipocyte-specific affinity and transdermal activity of the TDA1 were validated in vitro and targeting ability of the dermally administered TDA1 to visceral adipose tissue was also confirmed in vivo. TDA1 was effectively translocated into systemic circulation after dermal administration and selectively targeted visceral adipose tissue without any preference to other organs tested. Fluorescent microscopic analysis revealed that the TDA1 could be specifically localized in the hair follicles of the skin, as well as in the visceral adipose tissue. Thus, we inferred that dermally administered TDA1 would first access systemic circulation via hair follicles as its transdermal route and then could target visceral fat effectively. The overall results suggest that the TDA1 peptide could be potentially applied as a homing moiety for delivery of anti-obesity therapeutics to visceral fat through the convenient transdermal pathway.

  2. Polyvalent display and packing of peptides and proteins on semiconductor quantum dots: predicted versus experimental results.

    PubMed

    Prasuhn, Duane E; Deschamps, Jeffrey R; Susumu, Kimihiro; Stewart, Michael H; Boeneman, Kelly; Blanco-Canosa, Juan B; Dawson, Philip E; Medintz, Igor L

    2010-02-22

    Quantum dots (QDs) are loaded with a series of peptides and proteins of increasing size, including a <20 residue peptide, myoglobin, mCherry, and maltose binding protein, which together cover a range of masses from <2.2 to approximately 44 kDa. Conjugation to the surface of dihydrolipoic acid-functionalized QDs is facilitated by polyhistidine metal affinity coordination. Increasing ratios of dye-labeled peptides and proteins are self-assembled to the QDs and then the bioconjugates are separated and analyzed using agarose gel electrophoresis. Fluorescent visualization of both conjugated and unbound species allows determination of an experimentally derived maximum loading number. Molecular modeling utilizing crystallographic coordinates or space-filling structures of the peptides and proteins also allow the predicted maximum loadings to the QDs to be estimated. Comparison of the two sets of results provides insight into the nature of the QD surface and reflects the important role played by the nanoparticle's hydrophilic solubilizing surface ligands. It is found that for the larger protein molecules steric hindrance is the major packing constraint. In contrast, for the smaller peptides, the number of available QD binding sites is the principal determinant. These results can contribute towards an overall understanding of how to engineer designer bioconjugates for both QDs and other nanoparticle materials.

  3. A high affinity phage-displayed peptide as a recognition probe for the detection of Salmonella Typhimurium.

    PubMed

    Agrawal, Shailaja; Kulabhusan, Prabir Kumar; Joshi, Manali; Bodas, Dhananjay; Paknikar, Kishore M

    2016-08-10

    Salmonellosis is one of the most common and widely distributed foodborne diseases. A sensitive and robust detection method of Salmonella Typhimurium (S. Typhimurium) in food can critically prevent a disease outbreak. In this work, the use of phage displayed peptides was explored for the detection of S. Typhimurium. A phage-displayed random dodecapeptide library was subjected to biopanning against lipopolysaccharide (LPS) of S. Typhimurium. The peptide NFMESLPRLGMH (pep49) derived from biopanning displayed a high affinity (25.8nM) for the LPS of S. Typhimurium and low cross-reactivity with other strains of Salmonella and related Gram-negative bacteria. Molecular insights into the interaction of pep49 with the LPS of S. Typhimurium was gleaned using atomistic molecular dynamics simulations and docking. It was deduced that the specificity of pep49 with S. Typhimurium LPS originated from the interactions of pep49 with abequose that is found only in the O-antigen of S. Typhimurium. Further, pep49 was able to detect S. Typhimurium at a LOD of 10(3) CFU/mL using ELISA, and may be a potential cost efficient alternative to antibodies.

  4. Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display.

    PubMed

    Tammen, Harald; Schulte, Imke; Hess, Rudiger; Menzel, Christoph; Kellmann, Markus; Mohring, Thomas; Schulz-Knappe, Peter

    2005-08-01

    The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.

  5. Isolation of a peptide from Ph.D.-C7C phage display library for detection of Cry1Ab.

    PubMed

    Wang, Yun; Wang, Qian; Wu, Ai-Hua; Hao, Zhen-Ping; Liu, Xian-Jin

    2017-03-06

    Traditional ELISA methods of using animal immunity yield antibodies for detection Cry toxin. Not only is this incredibly harmful to the animals, but is also time-intensive. Here we developed a simple method to yield the recognition element. Using a critical selection strategy and immunoassay we confirmed a clone from the Ph.D-C7C phage library, which has displayed the most interesting Cry1Ab-binding characteristics examined in this study (Fig. 1). The current study indicates that isolating peptide is an alternative method for the preparation of a recognition element, and that the developed assay is a potentially useful tool for detecting Cry1Ab.

  6. Oyster hemocytes express a proline-rich peptide displaying synergistic antimicrobial activity with a defensin.

    PubMed

    Gueguen, Yannick; Bernard, Romestand; Julie, Fievet; Paulina, Schmitt; Delphine, Destoumieux-Garzón; Franck, Vandenbulcke; Philippe, Bulet; Evelyne, Bachère

    2009-02-01

    A cDNA sequence that encodes a 61-amino acid polypeptide precursor with homologies to proline-rich antimicrobial peptides (AMPs) was identified in the oyster Crassostrea gigas. After release of a hydrophobic signal peptide, the resulting 37-amino acid peptide, Cg-Prp, is composed of an acidic region and a cationic proline-rich region. To evaluate the biological properties of Cg-Prp, multiple proline-rich peptides corresponding to putative processing of the full-length Cg-Prp were synthesized. A limited antimicrobial activity was observed for two of them, which also showed strong synergistic antimicrobial activity with Cg-Def, a defensin from C. gigas. To our knowledge, this is the first evidence of synergy between a defensin and another AMP in an invertebrate. By in situ hybridization, the expression of Cg-prp was found to be restricted to hemocytes and induced following bacterial challenge. Cg-prp transcripts were also detected in hemocytes infiltrating mantle, where Cg-Def is expressed. Additionally, by immunocytochemistry, we showed that Cg-Prp or one of its variants is present in some hemocytes together with defensins. In conclusion, we described here the first proline-rich AMP from mollusk. From our study, it is likely to provide a first line of defense against bacterial invasion by acting through synergy with defensins.

  7. β-Amyloid peptides display protective activity against the human Alzheimer's disease-associated herpes simplex virus-1.

    PubMed

    Bourgade, Karine; Garneau, Hugo; Giroux, Geneviève; Le Page, Aurélie Y; Bocti, Christian; Dupuis, Gilles; Frost, Eric H; Fülöp, Tamàs

    2015-02-01

    Amyloid plaques, the hallmark of Alzheimer's disease (AD), contain fibrillar β-amyloid (Aβ) 1-40 and 1-42 peptides. Herpes simplex virus 1 (HSV-1) has been implicated as a risk factor for AD and found to co-localize within amyloid plaques. Aβ 1-40 and Aβ 1-42 display anti-bacterial, anti-yeast and anti-viral activities. Here, fibroblast, epithelial and neuronal cell lines were exposed to Aβ 1-40 or Aβ 1-42 and challenged with HSV-1. Quantitative analysis revealed that Aβ 1-40 and Aβ 1-42 inhibited HSV-1 replication when added 2 h prior to or concomitantly with virus challenge, but not when added 2 or 6 h after virus addition. In contrast, Aβ 1-40 and Aβ 1-42 did not prevent replication of the non-enveloped human adenovirus. In comparison, antimicrobial peptide LL-37 prevented HSV-1 infection independently of its sequence of addition. Our findings showed also that Aβ 1-40 and Aβ 1-42 acted directly on HSV-1 in a cell-free system and prevented viral entry into cells. The sequence homology between Aβ and a proximal transmembrane region of HSV-1 glycoprotein B suggested that Aβ interference with HSV-1 replication could involve its insertion into the HSV-1 envelope. Our data suggest that Aβ peptides represent a novel class of antimicrobial peptides that protect against neurotropic enveloped virus infections such as HSV-1. Overproduction of Aβ peptide to protect against latent herpes viruses and eventually against other infections, may contribute to amyloid plaque formation, and partially explain why brain infections play a pathogenic role in the progression of the sporadic form of AD.

  8. The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism.

    PubMed

    Flaherty, Briana R; Wang, Yuxiao; Trope, Edward C; Ho, Tienhuei G; Muralidharan, Vasant; Kennedy, Eileen J; Peterson, David S

    2015-01-01

    Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 μM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.

  9. Screening a Phage Display Library for Two Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against Vibrio mimicus

    PubMed Central

    Tao, Huizhu; Xiao, Ning; Li, Jinnian; Kong, Lingyan; Hou, Liting

    2016-01-01

    Vibrio mimicus is a pathogen that causes ascites disease in fish. We have previously demonstrated that the outer membrane protein U (OmpU) is an important adhesin in V. mimicus. Here eight specific OmpU-binding phage clones, which presented three different OmpU-binding peptides (designated P1, P2, P3), were screened from a commercially available phage displayed 12-mer peptide library using rOmpU protein as target. Then, synthetic OmpU-binding peptides were measured for their adhesion antagonistic activity and binding affinity via adhesion inhibition test and non-competitive ELISA, respectively. The results showed that after co-incubated with the mixture of rOmpU and P3, visible green fluorescence could be observed on the epithelioma papulosum cyprinidi (EPC) cells surface; while the EPC cells co-incubated with the mixture of rOmpU and P1/P2 exhibited little green fluorescence. The average adhesion number of V. mimicus 04–14 isolate before and after treatment with peptide was 21.4 ± 1.5, 20.8 ± 0.8 (irrelevant peptide), 20.2 ± 0.5 (P3), 5.1 ± 0.7 (P1) and 3.4 ± 0.8 (P2), respectively. There was a significant decrease in the adhesive level of 04–14 isolate treated with P1/ P2 compared to the untreated isolate (p<0.01). The affinity constants of P1 and P2 were (6.17 ± 0.19) × 108 L/mol and (1.24 ± 0.56) × 109 L/mol, respectively. Furthermore, protective effects of P1 and P2 on grass carps challenged with V. mimicus were preliminary detected. It was found there was delayed death of fish in the groups treated with P1/P2, and the survival rate of challenged fish improved with the increase of the dose of adhesion antagonistic peptide. Taken together, two novel OmpU-binding peptides, which possessed adhesion antagonistic activity, high affinity and a certain degree of antibacterial activity against V. mimicus, were screened and identified. PMID:27832083

  10. Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: A study using the hydatid vaccine EG95

    PubMed Central

    Read, A.J.; Gauci, C.G.; Lightowlers, M.W.

    2009-01-01

    The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95. PMID:19349218

  11. A muscle-targeting peptide displayed on AAV2 improves muscle tropism on systemic delivery.

    PubMed

    Yu, C-Y; Yuan, Z; Cao, Z; Wang, B; Qiao, C; Li, J; Xiao, X

    2009-08-01

    Adeno-associated virus (AAV) has become a leading gene transfer vector for striated muscles. However, the AAV vectors also exhibit broad tropisms after systemic delivery. In an attempt to improve muscle tropism, we inserted a 7-amino-acid (ASSLNIA) muscle-targeting peptide (MTP) in the capsids of AAV2 at residue 587 or 588, generating AAV(587)MTP and AAV(588)MTP. In vitro studies showed that both viruses diminished their infectivity on non-muscle cell lines as well as on un-differentiated myoblasts; however, preserved or enhanced their infectivity on differentiated myotubes. AAV(587)MTP, but not AAV(588)MTP, also abolished its heparin-binding capacity and infected myotubes in a heparin-independent manner. Furthermore, in vivo studies by intravenous vector administration in mice showed that AAV(587)MTP enhanced its tropism to various muscles and particularly to the heart (24.3-fold of unmodified AAV2), whereas reduced its tropism to the non-muscle tissues such as the liver, lungs, spleen and so on. This alteration of tissue tropism is not simply because of the loss of heparin-binding, as a mutant AAV2 (AAVHBSMut) containing heparin-binding site mutations lost infectivity on both non-muscle and muscle cells. Furthermore, free MTP peptide, but not the scrambled control peptide, competitively inhibited AAV(587)MTP infection on myotubes. These results suggest that AAV2 could be re-targeted to the striated muscles by a MTP inserted after residue 587 of the capsids. This proof of principle study showed first evidence of peptide-directed muscle targeting on systemic administration of AAV vectors.

  12. Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

    PubMed

    Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

    2014-08-01

    Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma.

  13. RGD peptide-displaying M13 bacteriophage/PLGA nanofibers as cell-adhesive matrices for smooth muscle cells

    NASA Astrophysics Data System (ADS)

    Shin, Yong Cheol; Lee, Jong Ho; Jin, Oh Seong; Lee, Eun Ji; Jin, Lin Hua; Kim, Chang-Seok; Hong, Suck Won; Han, Dong-Wook; Kim, Chuntae; Oh, Jin-Woo

    2015-01-01

    Extracellular matrices (ECMs) are network structures that play an essential role in regulating cellular growth and differentiation. In this study, novel nanofibrous matrices were fabricated by electrospinning M13 bacteriophage and poly(lactic- co-glycolic acid) (PLGA) and were shown to be structurally and functionally similar to natural ECMs. A genetically-engineered M13 bacteriophage was constructed to display Arg-Gly-Asp (RGD) peptides on its surface. The physicochemical properties of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage)/PLGA nanofibers were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. We used immunofluorescence staining to confirm that M13 bacteriophages were homogenously distributed in RGD-M13 phage/PLGA matrices. Furthermore, RGD-M13 phage/PLGA nanofibrous matrices, having excellent biocompatibility, can enhance the behaviors of vascular smooth muscle cells. This result suggests that RGD-M13 phage/PLGA nanofibrous matrices have potentials to serve as tissue engineering scaffolds.

  14. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    PubMed

    Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

    2012-01-01

    Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  15. Biomining with bacteriophage: selectivity of displayed peptides for naturally occurring sphalerite and chalcopyrite.

    PubMed

    Curtis, Susan B; Hewitt, Jeff; Macgillivray, Ross T A; Dunbar, W Scott

    2009-02-01

    During mineral processing, concentrates of sulfide minerals of economic interest are formed by froth flotation of fine ore particles. The method works well but recovery and selectivity can be poor for ores with complex mineralogy. There is considerable interest in methods that improve the selectivity of this process while avoiding the high costs of using flotation chemicals. Here we show the first application of phage biotechnology to the processing of economically important minerals in ore slurries. A random heptapeptide library was screened for peptide sequences that bind selectively to the minerals sphalerite (ZnS) and chalcopyrite (CuFeS2). After several rounds of enrichment, cloned phage containing the surface peptide loops KPLLMGS and QPKGPKQ bound specifically to sphalerite. Phage containing the peptide loop TPTTYKV bound to both sphalerite and chalcopyrite. By using an enzyme-linked immunosorbant assay (ELISA), the phage was characterized as strong binders compared to wild-type phage. Specificity of binding was confirmed by immunochemical visualization of phage bound to mineral particles but not to silica (a waste mineral) or pyrite. The current study focused primarily on the isolation of ZnS-specific phage that could be utilized in the separation of sphalerite from silica. At mining sites where sphalerite and chalcopyrite are not found together in natural ores, the separation of sphalerite from silica would be an appropriate enrichment step. At mining sites where sphalerite and chalcopyrite do occur together, more specific phage would be required. This bacteriophage has the potential to be used in a more selective method of mineral separation and to be the basis for advanced methods of mineral processing.

  16. Identification of an Immunogenic Mimic of a Conserved Epitope on the Plasmodium falciparum Blood Stage Antigen AMA1 Using Virus-Like Particle (VLP) Peptide Display

    PubMed Central

    Crossey, Erin; Frietze, Kathryn; Narum, David L.; Peabody, David S.; Chackerian, Bryce

    2015-01-01

    We have developed a peptide display platform based on VLPs of the RNA bacteriophage MS2 that combines the high immunogenicity of VLP display with affinity selection capabilities. Random peptides can be displayed on the VLP surface by genetically inserting sequences into a surface-exposed loop of the viral coat protein. VLP-displayed peptides can then be isolated by selection using antibodies, and the VLP selectants can then be used directly as immunogens. Here, we investigated the ability of this platform to identify mimotopes of a highly conserved conformational epitope present on the Plasmodium falciparum blood-stage protein AMA1. Using 4G2, a monoclonal antibody that binds to this epitope and is a potent inhibitor of erythrocyte invasion, we screened three different VLP-peptide libraries and identified specific VLPs that bound strongly to the selecting mAb. We then tested the ability of a handful of selected VLPs to elicit anti-AMA1 antibody responses in mice. Most of the selected VLPs failed to reliably elicit AMA1 specific antibodies. However, one VLP consistently induced antibodies that cross-reacted with AMA1. Surprisingly, this VLP bound to 4G2 more weakly than the other selectants we identified. Taken together, these data demonstrate that VLP-peptide display can identify immunogenic mimics of a complex conformational epitope and illustrate the promise and challenges of this approach. PMID:26147502

  17. Peptide mimics selected from immune sera using phage display technology can replace native antigens in the diagnosis of Epstein-Barr virus infection.

    PubMed

    Casey, J L; Coley, A M; Parisi, K; Foley, M

    2009-02-01

    There is an expanding area of small molecule discovery, especially in the area of peptide mimetics. Peptide sequences can be used to substitute for the entire native antigen for use in diagnostic assays. Our approach is to select peptides that mimic epitopes of the natural immune response to Epstein-Barr virus (EBV) that may be recognised by antibodies typically produced after infection with EBV. We screened a random peptide library on sera from rabbits immunised with a crude preparation of EBV and serum antibodies from a patient with a high titer of EBV antibodies. We selected four peptides (Eb1-4) with the highest relative binding affinity with immune rabbit sera and a single peptide with high affinity to human serum antibodies. The peptides were coupled to the carrier molecule BSA and the recognition of the peptides by IgM antibodies in clinical samples after infection with EBV was measured. The sensitivities were Eb1 94%, Eb2, 3, 4 88%, H1 81% and all had 100% specificity. This study illustrates that the phage display approach to select epitope mimics can be applied to polyclonal antibodies and peptides that represent several diagnostically important epitopes can be selected simultaneously. This panel of EBV peptides representing a wide coverage of immunodominant epitopes could replace crude antigen preparations currently used for capture in commercial diagnostic tests for EBV.

  18. Peptide mimics selected from immune sera using phage display technology can replace native antigens in the diagnosis of Epstein–Barr virus infection

    PubMed Central

    Casey, J.L.; Coley, A.M.; Parisi, K.; Foley, M.

    2009-01-01

    There is an expanding area of small molecule discovery, especially in the area of peptide mimetics. Peptide sequences can be used to substitute for the entire native antigen for use in diagnostic assays. Our approach is to select peptides that mimic epitopes of the natural immune response to Epstein–Barr virus (EBV) that may be recognised by antibodies typically produced after infection with EBV. We screened a random peptide library on sera from rabbits immunised with a crude preparation of EBV and serum antibodies from a patient with a high titer of EBV antibodies. We selected four peptides (Eb1–4) with the highest relative binding affinity with immune rabbit sera and a single peptide with high affinity to human serum antibodies. The peptides were coupled to the carrier molecule BSA and the recognition of the peptides by IgM antibodies in clinical samples after infection with EBV was measured. The sensitivities were Eb1 94%, Eb2, 3, 4 88%, H1 81% and all had 100% specificity. This study illustrates that the phage display approach to select epitope mimics can be applied to polyclonal antibodies and peptides that represent several diagnostically important epitopes can be selected simultaneously. This panel of EBV peptides representing a wide coverage of immunodominant epitopes could replace crude antigen preparations currently used for capture in commercial diagnostic tests for EBV. PMID:19073711

  19. Production and Evaluation of Antibodies and Phage Display-Derived Peptide Ligands for Immunomagnetic Separation of Mycobacterium bovis

    PubMed Central

    Stewart, Linda D.; McNair, James; McCallan, Lyanne; Thompson, Suzan; Kulakov, Leonid A.

    2012-01-01

    This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 105 CFU/ml) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis. PMID:22322353

  20. Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob

    PubMed Central

    Yamamoto, Yuki; Goto, Naoko; Miura, Kazuki; Narumi, Kenta; Ohnami, Shumpei; Uchida, Hiroaki; Miura, Yoshiaki; Yamamoto, Masato; Aoki, Kazunori

    2014-01-01

    Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 104 level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:24380399

  1. The HGF inhibitory peptide HGP-1 displays promising in vitro and in vivo efficacy for targeted cancer therapy.

    PubMed

    Chen, Lisha; Li, Chunlin; Zhu, Yimin

    2015-10-06

    HGF/MET pathway mediates cancer initiation and development. Thus, inhibition on HGF-initiated MET signaling pathway would provide a new approach to cancer targeted therapeutics. In our study, we identified a targeting peptide candidate binding to HGF which was named HGF binding peptide-1 (HGP-1) via bacterial surface display methods coupled with fluorescence-activated cell sorting (FACS). HGP-1 showed the moderate affinity when determined with surface plasmon resonance (SPR) technique and high specificity in binding to HGF while assessed by fluorescence-based ELISA assay. The results from MTT and in vitro migration assay indicated that HGF-dependent cell proliferation and migration could be inhibited by HGP-1. In vivo administration of HGP-1 led to an effective inhibitory effect on tumor growth in A549 tumor xenograft models. Moreover, findings from Western Blots revealed that HGP-1 could down-regulated the phosphorylation levels of MET and ERK1/2 initiated by HGF, which suggested that HGP-1 could disrupt the activation of HGF/MET signaling to influence the cell activity. All the data highlighted the potential of HGP-1 to be a potent inhibitor for HGF/MET signaling.

  2. Targeting essential Eimeria ninakohlyakimovae sporozoite ligands for caprine host endothelial cell invasion with a phage display peptide library.

    PubMed

    Ruiz, A; Pérez, D; Muñoz, M C; Molina, J M; Taubert, A; Jacobs-Lorena, M; Vega-Rodríguez, J; López, A M; Hermosilla, C

    2015-11-01

    Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe diarrhoea in young animals. Specific molecules that mediate E. ninakohlyakimovae host interactions and molecular mechanisms involved in the pathogenesis are still unknown. Although strong circumstantial evidence indicates that E. ninakohlyakimovae sporozoite interactions with caprine endothelial host cells (ECs) are specific, hardly any information is available about the interacting molecules that confer host cell specificity. In this study, we describe a novel method to identify surface proteins of caprine umbilical vein endothelial cells (CUVEC) using a phage display library. After several panning rounds, we identified a number of peptides that specifically bind to the surface of CUVEC. Importantly, caprine endothelial cell peptide 2 (PCEC2) and PCEC5 selectively reduced the infection rate by E. ninakohlyakimovae sporozoites. These preliminary data give new insight for the molecular identification of ligands involved in the interaction between E. ninakohlyakimovae sporozoites and host ECs. Further studies using this phage approach might be useful to identify new potential target molecules for the development of anti-coccidial drugs or even new vaccine strategies.

  3. Phage displayed peptides/antibodies recognizing growth factors and their tyrosine kinase receptors as tools for anti-cancer therapeutics.

    PubMed

    Ronca, Roberto; Benzoni, Patrizia; De Luca, Angela; Crescini, Elisabetta; Dell'era, Patrizia

    2012-01-01

    The basic idea of displaying peptides on a phage, introduced by George P. Smith in 1985, was greatly developed and improved by McCafferty and colleagues at the MRC Laboratory of Molecular Biology and, later, by Barbas and colleagues at the Scripps Research Institute. Their approach was dedicated to building a system for the production of antibodies, similar to a naïve B cell repertoire, in order to by-pass the standard hybridoma technology that requires animal immunization. Both groups merged the phage display technology with an antibody library to obtain a huge number of phage variants, each of them carrying a specific antibody ready to bind its target molecule, allowing, later on, rare phage (one in a million) to be isolated by affinity chromatography. Here, we will briefly review the basis of the technology and the therapeutic application of phage-derived bioactive molecules when addressed against key players in tumor development and progression: growth factors and their tyrosine kinase receptors.

  4. A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins

    PubMed Central

    1995-01-01

    Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*- peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD. PMID:7657703

  5. Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

    PubMed Central

    Zhao, Ruiming; Dai, Hui; Mendelman, Netanel; Cuello, Luis G.; Chill, Jordan H.; Goldstein, Steve A. N.

    2015-01-01

    Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with Ki of 1–4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors. PMID:26627718

  6. Phage-peptide display identifies the interferon-responsive, death-activated protein kinase family as a novel modifier of MDM2 and p21WAF1.

    PubMed

    Burch, Lindsay R; Scott, Mary; Pohler, Elizabeth; Meek, David; Hupp, Ted

    2004-03-12

    Phage-peptide display is a versatile tool for identifying novel protein-protein interfaces. Our previous work highlighted the selection of phage-peptides that bind to specific isoforms of MDM2 protein and in this work we subjected the putative MDM2-binding proteins to phage-peptide display to expand further on putative protein interaction maps. One peptide that bound MDM2 had significant homology to members of the death-activated protein kinase (DAPK) family, an enzyme family of no known direct link to the p53 pathway. We examined whether a nuclear member of the DAPK family named DAPK3 or ZIP kinase had direct links to the p53 pathway. ZIP kinase was cloned, purified, and the enzyme was able to phosphorylate MDM2 at Ser166, a site previously reported to be modified by Akt kinase, thus demonstrating that ZIP kinase is a bona fide MDM2-binding protein. Native ZIP kinase fractions were then subjected to phage-peptide display and one ZIP kinase consensus peptide motif was identified in p21(WAF1). ZIP kinase phosphorylates p21(WAF1) at Thr145 and alanine-substituted mutations in the p21(WAF1) phosphorylation site alter its ability to be phosphorylated by ZIP kinase. Thus, although ZIP kinase consensus sites were then defined as containing a minimal RKKx(T/S) consensus motif, alternate contacts in ZIP kinase binding are implicated, since amino acid residues surrounding the phospho-acceptor site can effect the specific activity of the kinase. Transfected ZIPK can promote the phosphorylation of p21(WAF1) at Thr145 in vivo and can increase the half-life of p21(WAF1), while the half-life of p21(WAF1[T145A]) is not effected by ZIP kinase. Thus, phage-peptide display identified an interferon-responsive protein kinase family as a novel modifier of two components of the p53 pathway, MDM2 and p21(WAF1), and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways.

  7. Ligand binding analyses of the putative peptide transporter YjdL from E. coli display a significant selectivity towards dipeptides

    SciTech Connect

    Ernst, Heidi A.; Pham, Antony; Hald, Helle; Kastrup, Jette S.; Rahman, Moazur; Mirza, Osman

    2009-11-06

    Proton-dependent oligopeptide transporters (POTs) are secondary active transporters that couple the inwards translocation of di- and tripeptides to inwards proton translocation. Escherichia coli contains four genes encoding the putative POT proteins YhiP, YdgR, YjdL and YbgH. We have over-expressed the previously uncharacterized YjdL and investigated the peptide specificity by means of uptake inhibition. The IC{sub 50} value for the dipeptide Ala-Ala was measured to 22 mM while Ala-Ala-Ala was not able to inhibit uptake. In addition, IC{sub 50} values of 0.3 mM and 1.5 mM were observed for Ala-Lys and Tyr-Ala, respectively, while the alanyl-extended tripeptides Ala-Lys-Ala, Ala-Ala-Lys, Ala-Tyr-Ala and Tyr-Ala-Ala displayed values of 8, >50, 31 and 31 mM, respectively. These results clearly indicate that unlike most POT members characterized to date, including YdgR and YhiP, YjdL shows significantly higher specificity towards dipeptides.

  8. Simulated digestion for testing the stability of edible vaccine based on Cucumber mosaic virus (CMV) chimeric particle display Hepatitis C virus (HCV) peptide.

    PubMed

    Vitti, Antonella; Nuzzaci, Maria; Condelli, Valentina; Piazzolla, Pasquale

    2014-01-01

    Edible vaccines must survive digestive process and preserve the specific structure of the antigenic peptide to elicit effective immune response. The stability of a protein to digestive process can be predicted by subjecting it to the in vitro assay with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Here, we describe the protocol of producing and using chimeric Cucumber mosaic virus (CMV) displaying Hepatitis C virus (HCV) derived peptide (R9) in double copy as an oral vaccine. Its stability after treatment with SGF and SIF and the preservation of the antigenic properties were verified by SDS-PAGE and immuno western blot techniques.

  9. cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA–protein fusions

    PubMed Central

    Yamaguchi, Junichi; Naimuddin, Mohammed; Biyani, Manish; Sasaki, Toru; Machida, Masayuki; Kubo, Tai; Funatsu, Takashi; Husimi, Yuzuru; Nemoto, Naoto

    2009-01-01

    We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA–protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a ‘ligation site’ for T4 RNA ligase, a ‘biotin site’ for solid-phase handling, a ‘reverse transcription primer site’ for the efficient and rapid conversion from an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a ‘restriction enzyme site’ for the release of a complex from the solid support. This enables not only stabilizing mRNA–protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications. PMID:19528071

  10. Novel miR-122 delivery system based on MS2 virus like particle surface displaying cell-penetrating peptide TAT for hepatocellular carcinoma

    PubMed Central

    Wang, Guojing; Jia, Tingting; Xu, Xixia; Chang, Le; Zhang, Rui; Fu, Yu; Li, Yulong; Yang, Xin; Zhang, Kuo; Lin, Guigao; Han, Yanxi; Li, Jinming

    2016-01-01

    Current treatments for hepatocellular carcinoma (HCC) have shown inadequate. MicroRNA-122 (miR-122) mediated RNA interference brings new prospects. A safe, efficient miRNA delivery system is an indispensable assurance. Previously, we developed an MS2 bacteriophage virus-like particle (VLP)-based microRNA delivery system crosslinked with the HIV TAT peptide, which served as an effective inhibitor in the treatments of systemic lupus erythematosus and osteoporosis. However, defects, such as low crosslinking efficiency, high cost, and potential toxicity of the crosslinking agent, needed to be confronted. Therefore, TAT peptide was designed to display on the surface of MS2 VLPs, instead of being chemically crosslinked, using the platform of phage surface display. The results reflected that MS2 VLPs displaying TAT could effectively penetrate the cytomembrane and deliver miR-122. Additionally, its inhibitory effects on HCC were significant in Hep3B, HepG2, and Huh7 cells and Hep3B related animal models. Thus, we have established a novel miR-122 delivery system based on MS2 VLPs surface displaying TAT peptide, which could effectively perform the function of penetrating cytomembrane and the inhibition of HCC. PMID:27449085

  11. Double Candida antarctica lipase B co-display on Pichia pastoris cell surface based on a self-processing foot-and-mouth disease virus 2A peptide.

    PubMed

    Sun, Yu-Fei; Lin, Ying; Zhang, Jun-Hui; Zheng, Sui-Ping; Ye, Yan-Rui; Liang, Xing-Xiang; Han, Shuang-Yan

    2012-12-01

    To develop a high efficiency Candida antarctica lipase B (CALB) yeast display system, we linked two CALB genes fused with Sacchromyces cerevisiae cell wall protein genes, the Sed1 and the 3'-terminal half of Sag1, separately by a 2A peptide of foot-and-mouth disease virus (FMDV) in a single open reading frame. The CALB copy number of recombinant strain KCSe2ACSa that harbored the ORF was identified, and the quantity of CALB displayed on the cell surface and the enzyme activity of the strain were measured. The results showed that the fusion of multiple genes linked by 2A peptide was translated into two independent proteins displayed on the cell surface of stain KCSe2ACSa. Judging from the data of immunolabeling assay, stain KCSe2ACSa displayed 94 % CALB-Sed1p compared with stain KCSe1 that harbored a single copy CALB-Sed1 and 64 % CALB-Sag1p compared with stain KCSa that harbored a single copy CALB-Sag1 on its surface. Besides, strain KCSe2ACSa possessed 170 % hydrolytic activity and 155 % synthetic activity compared with strain KCSe1 as well as 144 % hydrolytic activity and 121 % synthetic activity compared with strain KCSa. Strain KCSe2ACSa even owned 124 % hydrolytic activity compared with strain KCSe2 that harbored two copies CALB-Sed1. The heterogeneous glycosylphosphatidylinositol-anchored proteins co-displaying yeast system mediated by FMDV 2A peptide was shown to be an effective method for improving the efficiency of enzyme-displaying yeast biocatalysts.

  12. Peptides Derived from a Phage Display Library Inhibit Adhesion and Protect the Host against Infection by Paracoccidioides brasiliensis and Paracoccidioides lutzii

    PubMed Central

    de Oliveira, Haroldo C.; Michaloski, Jussara S.; da Silva, Julhiany F.; Scorzoni, Liliana; de Paula e Silva, Ana C. A.; Marcos, Caroline M.; Assato, Patrícia A.; Yamazaki, Daniella S.; Fusco-Almeida, Ana M.; Giordano, Ricardo J.; Mendes-Giannini, Maria J. S.

    2016-01-01

    Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and are the etiological agents of paracoccidioidomycosis (PCM). Adhesion is one of the most important steps in infections with Paracoccidioides and is responsible for the differences in the virulence of isolates of these fungi. Because of the importance of adhesion to the establishment of an infection, this study focused on the preliminary development of a new therapeutic strategy to inhibit adhesion by Paracoccidioides, thus inhibiting infection and preventing the disease. We used two phage display libraries to select peptides that strongly bind to the Paracoccidioides cell wall to inhibit adhesion to host cells and extracellular matrix (ECM) components (laminin, fibronectin, and type I and type IV collagen). This approach allowed us to identify four peptides that inhibited up to 64% of the adhesion of Paracoccidioides to pneumocytes in vitro and inhibited the adhesion to the ECM components by up to 57%. Encouraged by these results, we evaluated the ability of these peptides to protect Galleria mellonella from Paracoccidioides infection by treating G. mellonella larvae with the different peptides prior to infection with Paracoccidioides and observing larval survival. The results show that all of the peptides tested increased the survival of the larvae infected with P. brasiliensis by up to 64% and by up to 60% in those infected with P. lutzii. These data may open new horizons for therapeutic strategies to prevent PCM, and anti-adhesion therapy could be an important strategy. PMID:28066254

  13. Peptides Derived from a Phage Display Library Inhibit Adhesion and Protect the Host against Infection by Paracoccidioides brasiliensis and Paracoccidioides lutzii.

    PubMed

    de Oliveira, Haroldo C; Michaloski, Jussara S; da Silva, Julhiany F; Scorzoni, Liliana; de Paula E Silva, Ana C A; Marcos, Caroline M; Assato, Patrícia A; Yamazaki, Daniella S; Fusco-Almeida, Ana M; Giordano, Ricardo J; Mendes-Giannini, Maria J S

    2016-01-01

    Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and are the etiological agents of paracoccidioidomycosis (PCM). Adhesion is one of the most important steps in infections with Paracoccidioides and is responsible for the differences in the virulence of isolates of these fungi. Because of the importance of adhesion to the establishment of an infection, this study focused on the preliminary development of a new therapeutic strategy to inhibit adhesion by Paracoccidioides, thus inhibiting infection and preventing the disease. We used two phage display libraries to select peptides that strongly bind to the Paracoccidioides cell wall to inhibit adhesion to host cells and extracellular matrix (ECM) components (laminin, fibronectin, and type I and type IV collagen). This approach allowed us to identify four peptides that inhibited up to 64% of the adhesion of Paracoccidioides to pneumocytes in vitro and inhibited the adhesion to the ECM components by up to 57%. Encouraged by these results, we evaluated the ability of these peptides to protect Galleria mellonella from Paracoccidioides infection by treating G. mellonella larvae with the different peptides prior to infection with Paracoccidioides and observing larval survival. The results show that all of the peptides tested increased the survival of the larvae infected with P. brasiliensis by up to 64% and by up to 60% in those infected with P. lutzii. These data may open new horizons for therapeutic strategies to prevent PCM, and anti-adhesion therapy could be an important strategy.

  14. Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

    PubMed Central

    Xue, Mei; Shi, Xingming; Zhang, Jing; Zhao, Yan; Cui, Hongyu; Hu, Shunlei; Gao, Hongbo; Cui, Xianlan; Wang, Yun-Feng

    2012-01-01

    Background The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. Methods and Results This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched 213SVQYHPL219 of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that 213SVQYHPL219 was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. Conclusions and Significance We identified 213SVQYHPL219 as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group. PMID:23185456

  15. Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

    SciTech Connect

    Dai, Xiaoyong; Cai, Cuizan; Xiao, Fei; Xiong, Yaoling; Huang, Yadong; Zhang, Qihao; Xiang, Qi; Lou, Guofeng; Lian, Mengyang; Su, Zhijian; Zheng, Qing

    2014-03-21

    Highlights: • A specific aFGF-binding peptide AP8 was identified from a phage display library. • AP8 could inhibit aFGF-stimulated cell proliferation in a dose-dependent manner. • AP8 arrested the cell cycle at the G0/G1 phase by suppressing Cyclin D1. • AP8 could block the activation of Erk1/2 and Akt kinase. • AP8 counteracted proliferation and cell cycle via influencing PA2G4 and PCNA. - Abstract: It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.

  16. Phage display evolution of a peptide substrate for yeast biotin ligase and application to two-color quantum dot labeling of cell surface proteins.

    PubMed

    Chen, Irwin; Choi, Yoon-Aa; Ting, Alice Y

    2007-05-23

    Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to two-color imaging and yeast-based applications.

  17. Antibody binding site mapping of SARS-CoV spike protein receptor-binding domain by a combination of yeast surface display and phage peptide library screening.

    PubMed

    Zhang, Xiaoping; Wang, Jingxue; Wen, Kun; Mou, Zhirong; Zou, Liyun; Che, Xiaoyan; Ni, Bing; Wu, Yuzhang

    2009-12-01

    The receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays an important role in viral infection, and is a potential major neutralizing determinant. In this study, three hybridoma cell lines secreting specific monoclonal antibodies against the RBD of the S protein were generated and their exact binding sites were identified. Using yeast surface display, the binding sites of these antibodies were defined to two linear regions on the RBD: S(337-360) and S(380-399). Using these monoclonal antibodies in phage peptide library screening identified 10 distinct mimotopes 12 amino acids in length. Sequence comparison between native epitopes and these mimotopes further confirmed the binding sites, and revealed key amino acid residues involved in antibody binding. None of these antibodies could neutralize the murine leukemia virus pseudotyped expressing the SARS-CoV spike protein (MLV/SARS-CoV). However, these mAbs could be useful in the diagnosis of SARS-CoV due to their exclusive reactivity with SARS-CoV. Furthermore, this study established a feasible platform for epitope mapping. Yeast surface display combined with phage peptide library screening provides a convenient strategy for the identification of epitope peptides from certain antigenic proteins.

  18. Screening and evaluating the mimic peptides as a useful serum biomarker of ankylosing spondylitis using a phage display technique.

    PubMed

    Wang, Min; Li, Xianping; Chen, Jingwei; Zhou, Yong; Cao, Hong; Wu, Xiang; Jiang, Hongmin

    2011-08-01

    To screen specific serum biomarker for ankylosing spondylitis (AS) using a phage random peptide library. A phage random peptide library of random peptide 12-mers was immunoscreened with purified immunoglobulin (Ig) G from sera of AS patients. Positive clones obtained after three rounds of biopanning were detected with ELISA and sequenced. Reaction of the screened positive clones with sera from AS patients, systemic lupus erythematosus (SLE) patients, rheumatoid arthritis (RA) patients, osteoarthritis (OA) patients and healthy controls was detected using phage ELISA. Correlation among erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and the absorbance value of the positive clone in phage ELISA was examined in AS patients. Seventeen out of twenty randomly selected phage clones exhibited specific reaction with purified sera IgG from AS patients, among them seven coming from the same clone whose inserted peptide sequence was LALPPLAPNHHH (named "AS1"). Phage ELISA results showed that the positive reaction rate of the AS1 clone was 92.0% with AS patients, significantly different (P < 0.01) from those with SLE patients (56.7%), RA patients (50.0%), OA patients (13.3%), and healthy controls (14.0%). Absorbance value of the AS1 clone in phage ELISA was significantly higher than those in the other groups (P < 0.05). In addition, the absorbance value of the AS1 clone showed no statistically significant correlation with ESR and CRP in AS patients, suggesting that AS1 detects AS patients through a unique mechanism other than inflammation. The short peptide AS1 obtained through screening of a phage random peptide library with purified serum IgG from AS patients can specifically react with the sera of AS patients, and thereby may be a candidate of AS-specific serum biomarkers.

  19. Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

    NASA Astrophysics Data System (ADS)

    Steingroewer, Juliane; Bley, Thomas; Bergemann, Christian; Boschke, Elke

    2007-04-01

    Analyses of food-borne pathogens are of great importance in order to minimize the health risk for customers. Thus, very sensitive and rapid detection methods are required. Current conventional culture techniques are very time consuming. Modern immunoassays and biochemical analysis also require pre-enrichment steps resulting in a turnaround time of at least 24 h. Biomagnetic separation (BMS) is a promising more rapid method. In this study we describe the isolation of high affine and specific peptides from a phage-peptide library, which combined with BMS allows the detection of Salmonella spp. with a similar sensitivity as that of immunomagnetic separation using antibodies.

  20. A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches

    PubMed Central

    Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun

    2016-01-01

    Background Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. Methods and Results To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. Conclusions and Significance We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV. PMID:27191594

  1. Armadillidin H, a Glycine-Rich Peptide from the Terrestrial Crustacean Armadillidium vulgare, Displays an Unexpected Wide Antimicrobial Spectrum with Membranolytic Activity

    PubMed Central

    Verdon, Julien; Coutos-Thevenot, Pierre; Rodier, Marie-Helene; Landon, Celine; Depayras, Segolene; Noel, Cyril; La Camera, Sylvain; Moumen, Bouziane; Greve, Pierre; Bouchon, Didier; Berjeaud, Jean-Marc; Braquart-Varnier, Christine

    2016-01-01

    Antimicrobial peptides (AMPs) are key components of innate immunity and are widespread in nature, from bacteria to vertebrate animals. In crustaceans, there are currently 15 distinct AMP families published so far in the literature, mainly isolated from members of the Decapoda order. Up to now, armadillidin is the sole non-decapod AMP isolated from the haemocytes of Armadillidium vulgare, a crustacean isopod. Its first description demonstrated that armadillidin is a linear glycine-rich (47%) cationic peptide with an antimicrobial activity directed toward Bacillus megaterium. In the present work, we report identification of armadillidin Q, a variant of armadillidin H (earlier known as armadillidin), from crude haemocyte extracts of A. vulgare using LC-MS approach. We demonstrated that both armadillidins displayed broad spectrum antimicrobial activity against several Gram-positive and Gram-negative bacteria, fungi, but were totally inactive against yeasts. Membrane permeabilization assays, only performed with armadillidin H, showed that the peptide is membrane active against bacterial and fungal strains leading to deep changes in cell morphology. This damaging activity visualized by electronic microscopy correlates with a rapid decrease of cell viability leading to highly blebbed cells. In contrast, armadillidin H does not reveal cytotoxicity toward human erythrocytes. Furthermore, no secondary structure could be defined in this study [by circular dichroism (CD) and nuclear magnetic resonance (NMR)] even in a membrane mimicking environment. Therefore, armadillidins represent interesting candidates to gain insight into the biology of glycine-rich AMPs. PMID:27713732

  2. Armadillidin H, a Glycine-Rich Peptide from the Terrestrial Crustacean Armadillidium vulgare, Displays an Unexpected Wide Antimicrobial Spectrum with Membranolytic Activity.

    PubMed

    Verdon, Julien; Coutos-Thevenot, Pierre; Rodier, Marie-Helene; Landon, Celine; Depayras, Segolene; Noel, Cyril; La Camera, Sylvain; Moumen, Bouziane; Greve, Pierre; Bouchon, Didier; Berjeaud, Jean-Marc; Braquart-Varnier, Christine

    2016-01-01

    Antimicrobial peptides (AMPs) are key components of innate immunity and are widespread in nature, from bacteria to vertebrate animals. In crustaceans, there are currently 15 distinct AMP families published so far in the literature, mainly isolated from members of the Decapoda order. Up to now, armadillidin is the sole non-decapod AMP isolated from the haemocytes of Armadillidium vulgare, a crustacean isopod. Its first description demonstrated that armadillidin is a linear glycine-rich (47%) cationic peptide with an antimicrobial activity directed toward Bacillus megaterium. In the present work, we report identification of armadillidin Q, a variant of armadillidin H (earlier known as armadillidin), from crude haemocyte extracts of A. vulgare using LC-MS approach. We demonstrated that both armadillidins displayed broad spectrum antimicrobial activity against several Gram-positive and Gram-negative bacteria, fungi, but were totally inactive against yeasts. Membrane permeabilization assays, only performed with armadillidin H, showed that the peptide is membrane active against bacterial and fungal strains leading to deep changes in cell morphology. This damaging activity visualized by electronic microscopy correlates with a rapid decrease of cell viability leading to highly blebbed cells. In contrast, armadillidin H does not reveal cytotoxicity toward human erythrocytes. Furthermore, no secondary structure could be defined in this study [by circular dichroism (CD) and nuclear magnetic resonance (NMR)] even in a membrane mimicking environment. Therefore, armadillidins represent interesting candidates to gain insight into the biology of glycine-rich AMPs.

  3. Identification of two linear B-cell epitopes from West Nile virus NS1 by screening a phage-displayed random peptide library

    PubMed Central

    2011-01-01

    Background The West Nile virus (WNV) nonstructural protein 1 (NS1) is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. Results The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs) 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 (895LTATTEK901 and 925VVDGPETKEC934). Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that 896TATTEK901 and925VVDGPETKEC934 are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV) serocomplex. Conclusions We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex. PMID:21729328

  4. Screening a phage display library for a novel FGF8b-binding peptide with anti-tumor effect on prostate cancer

    SciTech Connect

    Wang, Wenhui; Chen, Xilei; Li, Tao; Li, Yanmei; Wang, Ruixue; He, Dan; Luo, Wu; Li, Xiaokun; Wu, Xiaoping

    2013-05-01

    Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163–169 (LLAVPAA) directly participating in ligand binding, carried the same charges as its corresponding motif (aa163–169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer. - Highlights: ► A novel FGF8b-binding peptide P12 was isolated from a phage display library. ► The mechanisms for P12 peptide inhibiting cell proliferation were proposed. ► P12 caused cell cycle arrest at G0/G1 phase via suppression of Cyclin D1 and PCNA. ► P12 suppressed FGF8b-induced activations of Akt and MAP kinases. ► P12 acting as an FGF8b antagonist may have therapeutic potential in prostate cancer.

  5. An integrated microfluidic system for screening of phage-displayed peptides specific to colon cancer cells and colon cancer stem cells.

    PubMed

    Che, Yu-Jui; Wu, Huei-Wen; Hung, Lien-Yu; Liu, Ching-Ann; Chang, Hwan-You; Wang, Kuan; Lee, Gwo-Bin

    2015-09-01

    Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.

  6. Analysis of the CD2 and spliceosomal Sm B/B' polyproline-arginine motifs defined by a monoclonal antibody using a phage-displayed random peptide library.

    PubMed

    Monos, Dimitri; Heliopoulos, John; Argyris, Elias; Cordopatis, Paul; Zompra, Aikaterini; Kamoun, Malek

    2006-01-01

    The cytoplasmic region of the CD2 receptor of lymphocytes contains proline-rich motifs, which are involved in T cell activation and interleukin-2 production. An intracellular CD2 binding protein, CD2BP2, interacts with two tandem PPPPGHR segments of the CD2 tail. CD2BP2 contains a GYF (glycine-tyrosine-phenylalanine) domain that confers binding to these proline-rich sequences. Monoclonal antibody 3E10 that was previously raised against a peptide containing the CD2 PPPPGHR segment reacts with the native CD2 molecule and spliceosomal Sm B/B' proteins. To identify the exact epitope on the CD2 peptide recognized by 3E10, a phage-displayed combinatorial peptide library was used. Analysis of the selected clones revealed that the mAb 3E10 binds preferentially to the motif PxxPPGxR. Experiments using amino acid substitutions with synthetic peptides confirmed the reactivity of mAb 3E10 with this motif. In addition, we show that several similarities exist between this motif and the CD2BP2-GFY recognition motif PPGxR/K. Binding of antibody 3E10 indicates some degree of degeneracy, which is consistent with its ability to recognize structurally related polyproline-arginine motifs found in intracellular proteins including Sm B/B' proteins and other RNA binding proteins. Thus, mAb 3E10 can be used to specifically identify a sub-class of proline-rich motifs, and as such can be used to study the potential role of these proline-rich sequences in mediating protein-protein interactions.

  7. Peptide sequences identified by phage display are immunodominant functional motifs of Pet and Pic serine proteases secreted by Escherichia coli and Shigella flexneri.

    PubMed

    Ulises, Hernández-Chiñas; Tatiana, Gazarian; Karlen, Gazarian; Guillermo, Mendoza-Hernández; Juan, Xicohtencatl-Cortes; Carlos, Eslava

    2009-12-01

    Plasmid-encoded toxin (Pet) and protein involved in colonization (Pic), are serine protease autotransporters of Enterobacteriaceae (SPATEs) secreted by enteroaggregative Escherichia coli (EAEC), which display the GDSGSG sequence or the serine motif. Our research was directed to localize functional sites in both proteins using the phage display method. From a 12mer linear and a 7mer cysteine-constrained (C7C) libraries displayed on the M13 phage pIII protein we selected different mimotopes using IgG purified from sera of children naturally infected with EAEC producing Pet and Pic proteins, and anti-Pet and anti-Pic IgG purified from rabbits immunized with each one of these proteins. Children IgG selected a homologous group of sequences forming the consensus sequence, motif, PQPxK, and the motifs PGxI/LN and CxPDDSSxC were selected by the rabbit anti-Pet and anti-Pic IgGs, respectively. Analysis of the amino terminal region of a panel of SPATEs showed the presence in all of them of sequences matching the PGxI/LN or CxPDDSSxC motifs, and in a three-dimensional model (Modeller 9v2) designed for Pet, both these motifs were found in the globular portion of the protein, close to the protease active site GDSGSG. Antibodies induced in mice by mimotopes carrying the three aforementioned motifs were reactive with Pet, Pic, and with synthetic peptides carrying the immunogenic mimotope sequences TYPGYINHSKA and LLPQPPKLLLP, thus confirming that the peptide moiety of the selected phages induced the antibodies specific for the toxins. The antibodies induced in mice to the PGxI/LN and CxPDDSSxC mimotopes inhibited fodrin proteolysis and macrophage chemotaxis biological activities of Pet. Our results showed that we were able to generate, by a phage display procedure, mimotopes with sequence motifs PGxI/LN and CxPDDSSxC, and to identify them as functional motifs of the Pet, Pic and other SPATEs involved in their biological activities.

  8. Screening of paclitaxel-binding molecules from a library of random peptides displayed on T7 phage particles using paclitaxel-photoimmobilized resin.

    PubMed

    Aoki, Sota; Morohashi, Kengo; Sunoki, Takashi; Kuramochi, Kouji; Kobayashi, Susumu; Sugawara, Fumio

    2007-01-01

    Paclitaxel (Taxol), an effective anticancer agent, is known to bind to tubulin and induce tubulin polymerization. Several other binding proteins of paclitaxel, such as Bcl-2, heat shock proteins, and NSC-1, have also been reported. Here, we describe a T7 phage-based display to screen for paclitaxel-binding molecules from a random peptide library using paclitaxel-photoimmobilized TentaGel resin. Specific phage particles that bind the paclitaxel-immobilized resin were obtained. Among them, two phage clones included the same consensus amino acid sequence (KACGRTRVTS). Analysis of the protein database using BLAST revealed that a portion of this sequence is conserved in the zinc finger domain of human NFX1. Binding affinity of paclitaxel against the partial recombinant protein of NFX1 (424aa-876aa) was confirmed by pull-down assays and surface plasmon resonance analyses.

  9. Mimotope peptides selected from phage display combinatorial library by serum antibodies of pigs experimentally infected with Taenia solium as leads to developing diagnostic antigens for human neurocysticercosis.

    PubMed

    Gazarian, Karlen; Rowlay, Merril; Gazarian, Tatiana; Vazquez Buchelli, Jorge Enrique; Hernández Gonzáles, Marisela

    2012-12-01

    Neurocysticercosis is caused by penetration of the tapeworm Taenia solium larvae into the central nervous system resulting in a diverse range of neurologic complications including epilepsy in endemic areas that globalization spreads worldwide. Sensitive and specific immunodiagnosis is needed for the early detection and elimination of the parasite, but the lack of standardized, readily obtainable antigens is a challenge. Here, we used the phage display for resolving the problem. The rationale of the strategy rests on the concept that the screening of combinatorial libraries with polyclonal serum to pathogens reveals families of peptides mimicking the pathogen most immunodominant epitopes indispensable for the successful diagnosis. The screening of a 7mer library with serum IgG of four pigs experimentally infected with parasite followed by computer aided segregation of the selected sequences resulted in the discovery of four clusters of homologous sequences of which one presented a family of ten mimotopes selected by three infected pig serum IgGs; the common motif sequence LSPF carried by the family was considered to be the core of an immunodominant epitope of the parasite critical for the binding with the antibody that selected the mimotopes. The immunoassay testing permitted to select a mimotope whose synthetic peptide free of the phage with the amino acid sequence Leu-Ser-Fen-Pro-Ser-Val-Val that distinguished well a panel of 21 cerebrospinal fluids of neurocysticercosis patients from the fluids of individuals with neurological complications of other etiology. This peptide is proposed as a lead for developing a novel molecularly defined diagnostic antigen(s) for the neurocysticercosis.

  10. Vaccination with prion peptide-displaying papillomavirus-like particles induces autoantibodies to normal prion protein that interfere with pathologic prion protein production in infected cells.

    PubMed

    Handisurya, Alessandra; Gilch, Sabine; Winter, Dorian; Shafti-Keramat, Saeed; Maurer, Dieter; Schätzl, Hermann M; Kirnbauer, Reinhard

    2007-04-01

    Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrP(Sc)). To date, there is no prophylaxis or therapy available for these transmissible encephalopathies. Passive immunization with monclonal antibodies recognizing the normal host-encoded prion protein (PrP(C)) has been reported to abolish PrP(Sc) infectivity and to delay onset of disease. Because of established immunologic tolerance against the widely expressed PrP(C), active immunization appears to be difficult to achieve. To overcome this limitation, papillomavirus-like particles were generated that display a nine amino acid B-cell epitope, DWEDRYYRE, of the murine/rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type 1. The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis. Immunization with PrP-virus-like particles induced high-titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects. As determined by peptide-specific ELISA, rabbit immune sera recognized the inserted murine/rat epitope and also cross-reacted with the homologous rabbit/human epitope differing in one amino acid residue. In contrast, rat immune sera recognized the murine/rat peptide only. Sera of both species reacted with PrP(C) in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence-activated cell sorting analysis. Importantly, rabbit anti-PrP serum contained high-affinity antibody that inhibited de novo synthesis of PrP(Sc) in prion-infected cells. If also effective in vivo, PrP-virus-like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable prion-mediated diseases.

  11. A peptide derived from phage display library exhibits anti-tumor activity by targeting GRP78 in gastric cancer multidrug resistance cells.

    PubMed

    Kang, Jianqin; Zhao, Guohong; Lin, Tao; Tang, Shanhong; Xu, Guanghui; Hu, Sijun; Bi, Qian; Guo, Changcun; Sun, Li; Han, Shuang; Xu, Qian; Nie, Yongzhan; Wang, Biaoluo; Liang, Shuhui; Ding, Jie; Wu, Kaichun

    2013-10-10

    Multidrug resistance (MDR) remains a significant challenge to the clinical treatment of gastric cancer (GC). In the present study, using a phage display approach combined with MTT assays, we screened a specific peptide GMBP1 (Gastric cancer MDR cell-specific binding peptide), ETAPLSTMLSPY, which could bind to the surface of GC MDR cells specifically and reverse their MDR phenotypes. Immunocytochemical staining showed that the potential receptor of GMBP1 was located at the membrane and cytoplasm of MDR cells. In vitro and in vivo drug sensitivity assays, FACS analysis and Western blotting confirmed that GMBP1 was able to re-sensitize MDR cells to chemical drugs. Western blotting and proteomic approaches were used to screen the receptor of GMBP1, and GRP78, a MDR-related protein, was identified as a receptor of GMBP1. This result was further supported by immunofluoresence microscopy and Western blot. Additionally, Western blotting demonstrated that pre-incubation of GMBP1 in MDR cells greatly diminished MDR1, Bcl-2 and GRP78 expression but increased the expression of Bax, whereas downregulation of GRP78, function as a receptor and directly target for GMBP1, only inhibited MDR1 expression. Our findings suggest that GMBP1 could re-sensitize GC MDR cells to a variety of chemotherapeutic agents and this role might be mediated partly through down-regulating GRP78 expression and then inhibiting MDR1 expression. These findings indicate that peptide GMBP1 likely recognizes a novel GRP78 receptor and mediates cellular activities associated with the MDR phenotype, which provides new insight into research on the management of MDR in gastric cancer cells.

  12. Cell-Adhesive Matrices Composed of RGD Peptide-Displaying M13 Bacteriophage/Poly(lactic-co-glycolic acid) Nanofibers Beneficial to Myoblast Differentiation.

    PubMed

    Shin, Yong Cheol; Lee, Jong Ho; Jin, Linhua; Kim, Min Jeong; Kim, Chuntae; Hong, Suck Won; Oh, Jin Woo; Han, Dong-Wook

    2015-10-01

    Recently, there has been considerable effort to develop suitable scaffolds for tissue engineering applications. Cell adhesion is a prerequisite for cells to survive. In nature, the extracellular matrix (ECM) plays this role. Therefore, an ideal scaffold should be structurally similar to the natural ECM and have biocompatibility and biodegradability. In addition, the scaffold should have biofunctionality, which provides the potent ability to enhance the cellular behaviors, such as adhesion, proliferation and differentiation. This study concentrates on fabricating cell-adhesive matrices composed of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) nanofibers. Long rod-shaped M13 bacteriophages are non-toxic and can express many desired proteins on their surface. A genetically engineered M13 phage was constructed to display RGD peptides on its surface. PLGA is a biodegradable polymer with excellent biocompatibility and suitable physicochemical property for adhesive matrices. In this study, RGD-M13 phage/PLGA hybrid nanofiber matrices were fabricated by electrospinning. The physicochemical properties of these matrices were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and contact angle measurement. In addition, the cellular behaviors, such as the initial attachment, proliferation and differentiation, were analyzed by a CCK-8 assay and immunofluorescence staining to evaluate the potential application of these matrices to tissue engineering scaffolds. The RGD-M13 phage/PLGA nanofiber matrices could enhance the cellular behaviors and promote the differentiation of C2C12 myoblasts. These results suggest that the RGD-M13 phage/PLGA nanofiber matrices are beneficial to myoblast differentiation and can serve as effective tissue engineering scaffolds.

  13. Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

    PubMed

    Kulabhusan, Prabir Kumar; Rajwade, Jyutika M; Sahul Hameed, A S; Paknikar, Kishore M

    2017-03-27

    White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (Kd,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 μg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

  14. Identification of a Conserved Linear B-Cell Epitope of Streptococcus dysgalactiae GapC Protein by Screening Phage-Displayed Random Peptide Library

    PubMed Central

    Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Wang, Xintong; Zhu, Zhanbo; Cui, Yudong

    2015-01-01

    The GapC of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce protective humoral immune response in animals. However, B-cell epitopes on the S. dysgalactiae GapC have not been well identified. In this study, a monoclonal antibody (mAb5B7) against the GapC1-150 protein was prepared. After passive transfer, mAb5B7 could partially protect mice against S. dysgalactiae infection. Eleven positive phage clones recognized by mAb5B7 were identified by screening phage-displayed random 12-peptide library, most of which matched the consensus motif DTTQGRFD. The motif sequence exactly matches amino acids 48-55 of the S. dysgalactiae GapC protein. In addition, the motif 48DTTQGRFD55 shows high homology among various streptococcus species. Site-directed mutagenic analysis further confirmed that residues D48, T50, Q51, G52 and F54 formed the core motif of 48DTTQGRFD55. This motif was the minimal determinant of the B-cell epitope recognized by the mAb5B7. As expected, epitope-peptide evoked protective immune response against S. dysgalactiae infection in immunized mice. Taken together, this identified conserved B-cell epitope within S. dysgalactiae GapC could provide very valuable insights for vaccine design against S. dysgalactiae infection. PMID:26121648

  15. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    SciTech Connect

    Gardner, S. N.

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  16. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F.W.; Rosenberg, A.H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

  17. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F. William; Rosenberg, Alan H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

  18. Phage display biopanning identifies the translation initiation and elongation factors (IF1α-3 and eIF-3) as components of Hsp70-peptide complexes in breast tumour cells.

    PubMed

    Siebke, Christina; James, Tharappel C; Cummins, Robert; O'Grady, Tony; Kay, Elaine; Bond, Ursula

    2012-03-01

    The heat shock protein, HSP70, is over-expressed in many tumours and acts at the crossroads of key intracellular processes in its role as a molecular chaperone. HSP70 associates with a vast array of peptides, some of which are antigenic and can mount adaptive immune responses against the tumour from which they are derived. The pool of peptides associated with HSP70 represents a unique barcode of protein metabolism in tumour cells. With a view to identifying unique protein targets that may be developed as tumour biomarkers, we used purified HSP70 and its associated peptide pool (HSP70-peptide complexes, HSP70-PCs) from different human breast tumour cell lines as targets for phage display biopanning. Our results show that HSP70-PCs from each cell line interact with unique sets of peptides within the phage display library. One of the peptides, termed IST, enriched in the biopanning process, was used in a 'pull-down' assay to identify the original protein from which the HSP70-associated peptides may have been derived. The eukaryotic translation initiation factor 3 (eIF-3), a member of the elongation factor EF1α family, and the HSP GRP78, were pulled down by the IST peptide. All of these proteins are known to be up-regulated in cancer cells. Immunohistochemical staining of tumour tissue microarrays showed that the peptide co-localised with HSP70 in breast tumour tissue. The data indicate that the reservoir of peptides associated with HSP70 can act as a unique indicator of cellular protein activity and a novel source of potential tumour biomarkers.

  19. Monocytes from Sjögren's syndrome patients display increased vasoactive intestinal peptide receptor 2 expression and impaired apoptotic cell phagocytosis

    PubMed Central

    Hauk, V; Fraccaroli, L; Grasso, E; Eimon, A; Ramhorst, R; Hubscher, O; Pérez Leirós, C

    2014-01-01

    Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by salivary and lacrimal gland dysfunction. Clinical observations and results from animal models of SS support the role of aberrant epithelial cell apoptosis and immune homeostasis loss in the glands as triggering factors for the autoimmune response. Vasoactive intestinal peptide (VIP) promotes potent anti-inflammatory effects in several inflammatory and autoimmune disease models, including the non-obese diabetic (NOD) mouse model of SS. With the knowledge that VIP modulates monocyte function through vasoactive intestinal peptide receptors (VPAC) and that immune homeostasis maintenance depends strongly upon a rapid and immunosuppressant apoptotic cell clearance by monocytes/macrophages, in this study we explored VPAC expression on monocytes from primary SS (pSS) patients and the ability of VIP to modulate apoptotic cell phagocytic function and cytokine profile. Monocytes isolated from individual pSS patients showed an increased expression of VPAC2 subtype of VIP receptors, absent in monocytes from control subjects, with no changes in VPAC1 expression. VPAC2 receptor expression could be induced further with lipopolysaccharide (LPS) in pSS monocytes and VIP inhibited the effect. Moreover, monocytes from pSS patients showed an impaired phagocytosis of apoptotic epithelial cells, as evidenced by reduced engulfment ability and the failure to promote an immunosuppressant cytokine profile. However, VIP neither modulated monocyte/macrophage phagocytic function nor did it reverse their inflammatory profile. We conclude that monocytes from pSS patients express high levels of VPAC2 and display a deficient clearance of apoptotic cells that is not modulated by VIP. PMID:24827637

  20. The kiwi fruit peptide kissper displays anti-inflammatory and anti-oxidant effects in in-vitro and ex-vivo human intestinal models.

    PubMed

    Ciacci, C; Russo, I; Bucci, C; Iovino, P; Pellegrini, L; Giangrieco, I; Tamburrini, M; Ciardiello, M A

    2014-03-01

    Literature reports describe kiwi fruit as a food with significant effects on human health, including anti-oxidant and anti-inflammatory activity. Fresh fruit or raw kiwi fruit extracts have been used so far to investigate these effects, but the molecule(s) responsible for these health-promoting activities have not yet been identified. Kissper is a kiwi fruit peptide displaying pore-forming activity in synthetic lipid bilayers, the composition of which is similar to that found in intestinal cells. The objective of this study was to investigate the kissper influence on intestinal inflammation using cultured cells and ex-vivo tissues from healthy subjects and Crohn's disease (CD) patients. The anti-oxidant and anti-inflammatory properties of kissper were tested on Caco-2 cells and on the colonic mucosa from 23 patients with CD, by challenging with the lipopolysaccharide from Escherichia coli (EC-LPS) and monitoring the appropriate markers by Western blot and immunofluorescence. EC-LPS challenge determined an increase in the intracellular concentration of calcium and reactive oxygen species (ROS). The peptide kissper was highly effective in preventing the increase of LPS-induced ROS levels in both the Caco-2 cells and CD colonic mucosa. Moreover, it controls the calcium increase, p65-nuclear factor (NF)-kB induction and transglutaminase 2 (TG2) activation inflammatory response in Caco-2 cells and CD colonic mucosa. Kissper efficiently counteracts the oxidative stress and inflammatory response in valuable model systems consisting of intestinal cells and CD colonic mucosa. This study reports the first evidence supporting a possible correlation between some beneficial effects of kiwi fruit and a specific protein molecule rather than generic nutrients.

  1. Development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study - Cumin seed.

    PubMed

    Siow, Hwee-Leng; Lim, Theam Soon; Gan, Chee-Yuen

    2017-01-01

    The main objective of this study was to develop an efficient workflow to discover α-amylase inhibitory peptides from cumin seed. A total of 56 unknown peptides was initially found in the cumin seed protein hydrolysate. They were subjected to 2 different in silico screenings and 6 peptides were shortlisted. The peptides were then subjected to in vitro selection using phage display technique and 3 clones (CSP3, CSP4 and CSP6) showed high affinity in binding α-amylase. These clones were subjected to the inhibitory test and only CSP4 and CSP6 exhibited high inhibitory activity. Therefore, these peptides were chemically synthesized for validation purposes. CSP4 exhibited inhibition of bacterial and human salivary α-amylases with IC50 values of 0.11 and 0.04μmol, respectively, whereas CSP6 was about 0.10 and 0.15μmol, respectively. Results showed that the strength of each protocol has been successfully combined as deemed fit to enhance the α-amylase inhibitor peptide discovery.

  2. Detection of immobilized amplicons by ELISA-like techniques.

    PubMed

    Oroskar, A A; Rasmussen, S E; Rasmussen, H N; Rasmussen, S R; Sullivan, B M; Johansson, A

    1996-09-01

    The NucleoLink surface is a physically modified, thermostable, optically clear resin. It allows the covalent binding of 5'-phosphorylated oligonucleotides. Target DNA amplification by polymerase chain reaction (PCR) is accomplished by asymmetric amplification on the covalently immobilized primer that develops into immobilized amplicons. A DNA fragment of bovine leukemia virus is used as a model system for the detection of immobilized amplicons by ELISA-like techniques. Covalently bound oligonucleotides are also utilized as capture probe in the hybridization-based signal amplification for detection of an infectious organism.

  3. A novel Omp25-binding peptide screened by phage display can inhibit Brucella abortus 2308 infection in vitro and in vivo

    PubMed Central

    Zhang, Junbo; Guo, Fei; Huang, Xiaoqiang; Zhang, Hui; Wang, Yuanzhi; Yin, Shuanghong; Li, Zhiqiang

    2014-01-01

    Brucellosis is a globally distributed zoonotic disease affecting animals and humans, and current antibiotic and vaccine strategies are not optimal. The surface-exposed protein Omp25 is involved in Brucella virulence and plays an important role in Brucella pathogenesis during infection, suggesting that Omp25 could be a useful target for selecting potential therapeutic molecules to inhibit Brucella pathogenesis. In this study, we identified, we believe for the first time, peptides that bind specifically to the Omp25 protein of pathogens, using a phage panning technique, After four rounds of panning, 42 plaques of eluted phages were subjected to pyrosequencing. Four phage clones that bound better than the other clones were selected following confirmation by ELISA and affinity constant determination. The peptides selected could significantly inhibit Brucella abortus 2308 (S2308) internalization and intracellular growth in RAW264.7 macrophages, and significantly induce secretion of TNF-α and IL-12 in peptide- and S2308-treated cells. Any observed peptide (OP11, OP27, OP35 or OP40) could significantly inhibit S2308 infection in BALB/c mice. Moreover, the peptide OP11 was the best candidate peptide for inhibiting S2308 infection in vitro and in vivo. These results suggest that peptide OP11 has potential for exploitation as a peptide drug in resisting S2308 infection. PMID:24722798

  4. Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems.

    PubMed

    Shaw, Jennifer L A; Monis, Paul; Weyrich, Laura S; Sawade, Emma; Drikas, Mary; Cooper, Alan J

    2015-09-01

    Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs.

  5. Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems

    PubMed Central

    Shaw, Jennifer L. A.; Weyrich, Laura S.; Sawade, Emma; Drikas, Mary; Cooper, Alan J.

    2015-01-01

    Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs. PMID:26162884

  6. Naturally processed peptides spanning the HPA-1a polymorphism are efficiently generated and displayed from platelet glycoprotein by HLA-DRB3*0101-positive antigen-presenting cells.

    PubMed

    Anani Sarab, Gholamreza; Moss, Michael; Barker, Robert N; Urbaniak, Stanislaw J

    2009-08-27

    In neonatal alloimmune thrombocytopenia, almost all human platelet antigen (HPA)-1b1b mothers who produce anti-HPA-1a antibody through carrying an HPA-1a fetus are human histocompatibility leukocyte antigen (HLA)-DRB3*0101 positive. It is predicted that the HPA-1a Leu(33) polymorphism forms part of an HLA-DRB3*0101-restricted T-helper epitope, and acts as an anchor residue for binding this class II molecule. However, it is not known whether any corresponding peptides are naturally processed and presented from platelet glycoprotein. In this study, peptides displayed by a homozygous HLA-DRB3*0101 antigen-presenting cell line were identified after pulsing with recombinant HPA-1a (Leu(33) plexin-semaphorin-integrin domain). The peptides were eluted from HLA-DR molecules, fractionated by high performance liquid chromatography, and analyzed by tandem mass spectrometry. A "nested set" of naturally presented HPA-1a-derived peptides, each containing the Trp(25)-Leu(33) core epitope, was identified, with the most abundant member being the 16-mer Met(22)-Arg(37). These peptides may provide the basis for novel treatments to tolerize the corresponding T-helper response in women at risk of neonatal alloimmune thrombocytopenia.

  7. Functional characterization of the 19q12 amplicon in grade III breast cancers

    PubMed Central

    2012-01-01

    Introduction The 19q12 locus is amplified in a subgroup of oestrogen receptor (ER)-negative grade III breast cancers. This amplicon comprises nine genes, including cyclin E1 (CCNE1), which has been proposed as its 'driver'. The aim of this study was to identify the genes within the 19q12 amplicon whose expression is required for the survival of cancer cells harbouring their amplification. Methods We investigated the presence of 19q12 amplification in a series of 313 frozen primary breast cancers and 56 breast cancer cell lines using microarray comparative genomic hybridisation (aCGH). The nine genes mapping to the smallest region of amplification on 19q12 were silenced using RNA interference in phenotypically matched breast cancer cell lines with (MDA-MB-157 and HCC1569) and without (Hs578T, MCF7, MDA-MB-231, ZR75.1, JIMT1 and BT474) amplification of this locus. Genes whose silencing was selectively lethal in amplified cells were taken forward for further validation. The effects of cyclin-dependent kinase 2 (CDK2) silencing and chemical inhibition were tested in cancer cells with and without CCNE1 amplification. Results 19q12 amplification was identified in 7.8% of ER-negative grade III breast cancer. Of the nine genes mapping to this amplicon, UQCRFS1, POP4, PLEKHF1, C19ORF12, CCNE1 and C19ORF2 were significantly over-expressed when amplified in primary breast cancers and/or breast cancer cell lines. Silencing of POP4, PLEKHF1, CCNE1 and TSZH3 selectively reduced cell viability in cancer cells harbouring their amplification. Cancer cells with CCNE1 amplification were shown to be dependent on CDK2 expression and kinase activity for their survival. Conclusions The 19q12 amplicon may harbour more than a single 'driver', given that expression of POP4, PLEKHF1, CCNE1 and TSZH3 is required for the survival of cancer cells displaying their amplification. The observation that cancer cells harbouring CCNE1 gene amplification are sensitive to CDK2 inhibitors provides a

  8. A novel microtubule de-stabilizing complementarity-determining region C36L1 peptide displays antitumor activity against melanoma in vitro and in vivo

    PubMed Central

    Figueiredo, Carlos R.; Matsuo, Alisson L.; Azevedo, Ricardo A.; Massaoka, Mariana H.; Girola, Natalia; Polonelli, Luciano; Travassos, Luiz R.

    2015-01-01

    Short peptide sequences from complementarity-determining regions (CDRs) of different immunoglobulins may exert anti-infective, immunomodulatory and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). In this sense, they resemble early molecules of innate immunity. C36L1 was identified as a bioactive light-chain CDR1 peptide by screening 19 conserved CDR sequences targeting murine B16F10-Nex2 melanoma. The 17-amino acid peptide is readily taken up by melanoma cells and acts on microtubules causing depolymerization, stress of the endoplasmic reticulum and intrinsic apoptosis. At low concentrations, C36L1 inhibited migration, invasion and proliferation of B16F10-Nex2 cells with cell cycle arrest at G2/M phase, by regulating the PI3K/Akt signaling axis involving Rho-GTPase and PTEN mediation. Peritumor injection of the peptide delayed growth of subcutaneously grafted melanoma cells. Intraperitoneal administration of C36L1 induced a significant immune-response dependent anti-tumor protection in a syngeneic metastatic melanoma model. Dendritic cells stimulated ex-vivo by the peptide and transferred to animals challenged with tumor cells were equally effective. The C36 VL CDR1 peptide is a promising microtubule-interacting drug that induces tumor cell death by apoptosis and inhibits metastases of highly aggressive melanoma cells. PMID:26391685

  9. DADA2: High resolution sample inference from Illumina amplicon data

    PubMed Central

    Callahan, Benjamin J; McMurdie, Paul J; Rosen, Michael J; Han, Andrew W; Johnson, Amy Jo A; Holmes, Susan P

    2016-01-01

    We present DADA2, a software package that models and corrects Illumina-sequenced amplicon errors. DADA2 infers sample sequences exactly, without coarse-graining into OTUs, and resolves differences of as little as one nucleotide. In several mock communities DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants. PMID:27214047

  10. DNA-based methods to prepare helper virus-free herpes amplicon vectors and versatile design of amplicon vector plasmids.

    PubMed

    Kasai, Kazue; Saeki, Yoshinaga

    2006-06-01

    The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle with a large transgene capacity (up to 150 kb) and the ability to infect a broad range of cell types. The vector system was originally developed by Frenkel and her colleagues in 1980. Ever since, a great deal of effort by various investigators has been directed at minimizing the toxicity associated with the inevitable contamination by helper virus. In 1996, Fraefel and his colleagues successfully devised a cosmid-based packaging system that was free of contamination by helper virus (so-called helper virus-free packaging), which utilized as helper a set of 5 overlapping cosmid clones that covered the entire HSV genome, which lacked the DNA packaging/cleavage signals. With the helper virus-free system, broader applications of the vector became possible. Cloning of the entire HSV genome in bacteria artificial chromosome (BAC) plasmids enabled stable maintenance and propagation of the helper HSV genome in bacteria. It also allowed for the development of BAC-based helper virus-free packaging systems. In this article, we review various versions of DNA-based methods to prepare HSV amplicon vectors free of helper virus contamination. We also examine recent advances in vector design, including methods of vector construction, hybrid amplicon vectors, and the infectious BAC system. Future directions in improving packaging systems and vector designs are discussed.

  11. The 5A apolipoprotein A-I mimetic peptide displays anti-inflammatory and antioxidant properties in vivo and in vitro

    PubMed Central

    Tabet, Fatiha; Remaley, Alan T.; Segaliny, Aude I.; Millet, Jonathan; Yan, Ling; Nakhla, Shirley; Barter, Philip J.; Rye, Kerry-Anne; Lambert, Gilles

    2010-01-01

    Objectives The apolipoprotein (apo) A-I mimetic peptide 5A is highly specific for ABCA1-transporter mediated cholesterol efflux. We investigated whether the 5A peptide shares other beneficial features of apoA-I, such as protection against inflammation and oxidation. Methods New-Zealand White rabbits received an infusion of apoA-I, reconstituted HDL containing apoA-I ((A-I)rHDL) or the 5A peptide complexed with phospholipids (PLPC), prior to inserting a collar around the carotid artery. Human coronary artery endothelial cells (HCAECs) were incubated with (A-I)rHDL or 5A/PLPC prior to TNFa stimulation. Results ApoA-I, (A-I)rHDL and 5A/PLPC reduced the collar mediated increase in (i) endothelial expression of cell adhesion molecules VCAM-1 and ICAM-1, (ii) O2− production as well as the expression of the Nox4 catalytic subunits of the NADPH oxidase, and (iii) infiltration of circulating neutrophils into the carotid intima-media. In HCAECs, both 5A/PLPC and (A-I)rHDL inhibited TNFa induced ICAM-1 and VCAM-1 expression as well as the NF-κB signalling cascade and O2− production. The effects of the 5A/PLPC complex were no longer apparent in HCAECs knocked down for ABCA1. Conclusion Like apoA-I, the 5A peptide inhibits acute inflammation and oxidative stress in rabbit carotids and HCAECs. In vitro, the 5A peptide exerts these beneficial effects through interaction with ABCA1. PMID:19965776

  12. Reduced pathology and improved behavioral performance in Alzheimer’s disease mice vaccinated with HSV amplicons expressing amyloid-beta and interleukin-4

    PubMed Central

    Frazer, Maria E.; Hughes, Jennifer E.; Mastrangelo, Michael A.; Tibbens, Jennifer L.; Federoff, Howard J.; Bowers, William J.

    2008-01-01

    Immunotherapeutics designed to dissolve existing amyloid plaques or to interrupt amyloid-beta (Aβ) accumulation may be feasible for treatment and/or prevention of Alzheimer’s disease (AD). “Shaping” immune responses elicited against Aβ is requisite to generate an efficacious and safe outcome by minimizing the possibility of deleterious inflammatory reactions in the brain as observed in clinical testing of Aβ peptide/adjuvant-based modalities. Herpes Simplex Virus (HSV)-based amplicons can co-express multiple antigens and/or immunomodulatory genes due to their large genetic size capacity, thereby facilitating antigen-specific immune response shaping. We have constructed an amplicon (HSVIEAβCMVIL-4) that co-delivers Aβ1-42 with interleukin-4, a cytokine that promotes the generation of Th2-like T cell responses, which are favored in the setting of AD immunotherapy. Triple-transgenic AD (3xTg-AD) mice, which progressively develop both amyloid and neurofibrillary tangle pathology, were vaccinated thrice with HSVIEAβCMVIL-4, or a set of control amplicon vectors. Increased Th2-related, Aβ-specific antibodies, improved learning and memory functioning, and prevention of AD-related amyloid and tau pathological progression were observed in HSVIEAβCMVIL-4 vaccinated mice as compared to the other experimental groups. Our study underscores the potential of Aβ immunotherapy for AD and highlights the potency of amplicons to facilitate immune response modulation to a disease-relevant antigen. PMID:18388924

  13. Low molecular weight oligomers of amyloid peptides display β-barrel conformations: A replica exchange molecular dynamics study in explicit solvent

    NASA Astrophysics Data System (ADS)

    De Simone, Alfonso; Derreumaux, Philippe

    2010-04-01

    The self-assembly of proteins and peptides into amyloid fibrils is connected to over 40 pathological conditions including neurodegenerative diseases and systemic amyloidosis. Diffusible, low molecular weight protein and peptide oligomers that form in the early steps of aggregation appear to be the harmful cytotoxic species in the molecular etiology of these diseases. So far, the structural characterization of these oligomers has remained elusive owing to their transient and dynamic features. We here address, by means of full atomistic replica exchange molecular dynamics simulations, the energy landscape of heptamers of the amyloidogenic peptide NHVTLSQ from the beta-2 microglobulin protein. The simulations totaling 5 μs show that low molecular weight oligomers in explicit solvent consist of β-barrels in equilibrium with amorphous states and fibril-like assemblies. The results, also accounting for the influence of the pH on the conformational properties, provide a strong evidence of the formation of transient β-barrel assemblies in the early aggregation steps of amyloid-forming systems. Our findings are discussed in terms of oligomers cytotoxicity.

  14. Low molecular weight oligomers of amyloid peptides display beta-barrel conformations: a replica exchange molecular dynamics study in explicit solvent.

    PubMed

    De Simone, Alfonso; Derreumaux, Philippe

    2010-04-28

    The self-assembly of proteins and peptides into amyloid fibrils is connected to over 40 pathological conditions including neurodegenerative diseases and systemic amyloidosis. Diffusible, low molecular weight protein and peptide oligomers that form in the early steps of aggregation appear to be the harmful cytotoxic species in the molecular etiology of these diseases. So far, the structural characterization of these oligomers has remained elusive owing to their transient and dynamic features. We here address, by means of full atomistic replica exchange molecular dynamics simulations, the energy landscape of heptamers of the amyloidogenic peptide NHVTLSQ from the beta-2 microglobulin protein. The simulations totaling 5 micros show that low molecular weight oligomers in explicit solvent consist of beta-barrels in equilibrium with amorphous states and fibril-like assemblies. The results, also accounting for the influence of the pH on the conformational properties, provide a strong evidence of the formation of transient beta-barrel assemblies in the early aggregation steps of amyloid-forming systems. Our findings are discussed in terms of oligomers cytotoxicity.

  15. A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma

    PubMed Central

    Kim, Julius W.; Kane, J. Robert; Young, Jacob S.; Chang, Alan L.; Kanojia, Deepak; Morshed, Ramin A.; Miska, Jason; Ahmed, Atique U.; Balyasnikova, Irina V.; Han, Yu; Zhang, Lingjiao; Curiel, David T.; Lesniak, Maciej S.

    2015-01-01

    The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as “GliomaFF.” We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy. PMID:26058317

  16. A quantitative SMRT cell sequencing method for ribosomal amplicons.

    PubMed

    Jones, Bethan M; Kustka, Adam B

    2017-04-01

    Advances in sequencing technologies continue to provide unprecedented opportunities to characterize microbial communities. For example, the Pacific Biosciences Single Molecule Real-Time (SMRT) platform has emerged as a unique approach harnessing DNA polymerase activity to sequence template molecules, enabling long reads at low costs. With the aim to simultaneously classify and enumerate in situ microbial populations, we developed a quantitative SMRT (qSMRT) approach that involves the addition of exogenous standards to quantify ribosomal amplicons derived from environmental samples. The V7-9 regions of 18S SSU rDNA were targeted and quantified from protistan community samples collected in the Ross Sea during the Austral summer of 2011. We used three standards of different length and optimized conditions to obtain accurate quantitative retrieval across the range of expected amplicon sizes, a necessary criterion for analyzing taxonomically diverse 18S rDNA molecules from natural environments. The ability to concurrently identify and quantify microorganisms in their natural environment makes qSMRT a powerful, rapid and cost-effective approach for defining ecosystem diversity and function.

  17. Herpes simplex virus type 1-derived recombinant and amplicon vectors.

    PubMed

    Fraefel, Cornel; Marconi, Peggy; Epstein, Alberto L

    2011-01-01

    Herpes simplex virus type 1 (HSV-1) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153 kbp double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes (1) the two approaches most commonly used to prepare recombinant vectors through homologous recombination, either in eukaryotic cells or in bacteria, and (2) the two methodologies currently used to generate helper-free amplicon vectors, either using a bacterial artificial chromosome (BAC)-based approach or a Cre/loxP site-specific recombination strategy.

  18. N-formyl peptide receptors in human neutrophils display distinct membrane distribution and lateral mobility when labeled with agonist and antagonist

    PubMed Central

    1993-01-01

    Receptors for bacterial N-formyl peptides are instrumental for neutrophil chemotactic locomotion and activation at sites of infection. As regulatory mechanisms for signal transduction, both rapid coupling of the occupied receptor to cytoskeletal components, and receptor lateral redistribution, have been suggested (Jesaitis et al., 1986, 1989). To compare the distribution and lateral diffusion of the nonactivated and activated neutrophil N-formyl-peptide receptor, before internalization, we used a new fluorescent N-formyl-peptide receptor antagonist, tertbutyloxycarbonyl-Phe(D)-Leu-Phe(D)-Leu-Phe-OH (Boc- FLFLF, 0.1-1 microM), and the fluorescent receptor agonist formyl-Nle- Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK, 0.1-1 microM). Fluorescent Boc-FLFLF did not elicit an oxidative burst in the neutrophil at 37 degrees C, as assessed by chemiluminescence and reduction of p-nitroblue tetrazolium chloride, but competed efficiently both with formyl-methionyl-leucyl- phenylalanine (fMLF) and fnLLFnLYK. It was not internalized, as evidenced by confocal microscopy and acid elution of surface bound ligand. The lateral mobility characteristics of the neutrophil fMLF receptor were investigated with the technique of FRAP. The diffusion coefficient (D) was similar for antagonist- and agonist-labeled receptors (D approximately 5 x 10(-10) cm2/s), but the fraction of mobile receptors was significantly lower in agonist- compared to antagonist-labeled cells, approximately 40% in contrast to approximately 60%. This reduction in receptor mobile fraction was slightly counteracted, albeit not significantly, by dihydrocytochalasin B (dhcB, 5 microM). To block internalization of agonist-labeled receptors, receptor mobility measurements were done at 14 degrees C. At this temperature, confocal microscopy revealed clustering of receptors in response to agonist binding, compared to a more uniform receptor distribution in antagonist-labeled cells. The pattern of agonist- induced receptor clustering was

  19. A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

    PubMed Central

    Uddin, Shah Mukim; Ibrahim, Fatimah; Sayad, Abkar Ahmed; Thiha, Aung; Pei, Koh Xiu; Mohktar, Mas S.; Hashim, Uda; Cho, Jongman; Thong, Kwai Lin

    2015-01-01

    In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment. PMID:25751077

  20. Evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization

    PubMed Central

    Abbineni, Gopal; Modali, Sita; Safiejko-Mroczka, Barbara; Petrenko, Valery A.; Mao, Chuanbin

    2010-01-01

    Filamentous phage as a bacteria-specific virus can be conjugated with an anti-cancer drug and has been proposed to serve as a carrier to deliver drugs to cancer cells for targeted therapy. However, how cell-targeting filamentous phage alone affects cancer cell biology is unclear. Phage libraries provide an inexhaustible reservoir of new ligands against tumor cells and tissues that have potential therapeutic and diagnostic applications in cancer treatment. Some of these identified ligands might stimulate various cell responses. Here we identified new cell internalizing peptides (and the phages with such peptides fused to each of ~3900 copies of their major coat protein) using landscape phage libraries and for the first time investigated the actin dynamics when selected phages are internalized into the SKBR-3 breast cancer cells. Our results show that phages harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into the SKBR-3 breast cancer cells with high affinity, and also show rapid involvement of membrane ruffling and re-arrangements of actin cytoskeleton during the phage entry. The actin dynamics was studied by using live cell and fluorescence imaging. The cell-targeting phages were found to enter breast cancer cells through energy dependent mechanism and phage entry interferes with actin dynamics, resulting in reorganization of actin filaments and increased membrane rufflings in SKBR-3 cells. These results suggest that, when phage enters epithelial cells, it triggers transient changes in the host cell actin cytoskeleton. This study also shows that using multivalent phage libraries considerably increases the repertoire of available cell-internalizing ligands with potential applications in targeted drug delivery, imaging, molecular monitoring and profiling of breast cancer cells. PMID:20735141

  1. Identification of a conserved JEV serocomplex B-cell epitope by screening a phage-display peptide library with a mAb generated against West Nile virus capsid protein

    PubMed Central

    2011-01-01

    Background The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported. Results In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae. Conclusion The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex. PMID:21375771

  2. Drugs derived from phage display

    PubMed Central

    Nixon, Andrew E; Sexton, Daniel J; Ladner, Robert C

    2014-01-01

    Phage display, one of today’s fundamental drug discovery technologies, allows identification of a broad range of biological drugs, including peptides, antibodies and other proteins, with the ability to tailor critical characteristics such as potency, specificity and cross-species binding. Further, unlike in vivo technologies, generating phage display-derived antibodies is not restricted by immunological tolerance. Although more than 20 phage display-derived antibody and peptides are currently in late-stage clinical trials or approved, there is little literature addressing the specific challenges and successes in the clinical development of phage-derived drugs. This review uses case studies, from candidate identification through clinical development, to illustrate the utility of phage display as a drug discovery tool, and offers a perspective for future developments of phage display technology. PMID:24262785

  3. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi.

    PubMed

    Lindner, Daniel L; Carlsen, Tor; Henrik Nilsson, R; Davey, Marie; Schumacher, Trond; Kauserud, Håvard

    2013-06-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3-5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.

  4. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

    PubMed Central

    Lindner, Daniel L; Carlsen, Tor; Henrik Nilsson, R; Davey, Marie; Schumacher, Trond; Kauserud, Håvard

    2013-01-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3–5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions. PMID:23789083

  5. Biodiscovery of aluminum binding peptides

    NASA Astrophysics Data System (ADS)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

    2013-05-01

    Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

  6. Construction of a cytomegalovirus-based amplicon: a vector with a unique transfer capacity.

    PubMed

    Borst, Eva Maria; Messerle, Martin

    2003-07-01

    Cytomegalovirus (CMV) has a number of interesting properties that qualifies it as a vector for gene transfer. Especially appealing is the ability of the CMV genome to persist in hematopoietic progenitor cells and the packaging capacity of the viral capsid that accommodates a DNA genome of 230 kbp. In order to exploit the packaging capacity of the CMV capsid we investigated whether the principles of an amplicon vector can be applied to CMV. Amplicons are herpesviral vectors, which contain only the cis-active sequences required for replication and packaging of the vector genome. For construction of a CMV amplicon the sequences comprising the lytic origin of replication (orilyt) and the cleavage packaging recognition sites (pac) of human CMV were cloned onto a plasmid. A gene encoding the green fluorescent protein was used as a model transgene. The amplicon plasmid replicated in the presence of a CMV helper virus and was packaged into CMV particles, with replication and packaging being dependent on the presence of the orilyt and pac sequences. The packaged amplicon could be transferred to recipient cells and reisolated from the transduced cells. Analysis of the DNA isolated from CMV capsids revealed that the CMV amplicon was packaged as a concatemer with a size of approximately 210 kbp. The CMV amplicon vector has the potential to transfer therapeutic genes with a size of more than 200 kbp and thus provides a unique transfer capacity among viral vectors.

  7. Quantitative assessment of short amplicons in FFPE-derived long-chain RNA

    PubMed Central

    Kong, Hui; Zhu, Mengou; Cui, Fengyun; Wang, Shuyang; Gao, Xue; Lu, Shaohua; Wu, Ying; Zhu, Hongguang

    2014-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are important resources for molecular medical research. However, long-chain RNA analysis is restricted in FFPE tissues due to high levels of degradation. To explore the possibility of long RNA quantification in FFPE tissues, we selected 14 target RNAs (8 mRNAs and 6 long noncoding RNAs) from literatures, and designed short (~60 bp) and long (~200 bp) amplicons for each of them. Colorectal carcinomas with adjacent normal tissues were subjected to quantitative reverse-transcription PCR (quantitative RT-PCR) in 3 cohorts, including 18 snap-frozen and 83 FFPE tissues. We found that short amplicons were amplified more efficiently than long amplicons both in snap-frozen (P = 0.0006) and FFPE (P = 0.0152) tissues. Nonetheless, comparison of colorectal carcinomas with their adjacent normal tissues demonstrated that the consistency of fold-change trends in a single short amplicon between snap-frozen and FFPE tissues was only 36%. Therefore, we innovatively performed quantitative RT-PCR with 3 non-overlapping short amplicons for 14 target RNAs in FFPE tissues. All target RNAs showed a concordance of 100% of fold-change trends in at least two short amplicons, which offers sufficient information for accurate quantification of target RNAs. Our findings demonstrated the possibility of long-chain RNA analysis with 3 non-overlapping short amplicons in standardized-preserved FFPE tissues. PMID:25430878

  8. Display Tactics

    ERIC Educational Resources Information Center

    Tetlow, Linda

    2009-01-01

    Display took a wide variety of forms ranging from students presenting their initial planning and thought processes, to displays of their finished work, and their suggestions for extending the task should they, or others, have time to return to it in the future. A variety of different media were used from traditional posters in many shapes and…

  9. 16S rRNA Amplicon Sequencing for Epidemiological Surveys of Bacteria in Wildlife

    PubMed Central

    Razzauti, Maria; Bard, Emilie; Bernard, Maria; Brouat, Carine; Charbonnel, Nathalie; Dehne-Garcia, Alexandre; Loiseau, Anne; Tatard, Caroline; Tamisier, Lucie; Vayssier-Taussat, Muriel; Vignes, Helene

    2016-01-01

    ABSTRACT The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial

  10. Biodiscovery of Aluminum Binding Peptides

    DTIC Science & Technology

    2013-08-01

    display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high...scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the self- sustaining peptide libraries to be rapidly screened for high...removal. An eCPX peptide display library was grown and induced as described in the paragraph above. After rinsing samples briefly in PBS, the aluminum

  11. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification.

    PubMed

    Mauk, Michael G; Liu, Changchun; Song, Jinzhao; Bau, Haim H

    2015-10-20

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of "lab on a chip" NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase ("membrane") to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 10³ virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  12. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    PubMed Central

    Mauk, Michael G.; Liu, Changchun; Song, Jinzhao; Bau, Haim H.

    2015-01-01

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed. PMID:27600235

  13. Taxonomic Assessment of Rumen Microbiota Using Total RNA and Targeted Amplicon Sequencing Approaches

    PubMed Central

    Li, Fuyong; Henderson, Gemma; Sun, Xu; Cox, Faith; Janssen, Peter H.; Guan, Le Luo

    2016-01-01

    Taxonomic characterization of active gastrointestinal microbiota is essential to detect shifts in microbial communities and functions under various conditions. This study aimed to identify and quantify potentially active rumen microbiota using total RNA sequencing and to compare the outcomes of this approach with the widely used targeted RNA/DNA amplicon sequencing technique. Total RNA isolated from rumen digesta samples from five beef steers was subjected to Illumina paired-end sequencing (RNA-seq), and bacterial and archaeal amplicons of partial 16S rRNA/rDNA were subjected to 454 pyrosequencing (RNA/DNA Amplicon-seq). Taxonomic assessments of the RNA-seq, RNA Amplicon-seq, and DNA Amplicon-seq datasets were performed using a pipeline developed in house. The detected major microbial phylotypes were common among the three datasets, with seven bacterial phyla, fifteen bacterial families, and five archaeal taxa commonly identified across all datasets. There were also unique microbial taxa detected in each dataset. Elusimicrobia and Verrucomicrobia phyla; Desulfovibrionaceae, Elusimicrobiaceae, and Sphaerochaetaceae families; and Methanobrevibacter woesei were only detected in the RNA-Seq and RNA Amplicon-seq datasets, whereas Streptococcaceae was only detected in the DNA Amplicon-seq dataset. In addition, the relative abundances of four bacterial phyla, eight bacterial families and one archaeal taxon were different among the three datasets. This is the first study to compare the outcomes of rumen microbiota profiling between RNA-seq and RNA/DNA Amplicon-seq datasets. Our results illustrate the differences between these methods in characterizing microbiota both qualitatively and quantitatively for the same sample, and so caution must be exercised when comparing data. PMID:27446027

  14. "COV'COP" allows to detect CNVs responsible for inherited diseases among amplicons sequencing data.

    PubMed

    Derouault, P; Parfait, B; Moulinas, R; Barrot, C-C; Sturtz, F; Merillou, S; Lia, A-S

    2017-01-30

    In order to help molecular geneticists to rapidly identify CNVs responsible for inherited diseases among amplicons sequencing data generated by NGS, we designed a user-friendly tool "Cov'Cop". Using the run's coverage file provided by the sequencer, Cov'Cop simultaneously analyzes all the patients of the run using a two-stage algorithm containing correction and normalization levels and provides an easily understandable output, showing with various colors, potentially deleted and duplicated amplicons.

  15. Plasma displays

    SciTech Connect

    Sobel, A.

    1991-12-01

    Plasma displays make use of lightly ionized glow discharges to produce light, perform switching and selection functions, or both. Both the negative glow and the positive column are used. Color can be attained by using UV from the discharge to stimulate phosphors. The adroit use of priming can reduce the number of drive circuits required - an advantage unique in the display art to plasma devices. Short voltage pulses can improve the efficacy of positive-column devices. Short voltage pulses can improve the efficacy of positive-column devices. The gas discharge can be used as a source of electrons, which can then excite cathodoluminescent phosphors in a variety of colors. It can also be used as a selection means for liquid-crystal displays. In this paper a wide variety of device configurations, using both unidirectional and bidirectional pulse excitations, is described.

  16. Phage display technology: clinical applications and recent innovations.

    PubMed

    Azzazy, Hassan M E; Highsmith, W Edward

    2002-09-01

    Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target. A crucial advantage of this technology is the direct link that exists between the experimental phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized molecules. Phage display facilitates engineering of antibodies with regard to their size, valency, affinity, and effector functions. The selection of antibodies and peptides from libraries displayed on the surface of filamentous phage has proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications. This review serves as an introduction to phage display, antibody engineering, the development of phage-displayed peptides and antibody fragments into viable diagnostic reagents, and recent trends in display technology.

  17. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms

    PubMed Central

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/ PMID:26424080

  18. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.

    PubMed

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/.

  19. Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia.

    PubMed

    Williams, Anna V; Miller, Joseph T; Small, Ian; Nevill, Paul G; Boykin, Laura M

    2016-03-01

    Combining whole genome data with previously obtained amplicon sequences has the potential to increase the resolution of phylogenetic analyses, particularly at low taxonomic levels or where recent divergence, rapid speciation or slow genome evolution has resulted in limited sequence variation. However, the integration of these types of data for large scale phylogenetic studies has rarely been investigated. Here we conduct a phylogenetic analysis of the whole chloroplast genome and two nuclear ribosomal loci for 65 Acacia species from across the most recent Acacia phylogeny. We then combine this data with previously generated amplicon sequences (four chloroplast loci and two nuclear ribosomal loci) for 508 Acacia species. We use several phylogenetic methods, including maximum likelihood bootstrapping (with and without constraint) and ExaBayes, in order to determine the success of combining a dataset of 4000bp with one of 189,000bp. The results of our study indicate that the inclusion of whole genome data gave a far better resolved and well supported representation of the phylogenetic relationships within Acacia than using only amplicon sequences, with the greatest support observed when using a whole genome phylogeny as a constraint on the amplicon sequences. Our study therefore provides methods for optimal integration of genomic and amplicon sequences.

  20. Formation of Linear Amplicons with Inverted Duplications in Leishmania Requires the MRE11 Nuclease

    PubMed Central

    Laffitte, Marie-Claude N.; Genois, Marie-Michelle; Mukherjee, Angana; Légaré, Danielle; Masson, Jean-Yves; Ouellette, Marc

    2014-01-01

    Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11−/− parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment. PMID:25474106

  1. Electroanalytical study of proflavine intercalation in 5-methyl or inosine-containing amplicons.

    PubMed

    Alexiadou, Despina K; Ioannou, Andrea K; Kouidou-Andreou, Sofia A; Voulgaropoulos, Anastasios N; Girousi, Stella Th

    2008-10-01

    Amplicons corresponding to the GC-rich p53 exon 5 and its analogues, synthesized by substituting 60% of cytosine by 5-methyl-cytosine, or 60% of guanosine by inosine and GC-poor p53 exon 6 were synthesized and investigated electrochemically, in the presence and absence of proflavine, by differential pulse voltammetry (DPV). Incorporation of base analogues and the thermal stability of the resulting amplicons were tested in the presence of a fluorescent probe (Sybr-Green). Peak current at 1.0 V was lower for methylated than for unmethylated PCR amplicons and was similarly affected by proflavine intercalation. In contrast, considerable peak current differences were observed in the presence of proflavine for unmodified exon 5 v.s. exon 6 or inosine-containing amplicons. Thermal analysis verified the expected shifts in melting temperature (T (m)) due to the base analogue incorporation and GC-content variations. In conclusion, methylated and unmethylated PCR amplicons could be distinguished in model DNA systems using differential pulse voltammetry (DPV) and use of proflavine could serve as an electrochemical probe for identifying different DNA conformations.

  2. Sequence-specific electrochemical detection of asymmetric PCR amplicons of traditional Chinese medicinal plant DNA.

    PubMed

    Lee, Thomas M H; Hsing, I-Ming

    2002-10-01

    In this study, an electrochemistry-based approach to detect nucleic acid amplification products of Chinese herbal genes is reported. Using asymmetric polymerase chain reaction and electrochemical techniques, single-stranded target amplicons are produced from trace amounts of DNA sample and sequence-specific electrochemical detection based on the direct hybridization of the crude amplicon mix and immobilized DNA probe can be achieved. Electrochemically active intercalator Hoechst 33258 is bound to the double-stranded duplex formed by the target amplicon hybridized with the 5'-thiol-derivated DNA probe (16-mer) on the gold electrode surface. The electrochemical current signal of the hybridization event is measured by linear sweep voltammetry, the response of which can be used to differentiate the sequence complementarities of the target amplicons. To improve the reproducibility and sensitivity of the current signal, issues such as electrode surface cleaning, probe immobilization, and target hybridization are addressed. Factors affecting hybridization efficiency including the length and binding region of the target amplicon are discussed. Using our approach, differentiation of Chinese herbal species Fritillaria (F. thunbergii and F. cirrhosa) based on the 16-mer unique sequences in the spacer region of the 5S-rRNA is demonstrated. The ability to detect PCR products using a nonoptical electrochemical detection technique is an important step toward the realization of portable biomicrodevices for on-spot bacterial and viral detections.

  3. Polycyclic peptide therapeutics.

    PubMed

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases.

  4. Sex determination in beef by melting curve analysis of PCR amplicons from the amelogenin locus.

    PubMed

    Ballin, Nicolai Z; Madsen, Knud G

    2007-11-01

    Sex determination of beef is important to meet the rules of the Commission Regulation (EC) 765/2002 that qualify for export refunds. A SYBR Green sex identification assay based on melting curve analysis of PCR amplicons from the amelogenin locus (AMELX and AMELY) was developed. The PCR amplicons of 130/130 and 130/67 base pairs produced from female and male beef, respectively, are easily distinguished by both melting curve analysis and gel electrophoresis. Results from the melting curve analysis of amplicons are ready in less than three minutes, and requires no additional work in addition to the PCR setup. Applicability of the sex determination assay was studied by analysis of 12 unknown beef samples and the results were compared to an accredited method based on gel electrophoresis. In addition, six different cattle breeds were examined. All test results were correct in respect to sex.

  5. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors

    PubMed Central

    Falcicchia, Chiara; Trempat, Pascal; Binaschi, Anna; Perrier-Biollay, Coline; Roncon, Paolo; Soukupova, Marie; Berthommé, Hervé; Simonato, Michele

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies. PMID:26954758

  6. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

    PubMed

    Falcicchia, Chiara; Trempat, Pascal; Binaschi, Anna; Perrier-Biollay, Coline; Roncon, Paolo; Soukupova, Marie; Berthommé, Hervé; Simonato, Michele

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

  7. Glioma-specific and cell cycle-regulated herpes simplex virus type 1 amplicon viral vector.

    PubMed

    Ho, Ivy A W; Hui, Kam M; Lam, Paula Y P

    2004-05-01

    We have engineered a novel herpes simplex virus type 1 (HSV-1)-based amplicon viral vector, whereby gene expression is controlled by cell cycle events. In nondividing cells, trans-activation of the cyclin A promoter via interaction of the Gal4/NF-YA fusion protein with the Gal4-binding sites is prevented by the presence of a repressor protein, cell cycle-dependent factor 1 (CDF-1). CDF-1 is specifically expressed during the G(0)/G(1) phase of the cell cycle and its binding site is located within the cyclin A promoter. In actively proliferating cells, trans-activation could take place because of the absence of CDF-1. Our results showed that when all these cell cycle-specific regulatory elements are incorporated in cis into a single HSV-1 amplicon plasmid vector backbone (pC8-36), reporter luciferase activity is greatly enhanced. Transgene expression mediated by this series of HSV-1 amplicon plasmid vectors and amplicon viral vectors could be regulated in a cell cycle-dependent manner in a variety of cell lines. In a further attempt to target transgene expression to a selected group of actively proliferating cells such as glial cells, we have replaced the cytomegalovirus promoter of the pC8-36 amplicon plasmid with the glial cell-specific GFAP enhancer element. With this latter viral construct, cell type-specific and cell cycle-dependent transgene expression could subsequently be demonstrated specifically in glioma-bearing animals. Taken together, our results suggest that this series of cell cycle-regulatable HSV-1 amplicon viral vectors could potentially be adapted as useful tools for the treatment of human cancers.

  8. Use of AmpliWax to optimize amplicon sterilization by isopsoralen.

    PubMed Central

    De la Viuda, M; Fille, M; Ruiz, J; Aslanzadeh, J

    1996-01-01

    The photochemical inactivation of amplicons by isopsoralen (IP-10) has been suggested as a possible means to prevent PCR carryover contamination. To evaluate the technique, serial dilutions of amplicons (10(11) to 10(3)) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 micrograms of IP-10 per ml for 45 cycles. The PCR products were exposed to UV light for 15 min to activate IP-10 and sterilize the amplicons. One microliter of each sterilized sample was reamplified for an additional 45 cycles. The PCR products were then resolved in an agarose gel, blotted onto a nylon membrane, and probed with an alkaline phosphatase-conjugated chemiluminescent probe. Although IP-10 at concentrations of 50 and 100 micrograms/ml effectively sterilized up to 10(11) amplicons, the compound was inhibitory to PCR. IP-10 at a concentration of 25 micrograms/ml had slight inhibitory effect on PCR and did not completely sterilized all of the amplicons. Therefore, in subsequent experiments AmpliWax was substituted for mineral oil, and PCR was performed on 10(9) to 10(3) amplicons as described above. Following the amplification, the PCR tubes were cooled to solidify the AmpliWax and inoculated with various concentrations of IP-10. With this technique, PCR products produced from as many as 10(9) target amplicons were effectively sterilized with 200 micrograms of IP-10 per ml. Similarly, the addition of IP-10 (50 micrograms/ml) before and after PCR was evaluated for the detection of B. burgdorferi in 62 ticks from a region of Southern Connecticut where the organism is highly endemic. PCR performed in the presence of 50 micrograms of IP-10 per ml detected B. burgdorferi-specific DNA in 17 of 62 ticks (27%) following gel electrophoresis and in 34 of 62 ticks (55%) following Southern blot hybridization of the PCR products. In contrast, post-PCR addition of IP-10 detected borrelia-specific DNA in 31 of 62 ticks (50%) following gel electrophoresis and in

  9. Epitope Mapping with Random Phage Display Library

    PubMed Central

    Midoro-Horiuti, Terumi; Goldblum, Randall M.

    2017-01-01

    Random phage display library is used to map conformational as well as linear epitopes. These libraries are available in varying lengths and with circularization. We provide here a protocol conveying our experience using a commercially available peptide phage display library, which in our hands provides good results. PMID:24515483

  10. Macrocyclization of Unprotected Peptide Isocyanates.

    PubMed

    Vinogradov, Alexander A; Choo, Zi-Ning; Totaro, Kyle A; Pentelute, Bradley L

    2016-03-18

    A chemistry for the facile two-component macrocyclization of unprotected peptide isocyanates is described. Starting from peptides containing two glutamic acid γ-hydrazide residues, isocyanates can be readily accessed and cyclized with hydrazides of dicarboxylic acids. The choice of a nucleophilic linker allows for the facile modulation of biochemical properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues were synthesized using the described method and displayed a range of biological activities.

  11. Cell surface display of functional human MHC class II proteins: yeast display versus insect cell display

    PubMed Central

    Wen, Fei; Sethi, Dhruv K.; Wucherpfennig, Kai W.; Zhao, Huimin

    2011-01-01

    Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2–MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs. PMID:21752831

  12. Characterizing partial AZFc deletions of the Y chromosome with amplicon-specific sequence markers

    PubMed Central

    Navarro-Costa, Paulo; Pereira, Luísa; Alves, Cíntia; Gusmão, Leonor; Proença, Carmen; Marques-Vidal, Pedro; Rocha, Tiago; Correia, Sónia C; Jorge, Sónia; Neves, António; Soares, Ana P; Nunes, Joaquim; Calhaz-Jorge, Carlos; Amorim, António; Plancha, Carlos E; Gonçalves, João

    2007-01-01

    Background The AZFc region of the human Y chromosome is a highly recombinogenic locus containing multi-copy male fertility genes located in repeated DNA blocks (amplicons). These AZFc gene families exhibit slight sequence variations between copies which are considered to have functional relevance. Yet, partial AZFc deletions yield phenotypes ranging from normospermia to azoospermia, thwarting definite conclusions on their real impact on fertility. Results The amplicon content of partial AZFc deletion products was characterized with novel amplicon-specific sequence markers. Data indicate that partial AZFc deletions are a male infertility risk [odds ratio: 5.6 (95% CI: 1.6–30.1)] and although high diversity of partial deletion products and sequence conversion profiles were recorded, the AZFc marker profiles detected in fertile men were also observed in infertile men. Additionally, the assessment of rearrangement recurrence by Y-lineage analysis indicated that while partial AZFc deletions occurred in highly diverse samples, haplotype diversity was minimal in fertile men sharing identical marker profiles. Conclusion Although partial AZFc deletion products are highly heterogeneous in terms of amplicon content, this plasticity is not sufficient to account for the observed phenotypical variance. The lack of causative association between the deletion of specific gene copies and infertility suggests that AZFc gene content might be part of a multifactorial network, with Y-lineage evolution emerging as a possible phenotype modulator. PMID:17903263

  13. High-throughput amplicon sequencing reveals distinct communities within a corroding concrete sewer system.

    PubMed

    Cayford, Barry I; Dennis, Paul G; Keller, Jurg; Tyson, Gene W; Bond, Philip L

    2012-10-01

    Microbially induced concrete corrosion (MICC) is an important problem in sewers. Here, small-subunit (SSU) rRNA gene amplicon pyrosequencing was used to characterize MICC communities. Microbial community composition differed between wall- and ceiling-associated MICC layers. Acidithiobacillus spp. were present at low abundances, and the communities were dominated by other sulfur-oxidizing-associated lineages.

  14. High-Throughput Amplicon Sequencing Reveals Distinct Communities within a Corroding Concrete Sewer System

    PubMed Central

    Dennis, Paul G.; Keller, Jurg; Tyson, Gene W.

    2012-01-01

    Microbially induced concrete corrosion (MICC) is an important problem in sewers. Here, small-subunit (SSU) rRNA gene amplicon pyrosequencing was used to characterize MICC communities. Microbial community composition differed between wall- and ceiling-associated MICC layers. Acidithiobacillus spp. were present at low abundances, and the communities were dominated by other sulfur-oxidizing-associated lineages. PMID:22843532

  15. Advance in phage display technology for bioanalysis.

    PubMed

    Tan, Yuyu; Tian, Tian; Liu, Wenli; Zhu, Zhi; J Yang, Chaoyong

    2016-06-01

    Phage display technology has emerged as a powerful tool for target gene expression and target-specific ligand selection. It is widely used to screen peptides, proteins and antibodies with the advantages of simplicity, high efficiency and low cost. A variety of targets, including ions, small molecules, inorganic materials, natural and biological polymers, nanostructures, cells, bacteria, and even tissues, have been demonstrated to generate specific binding ligands by phage display. Phages and target-specific ligands screened by phage display have been widely used as affinity reagents in therapeutics, diagnostics and biosensors. In this review, comparisons of different types of phage display systems are first presented. Particularly, microfluidic-based phage display, which enables screening with high throughput, high efficiency and integration, is highlighted. More importantly, we emphasize the advances in biosensors based on phages or phage-derived probes, including nonlytic phages, lytic phages, peptides or proteins screened by phage display, phage assemblies and phage-nanomaterial complexes. However, more efficient and higher throughput phage display methods are still needed to meet an explosion in demand for bioanalysis. Furthermore, screening of cyclic peptides and functional peptides will be the hotspot in bioanalysis.

  16. Use of Peptide Libraries for Identification and Optimization of Novel Antimicrobial Peptides.

    PubMed

    Ashby, Martin; Petkova, Asya; Gani, Jurnorain; Mikut, Ralf; Hilpert, Kai

    2017-01-01

    The increasing rates of resistance among bacteria and to a lesser extent fungi have resulted in an urgent need to find new molecules that hold therapeutic promise against multidrug-resistant strains. Antimicrobial peptides have proven very effective against a variety of multidrug-resistant bacteria. Additionally, the low levels of resistance reported towards these molecules are an attractive feature for antimicrobial drug development. Here we summarise information on diverse peptide libraries used to discover or to optimize antimicrobial peptides. Chemical synthesized peptide libraries, for example split and mix method, tea bag method, multi-pin method and cellulose spot method are discussed. In addition biological peptide library screening methods are summarized, like phage display, bacterial display, mRNA-display and ribosomal display. A few examples are given for small peptide libraries, which almost exclusively follow a rational design of peptides of interest rather than a combinatorial approach.

  17. Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line.

    PubMed

    Ma, C; Looney, J E; Leu, T H; Hamlin, J L

    1988-06-01

    We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

  18. [Hydrolysis of peptides by immobilized bacterial peptide hydrolases].

    PubMed

    Nekliudov, A D; Deniakina, E K

    2004-01-01

    The feasibility of hydrolysis of a mixture of peptides with an enzyme from the bacterium Xanthomonas rubrilineans, displaying a peptidase activity and immobilized on aluminum oxide, was studied. Kinetic schemes and equations allowing for approaching quantitative description of peptide hydrolysis in complex mixtures containing free amino acids and peptides were obtained. It was demonstrated that as a result of hydrolysis, the content of free amino acids in hydrolysates decreased 2.5- to 3-fold and the molecular weight of the constituent peptides, 2-fold.

  19. Potential of pmoA amplicon pyrosequencing for methanotroph diversity studies.

    PubMed

    Lüke, Claudia; Frenzel, Peter

    2011-09-01

    We analyzed the potential of pmoA amplicon pyrosequencing compared to that of Sanger sequencing with paddy soils as a model environment. We defined operational taxonomic unit (OTU) cutoff values of 7% and 18%, reflecting methanotrophic species and major phylogenetic pmoA lineages, respectively. Major lineages were already well covered by clone libraries; nevertheless, pyrosequencing provided a higher level of diversity at the species level.

  20. Fast, accurate and easy-to-pipeline methods for amplicon sequence processing

    NASA Astrophysics Data System (ADS)

    Antonielli, Livio; Sessitsch, Angela

    2016-04-01

    Next generation sequencing (NGS) technologies established since years as an essential resource in microbiology. While on the one hand metagenomic studies can benefit from the continuously increasing throughput of the Illumina (Solexa) technology, on the other hand the spreading of third generation sequencing technologies (PacBio, Oxford Nanopore) are getting whole genome sequencing beyond the assembly of fragmented draft genomes, making it now possible to finish bacterial genomes even without short read correction. Besides (meta)genomic analysis next-gen amplicon sequencing is still fundamental for microbial studies. Amplicon sequencing of the 16S rRNA gene and ITS (Internal Transcribed Spacer) remains a well-established widespread method for a multitude of different purposes concerning the identification and comparison of archaeal/bacterial (16S rRNA gene) and fungal (ITS) communities occurring in diverse environments. Numerous different pipelines have been developed in order to process NGS-derived amplicon sequences, among which Mothur, QIIME and USEARCH are the most well-known and cited ones. The entire process from initial raw sequence data through read error correction, paired-end read assembly, primer stripping, quality filtering, clustering, OTU taxonomic classification and BIOM table rarefaction as well as alternative "normalization" methods will be addressed. An effective and accurate strategy will be presented using the state-of-the-art bioinformatic tools and the example of a straightforward one-script pipeline for 16S rRNA gene or ITS MiSeq amplicon sequencing will be provided. Finally, instructions on how to automatically retrieve nucleotide sequences from NCBI and therefore apply the pipeline to targets other than 16S rRNA gene (Greengenes, SILVA) and ITS (UNITE) will be discussed.

  1. Characterization of Novel Genes Within 8P11-12 Amplicon in Breast Cancer

    DTIC Science & Technology

    2007-06-01

    C-myc amplification in breast cancer: a meta - analysis of its occurrence and prognostic relevance. Br J Cancer, 83: 1688-1695, 2000. 2. Hui, R...Nass SJ, Dickson RB, Trock BJ. C-myc amplification in breast cancer: a meta - analysis of its occurrence and prognostic relevance. Br J Cancer 2000;83...a detailed genomic and expression analysis of the 8p11-p12 amplicon in breast cancer cell lines and identified several novel candidate genes

  2. Functional Analysis of Frequent Lung Cancer Amplicons Under Current Genome Annotation

    DTIC Science & Technology

    2015-12-01

    Exemplary groups of such genes include many non-protein-coding genes like long non-coding RNAs and microRNAs (miRNAs). In study, we examined the PVT1...lung tumorigenesis. The implication is that future studies of the 8q24 amplicon should concentrate on characterizing the PVT1 RNA transcript. 15...SUBJECT TERMS Noncoding RNA , microRNA, lung cancer, Genome annotation 16. SECURITY CLASSIFICATION OF: U 17. LIMITATION OF ABSTRACT 18. NUMBER OF

  3. Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons.

    PubMed

    Kim, Eun Hye; Lee, Hwan Young; Yang, In Seok; Jung, Sang-Eun; Yang, Woo Ick; Shin, Kyoung-Jin

    2016-05-01

    The next-generation sequencing (NGS) method has been utilized to analyze short tandem repeat (STR) markers, which are routinely used for human identification purposes in the forensic field. Some researchers have demonstrated the successful application of the NGS system to STR typing, suggesting that NGS technology may be an alternative or additional method to overcome limitations of capillary electrophoresis (CE)-based STR profiling. However, there has been no available multiplex PCR system that is optimized for NGS analysis of forensic STR markers. Thus, we constructed a multiplex PCR system for the NGS analysis of 18 markers (13CODIS STRs, D2S1338, D19S433, Penta D, Penta E and amelogenin) by designing amplicons in the size range of 77-210 base pairs. Then, PCR products were generated from two single-sources, mixed samples and artificially degraded DNA samples using a multiplex PCR system, and were prepared for sequencing on the MiSeq system through construction of a subsequent barcoded library. By performing NGS and analyzing the data, we confirmed that the resultant STR genotypes were consistent with those of CE-based typing. Moreover, sequence variations were detected in targeted STR regions. Through the use of small-sized amplicons, the developed multiplex PCR system enables researchers to obtain successful STR profiles even from artificially degraded DNA as well as STR loci which are analyzed with large-sized amplicons in the CE-based commercial kits. In addition, successful profiles can be obtained from mixtures up to a 1:19 ratio. Consequently, the developed multiplex PCR system, which produces small size amplicons, can be successfully applied to STR NGS analysis of forensic casework samples such as mixtures and degraded DNA samples.

  4. Characterization of FGFR1 Locus in sqNSCLC Reveals a Broad and Heterogeneous Amplicon.

    PubMed

    Rooney, Claire; Geh, Catherine; Williams, Victoria; Heuckmann, Johannes M; Menon, Roopika; Schneider, Petra; Al-Kadhimi, Katherine; Dymond, Michael; Smith, Neil R; Baker, Dawn; French, Tim; Smith, Paul D; Harrington, Elizabeth A; Barrett, J Carl; Kilgour, Elaine

    2016-01-01

    FGFR1 amplification occurs in ~20% of sqNSCLC and trials with FGFR inhibitors have selected FGFR1 amplified patients by FISH. Lung cancer cell lines were profiled for sensitivity to AZD4547, a potent, selective inhibitor of FGFRs 1-3. Sensitivity to FGFR inhibition was associated with but not wholly predicted by increased FGFR1 gene copy number. Additional biomarker assays evaluating expression of FGFRs and correlation between amplification and expression in clinical tissues are therefore warranted. We validated nanoString for mRNA expression analysis of 194 genes, including FGFRs, from clinical tumour tissue. In a panel of sqNSCLC tumours 14.4% (13/90) were FGFR1 amplified by FISH. Although mean FGFR1 expression was significantly higher in amplified samples, there was significant overlap in the range of expression levels between the amplified and non-amplified cohorts with several non-amplified samples expressing FGFR1 to levels equivalent to amplified samples. Statistical analysis revealed increased expression of FGFR1 neighboring genes on the 8p12 amplicon (BAG4, LSM1 and WHSC1L1) in FGFR1 amplified tumours, suggesting a broad rather than focal amplicon and raises the potential for codependencies. High resolution aCGH analysis of pre-clinical and clinical samples supported the presence of a broad and heterogeneous amplicon around the FGFR1 locus. In conclusion, the range of FGFR1 expression levels in both FGFR1 amplified and non-amplified NSCLC tissues, together with the breadth and intra-patient heterogeneity of the 8p amplicon highlights the need for gene expression analysis of clinical samples to inform the understanding of determinants of response to FGFR inhibitors. In this respect the nanoString platform provides an attractive option for RNA analysis of FFPE clinical samples.

  5. Evaluating sequence-derived mtDNA length heteroplasmy by amplicon size analysis

    PubMed Central

    Berger, C.; Hatzer-Grubwieser, P.; Hohoff, C.; Parson, W.

    2011-01-01

    Length heteroplasmy (LH) in mitochondrial (mt)DNA is usually observed in homopolymeric tracts and manifest as mixture of various length variants. The generally used difference-coded annotation to report mtDNA haplotypes does not express the degree of LH variation present in a sample, even more so, it is sometimes difficult to establish which length variants are present and clearly distinguishable from background noise. It has therefore become routine practice for some researchers to call the dominant type, the “major molecule”, which represents the LH variant that is most abundant in a DNA extract. In the majority of cases a clear single dominant variant can be identified. However, in some samples this interpretation is difficult, i.e. when (almost) equally quantitative LH variants are present or when multiple sequencing primers result in the presentation of different dominant types. To better understand those cases we designed amplicon sizing assays for the five most relevant LH regions in the mtDNA control region (around ntps 16,189, 310, 460, 573, and the AC-repeat between 514 and 524) to determine the ratio of the LH variants by fluorescence based amplicon sizing assays. For difficult LH constellations derived by Sanger sequencing (with Big Dye terminators) these assays mostly gave clear and unambiguous results. In the vast majority of cases we found agreement between the results of the sequence and amplicon analyses and propose this alternative method in difficult cases. PMID:21067985

  6. Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

    PubMed Central

    Blanc, Andrea Maria; Berois, Mabel Beatriz; Tomé, Lorena Magalí; Epstein, Alberto L.

    2012-01-01

    Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease. PMID:22437537

  7. From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows

    PubMed Central

    Murray, Dáithí C.; Coghlan, Megan L.; Bunce, Michael

    2015-01-01

    Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose. PMID:25902146

  8. Phage display: concept, innovations, applications and future.

    PubMed

    Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K

    2010-01-01

    Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.

  9. Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

    PubMed Central

    Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

    2013-01-01

    We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. PMID:23178437

  10. Mining environmental high-throughput sequence data sets to identify divergent amplicon clusters for phylogenetic reconstruction and morphotype visualization.

    PubMed

    Gimmler, Anna; Stoeck, Thorsten

    2015-08-01

    Environmental high-throughput sequencing (envHTS) is a very powerful tool, which in protistan ecology is predominantly used for the exploration of diversity and its geographic and local patterns. We here used a pyrosequenced V4-SSU rDNA data set from a solar saltern pond as test case to exploit such massive protistan amplicon data sets beyond this descriptive purpose. Therefore, we combined a Swarm-based blastn network including 11 579 ciliate V4 amplicons to identify divergent amplicon clusters with targeted polymerase chain reaction (PCR) primer design for full-length small subunit of the ribosomal DNA retrieval and probe design for fluorescence in situ hybridization (FISH). This powerful strategy allows to benefit from envHTS data sets to (i) reveal the phylogenetic position of the taxon behind divergent amplicons; (ii) improve phylogenetic resolution and evolutionary history of specific taxon groups; (iii) solidly assess an amplicons (species') degree of similarity to its closest described relative; (iv) visualize the morphotype behind a divergent amplicons cluster; (v) rapidly FISH screen many environmental samples for geographic/habitat distribution and abundances of the respective organism and (vi) to monitor the success of enrichment strategies in live samples for cultivation and isolation of the respective organisms.

  11. Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene.

    PubMed

    Suzuki, Masataka; Chiocca, E Antonio; Saeki, Yoshinaga

    2007-11-01

    The herpes simplex virus (HSV) amplicon vector is a powerful gene delivery vehicle that can accommodate up to 150 kilobase of exogenous DNA. However, amplicon-mediated transgene expression is often transient outside the nervous system. In order to define the role of host immune responses in silencing amplicon-encoded transgenes, we evaluated the kinetics of cytokine-/chemokine-expression after tail-vein injection of a luciferase-encoding amplicon into mice. Type I interferons (IFNs) were induced earliest, within an hour after injection, and several other cytokines/chemokines were subsequently upregulated in the livers of wild-type (WT) mice. When the same experiment was performed in signal transducers and activators of transcription 1 (STAT1)-knockout (KO) mice, the levels of type I IFN expression were significantly lower and chemokine induction was almost non-existent. Importantly, STAT1-KO mice exhibited significantly higher and more sustained luciferase activity than did the WT mice, which is attributable to increased transcriptional activity rather than increased copy numbers of luciferase-encoding vector DNA. Further studies using primary cultured fibroblasts derived from WT and STAT1-KO mice revealed the significance of STAT1 signaling in transcriptional silencing of the amplicon-encoded transgene in vitro. Our results indicate that type I IFNs induced by systemic delivery of HSV amplicon vectors initiate a cascade of immune responses and suppress transgene expression at the transcriptional level by activation of STAT1.

  12. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

    PubMed

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

    2015-08-01

    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31).

  13. Regional effects on chimera formation in 454 pyrosequenced amplicons from a mock community.

    PubMed

    Shin, Sunguk; Lee, Tae Kwon; Han, Jung Min; Park, Joonhong

    2014-07-01

    Chimeras are a frequent artifact in polymerase chain reaction and could be the underlying causes of erroneous taxonomic identifications, overestimated microbial diversity, and spurious sequences. However, little is known about the regional effects on chimera formation. Therefore, we investigated the chimera formation rates in different regions of phylogenetically important biomarker genes to test the regional effects on chimera formation. An empirical study of chimera formation rates was performed using the Roche GSFLX™ system with sequences of the V1/V2/V3 and V4/V5 regions of the 16S rRNA gene and sequences of the nifH gene from a mock microbial community. The chimera formation rates for the 16S V1/V2/V3 region, V4/V5 region, and nifH gene were 22.1-38.5%, 3.68-3.88%, and 0.31-0.98%, respectively. Some amplicons from the V1/V2/V3 regions were shorter than the typical length (∼7-31%), reflecting incomplete extension. In the V1/V2/V3 and V4/V5 regions, conserved and hypervariable regions were identified. Chimeric hot spots were located in parts of conserved regions near the ends of the amplicons. The 16S V1/V2/V3 region had the highest chimera formation rate, likely because of long template lengths and incomplete extension. The amplicons of the nifH gene had the lowest frequency of chimera formation most likely because of variations in their wobble positions in triplet codons. Our results suggest that the main reasons for chimera formation are sequence similarity and premature termination of DNA extension near primer regions. Other housekeeping genes can be a good substitute for 16S rRNA genes in molecular microbial studies to reduce the effects of chimera formation.

  14. Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers

    PubMed Central

    Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M.; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

    2016-01-01

    Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage. TABSAT is freely

  15. Identification of tissue-specific targeting peptide

    NASA Astrophysics Data System (ADS)

    Jung, Eunkyoung; Lee, Nam Kyung; Kang, Sang-Kee; Choi, Seung-Hoon; Kim, Daejin; Park, Kisoo; Choi, Kihang; Choi, Yun-Jaie; Jung, Dong Hyun

    2012-11-01

    Using phage display technique, we identified tissue-targeting peptide sets that recognize specific tissues (bone-marrow dendritic cell, kidney, liver, lung, spleen and visceral adipose tissue). In order to rapidly evaluate tissue-specific targeting peptides, we performed machine learning studies for predicting the tissue-specific targeting activity of peptides on the basis of peptide sequence information using four machine learning models and isolated the groups of peptides capable of mediating selective targeting to specific tissues. As a representative liver-specific targeting sequence, the peptide "DKNLQLH" was selected by the sequence similarity analysis. This peptide has a high degree of homology with protein ligands which can interact with corresponding membrane counterparts. We anticipate that our models will be applicable to the prediction of tissue-specific targeting peptides which can recognize the endothelial markers of target tissues.

  16. Potential of novel antimicrobial peptide P3 from bovine erythrocytes and its analogs to disrupt bacterial membranes in vitro and display activity against drug-resistant bacteria in a mouse model.

    PubMed

    Zhang, Qinghua; Xu, Yanzhao; Wang, Qing; Hang, Bolin; Sun, Yawei; Wei, Xiaoxiao; Hu, Jianhe

    2015-05-01

    With the emergence of many antibiotic-resistant strains worldwide, antimicrobial peptides (AMPs) are being evaluated as promising alternatives to conventional antibiotics. P3, a novel hemoglobin peptide derived from bovine erythrocytes, exhibited modest antimicrobial activity in vitro. We evaluated the antimicrobial activities of P3 and an analog, JH-3, both in vitro and in vivo. The MICs of P3 and JH-3 ranged from 3.125 μg/ml to 50 μg/ml when a wide spectrum of bacteria was tested, including multidrug-resistant strains. P3 killed bacteria within 30 min by disrupting the bacterial cytoplasmic membrane and disturbing the intracellular calcium balance. Circular dichroism (CD) spectrometry showed that P3 assumed an α-helical conformation in bacterial lipid membranes, which was indispensable for antimicrobial activity. Importantly, the 50% lethal dose (LD50) of JH-3 was 180 mg/kg of mouse body weight after intraperitoneal (i.p.) injection, and no death was observed at any dose up to 240 mg/kg body weight following subcutaneous (s.c.) injection. Furthermore, JH-3 significantly decreased the bacterial count and rescued infected mice in a model of mouse bacteremia. In conclusion, P3 and an analog exhibited potent antimicrobial activities and relatively low toxicities in a mouse model, indicating that they may be useful for treating infections caused by drug-resistant bacteria.

  17. Display formats manual

    NASA Technical Reports Server (NTRS)

    Runnels, R. L.

    1973-01-01

    The standards and procedures for the generation of operational display formats to be used in the Mission Control Center (MCC) display control system are presented. The required effort, forms, and fundamentals for the design, specifications, and production of display formats are identified. The principles of display design and system constraints controlling the creation of optimum operational displays for mission control are explained. The basic two types of MCC display systems for presenting information are described.

  18. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    PubMed

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection.

  19. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis.

    PubMed

    Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum; Delay, Catherine E; Yang, Zhang; Niola, Francesco; Duda, Katarzyna; Ober, Elke A; Clausen, Henrik; Wandall, Hans H; Hansen, Steen H; Bennett, Eric P; Frödin, Morten

    2017-03-01

    This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.

  20. Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform

    PubMed Central

    Schirmer, Melanie; Ijaz, Umer Z.; D'Amore, Rosalinda; Hall, Neil; Sloan, William T.; Quince, Christopher

    2015-01-01

    With read lengths of currently up to 2 × 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%. PMID:25586220

  1. Multiplex amplicon sequencing for microbe identification in community-based culture collections

    PubMed Central

    Armanhi, Jaderson Silveira Leite; de Souza, Rafael Soares Correa; de Araújo, Laura Migliorini; Okura, Vagner Katsumi; Mieczkowski, Piotr; Imperial, Juan; Arruda, Paulo

    2016-01-01

    Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC. PMID:27404280

  2. ThermoAlign: a genome-aware primer design tool for tiled amplicon resequencing

    PubMed Central

    Francis, Felix; Dumas, Michael D.; Wisser, Randall J.

    2017-01-01

    Isolating and sequencing specific regions in a genome is a cornerstone of molecular biology. This has been facilitated by computationally encoding the thermodynamics of DNA hybridization for automated design of hybridization and priming oligonucleotides. However, the repetitive composition of genomes challenges the identification of target-specific oligonucleotides, which limits genetics and genomics research on many species. Here, a tool called ThermoAlign was developed that ensures the design of target-specific primer pairs for DNA amplification. This is achieved by evaluating the thermodynamics of hybridization for full-length oligonucleotide-template alignments — thermoalignments — across the genome to identify primers predicted to bind specifically to the target site. For amplification-based resequencing of regions that cannot be amplified by a single primer pair, a directed graph analysis method is used to identify minimum amplicon tiling paths. Laboratory validation by standard and long-range polymerase chain reaction and amplicon resequencing with maize, one of the most repetitive genomes sequenced to date (≈85% repeat content), demonstrated the specificity-by-design functionality of ThermoAlign. ThermoAlign is released under an open source license and bundled in a dependency-free container for wide distribution. It is anticipated that this tool will facilitate multiple applications in genetics and genomics and be useful in the workflow of high-throughput targeted resequencing studies. PMID:28300202

  3. AMPLISAS: a web server for multilocus genotyping using next-generation amplicon sequencing data.

    PubMed

    Sebastian, Alvaro; Herdegen, Magdalena; Migalska, Magdalena; Radwan, Jacek

    2016-03-01

    Next-generation sequencing (NGS) technologies are revolutionizing the fields of biology and medicine as powerful tools for amplicon sequencing (AS). Using combinations of primers and barcodes, it is possible to sequence targeted genomic regions with deep coverage for hundreds, even thousands, of individuals in a single experiment. This is extremely valuable for the genotyping of gene families in which locus-specific primers are often difficult to design, such as the major histocompatibility complex (MHC). The utility of AS is, however, limited by the high intrinsic sequencing error rates of NGS technologies and other sources of error such as polymerase amplification or chimera formation. Correcting these errors requires extensive bioinformatic post-processing of NGS data. Amplicon Sequence Assignment (AMPLISAS) is a tool that performs analysis of AS results in a simple and efficient way, while offering customization options for advanced users. AMPLISAS is designed as a three-step pipeline consisting of (i) read demultiplexing, (ii) unique sequence clustering and (iii) erroneous sequence filtering. Allele sequences and frequencies are retrieved in excel spreadsheet format, making them easy to interpret. AMPLISAS performance has been successfully benchmarked against previously published genotyped MHC data sets obtained with various NGS technologies.

  4. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing

    PubMed Central

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S.; Perkins, David L.

    2016-01-01

    The human microbiome has emerged as a major player in regulating human health and disease. Translation studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using shotgun whole genome sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1×106 reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that shotgun whole genome sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  5. Application of novel "mini-amplicon" STR multiplexes to high volume casework on degraded skeletal remains.

    PubMed

    Parsons, Thomas J; Huel, Rene; Davoren, Jon; Katzmarzyk, Cheryl; Milos, Ana; Selmanović, Arijana; Smajlović, Lejla; Coble, Michael D; Rizvić, Adnan

    2007-06-01

    The International Commission on Missing Persons (ICMP) conducts high throughput STR profiling on degraded skeletal remains, primarily recovered from mass graves relating to conflicts from 1992 to 1999 in the former Yugoslavia. To date, over 11,000 individuals have been identified through comparison of bone profiles to a large database of profiles from family members of the missing. To increase success rates in STR recovery, three short amplicon STR multiplexes (a 7-plex, a 6-plex, and a 5-plex) have been devised and implemented. These target loci from large commercial multiplexes, with an average decrease in amplicon size of 144 bp. The ICMP "miniplexes" have proven to provide substantially greater recovery of DNA data from a certain subset of difficult samples. However, the circumstances under which miniplexes provide additional data are restricted, and their advantages do not outweigh those of large commercial multiplexes for a majority of cases. The miniplexes, however, also have a very powerful use in DNA testing to support large scale reassociation of commingled, partial skeletons recovered from secondary mass graves.

  6. Bioinformatic Amplicon Read Processing Strategies Strongly Affect Eukaryotic Diversity and the Taxonomic Composition of Communities

    PubMed Central

    Majaneva, Markus; Hyytiäinen, Kirsi; Varvio, Sirkka Liisa; Nagai, Satoshi; Blomster, Jaanika

    2015-01-01

    Amplicon read sequencing has revolutionized the field of microbial diversity studies. The technique has been developed for bacterial assemblages and has undergone rigorous testing with mock communities. However, due to the great complexity of eukaryotes and the numbers of different rDNA copies, analyzing eukaryotic diversity is more demanding than analyzing bacterial or mock communities, so studies are needed that test the methods of analyses on taxonomically diverse natural communities. In this study, we used 20 samples collected from the Baltic Sea ice, slush and under-ice water to investigate three program packages (UPARSE, mothur and QIIME) and 18 different bioinformatic strategies implemented in them. Our aim was to assess the impact of the initial steps of bioinformatic strategies on the results when analyzing natural eukaryotic communities. We found significant differences among the strategies in resulting read length, number of OTUs and estimates of diversity as well as clear differences in the taxonomic composition of communities. The differences arose mainly because of the variable number of chimeric reads that passed the pre-processing steps. Singleton removal and denoising substantially lowered the number of errors. Our study showed that the initial steps of the bioinformatic amplicon read processing strategies require careful consideration before applying them to eukaryotic communities. PMID:26047335

  7. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry

    PubMed Central

    Xu, Xiangming; Passey, Thomas; Wei, Feng; Saville, Robert; Harrison, Richard J.

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites had a yield-decline problem, the other two did not. More than 2000 fungal or bacterial operational taxonomy units (OTUs) were found in these samples. Relative abundance of individual OTUs was statistically compared for differences between samples from sites with or without yield decline. A total of 721 individual comparisons were statistically significant – involving 366 unique bacterial and 44 unique fungal OTUs. Based on further selection criteria, we focused on 34 bacterial and 17 fungal OTUs and found that yield decline resulted probably from one or more of the following four factors: (1) low abundance of Bacillus and Pseudomonas populations, which are well known for their ability of supressing pathogen development and/or promoting plant growth; (2) lack of the nematophagous fungus (Paecilomyces species); (3) a high level of two non-specific fungal root rot pathogens; and (4) wet soil conditions. This study demonstrated the usefulness of an amplicon-based metagenomics approach to profile soil microbiota and to detect differential abundance in microbes. PMID:26504572

  8. ICO amplicon NGS data analysis: a Web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS Junior next-generation sequencing.

    PubMed

    Lopez-Doriga, Adriana; Feliubadaló, Lídia; Menéndez, Mireia; Lopez-Doriga, Sergio; Morón-Duran, Francisco D; del Valle, Jesús; Tornero, Eva; Montes, Eva; Cuesta, Raquel; Campos, Olga; Gómez, Carolina; Pineda, Marta; González, Sara; Moreno, Victor; Capellá, Gabriel; Lázaro, Conxi

    2014-03-01

    Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.

  9. Peptide array-based characterization and design of ZnO-high affinity peptides.

    PubMed

    Okochi, Mina; Sugita, Tomoya; Furusawa, Seiji; Umetsu, Mitsuo; Adschiri, Tadafumi; Honda, Hiroyuki

    2010-08-15

    Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self-assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO-binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO-binding peptide, 125 spot-synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO-binding sites were found as 6-mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles.

  10. Stereoscopic Flat Panel Display

    DTIC Science & Technology

    2004-12-01

    the display of stereo imagery have been demonstrated. Stereoscopic displays typically require the user to wear special headgear. Autostereoscopic ...components and the resulting changes in the encoding algorithm. Keywords: Stereoscopic display, LCD, 3D , polarization encoding, flat panel 1...panel display when viewing non-stereoscopic imagery or data. Remotely operated vehicles do not represent the only potential application for 3D

  11. Electrochromic display device

    NASA Astrophysics Data System (ADS)

    Nicholson, M. M.

    1984-07-01

    This invention relates to electrochromic devices. In one aspect it relates to electrically controllable display devices. In another aspect it relates to electrically tunable optical or light filters. In yet another aspect it relates to a chemical sensor device which employs a color changing film. There are many uses for electrically controllable display devices. A number of such devices have been in commercial use for some time. These display devices include liquid crystal displays, light emitting diode displays, plasma displays, and the like. Light emitting diode displays and plasma display panels both suffer from the fact that they are active. Light emissive devices which require substantial power for their operation, In addition, it is difficult to fabricate light emitting diode displays in a manner which renders them easily distinguishable under bright ambient illumination. Liquid crystal displays suffer from the disadvantage that they are operative only over a limited temperature range and have substantially no memory within the liquid crystal material.

  12. System status display information

    NASA Technical Reports Server (NTRS)

    Summers, L. G.; Erickson, J. B.

    1984-01-01

    The system Status Display is an electronic display system which provides the flight crew with enhanced capabilities for monitoring and managing aircraft systems. Guidelines for the design of the electronic system displays were established. The technical approach involved the application of a system engineering approach to the design of candidate displays and the evaluation of a Hernative concepts by part-task simulation. The system engineering and selection of candidate displays are covered.

  13. Ultra-Deep Pyrosequencing (UDPS) Data Treatment to Study Amplicon HCV Minor Variants

    PubMed Central

    Gregori, Josep; Esteban, Juan I.; Cubero, María; Garcia-Cehic, Damir; Perales, Celia; Casillas, Rosario; Alvarez-Tejado, Miguel; Rodríguez-Frías, Francisco; Guardia, Jaume; Domingo, Esteban; Quer, Josep

    2013-01-01

    We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity. PMID:24391758

  14. The Bias Associated with Amplicon Sequencing Does Not Affect the Quantitative Assessment of Bacterial Community Dynamics

    PubMed Central

    Figuerola, Eva L. M.; Erijman, Leonardo

    2014-01-01

    The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1–V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1–V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1–V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa–time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1–V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point. PMID:24923665

  15. Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR.

    PubMed

    El-Sherbini, Yasser M; Stevenson, Mark M; Seymour, Leonard W; Wade-Martins, Richard

    2009-08-01

    Herpes simplex virus type 1 (HSV-1) amplicon preparations are usually quantified as transducing units/ml (TU/ml), with little information on genomic copy/TU ratios. In the present study, two HSV-1 amplicons expressing enhanced green fluorescent protein (EGFP) were analysed by quantitative PCR (qPCR) and transducing activity to obtain genomic copy/TU ratios. One vector (pHSV-GL) contains the HSV-1 packaging signal (pac) and origin of replication (oriS) and the other (pHSV/EBV-GL) includes Epstein-Barr virus (EBV) episomal maintenance elements. The pHSV-GL and pHSV/EBV-GL amplicons were prepared at titres of 7.55x10(7) and 7.24x10(7)TU/ml, containing 2.56x10(9) and 1.33x10(9) genomic copies/ml respectively. This produced preliminary estimates of genomic copy/TU ratios of 34:1 and 18:1. However standard transduction conditions did not deplete fully the supernatant of transducing particles since the same supernatant was subsequently able to achieve 25% the initial transduction efficiency, although centrifugation of amplicon particles onto cells improved infectivity by 1.8-fold. Finally, qPCR analysis of FACS-purified EGFP-expressing cells showed the presence of approximately 3 amplicon genomes/transduced cell, independent of the infection dose. Accordingly, the initial estimated genomic copy/TU ratio for pHSV-GL was revised to 6.3:1. Measuring the genomic copy/TU ratios is an important parameter for comparing the quality of amplicon preparations and standardizing experimental conditions.

  16. Peptide -- Silica Hybrid Networks

    NASA Astrophysics Data System (ADS)

    Altunbas, Aysegul; Sharma, Nikhil; Nagarkar, Radhika; Schneider, Joel; Pochan, Darrin

    2010-03-01

    In this study, a bio-inspired route was used to fabricate scaffolds that display hierarchical organization of an inorganic layer around an organic self-assembled peptide fibril template. The 20 amino acid peptide used in this study intramolecular folds into a beta-hairpin conformation on addition of a desired solution stimulus. This intramolecular folding is followed by intermolecular self-assembly of the peptides into a three dimensional network of entangled fibrils rich in beta-sheet with a high density of lysine groups exposed on the fibril-surfaces. The lysine-rich surface chemistry was utilized to create a silica shell around the fibrils. The mineralization process of the fibrils results in a rigid, porous silica network that retains the microscale and nanoscale structure of the peptide fibril network. Structural characterization via Transmission Electron Microscopy, cryogenic-Scanning Electron Microscopy, mechanical characterization via oscillatory rheology, Small Angle X-ray and Neutron Scattering of the silicified hydrogels will be presented.

  17. Thin optical display panel

    DOEpatents

    Veligdan, James Thomas

    1997-01-01

    An optical display includes a plurality of optical waveguides each including a cladding bound core for guiding internal display light between first and second opposite ends by total internal reflection. The waveguides are stacked together to define a collective display thickness. Each of the cores includes a heterogeneous portion defining a light scattering site disposed longitudinally between the first and second ends. Adjacent ones of the sites are longitudinally offset from each other for forming a longitudinal internal image display over the display thickness upon scattering of internal display light thereagainst for generating a display image. In a preferred embodiment, the waveguides and scattering sites are transparent for transmitting therethrough an external image in superposition with the display image formed by scattering the internal light off the scattering sites for defining a heads up display.

  18. Seamless tiled display system

    NASA Technical Reports Server (NTRS)

    Dubin, Matthew B. (Inventor); Larson, Brent D. (Inventor); Kolosowsky, Aleksandra (Inventor)

    2006-01-01

    A modular and scalable seamless tiled display apparatus includes multiple display devices, a screen, and multiple lens assemblies. Each display device is subdivided into multiple sections, and each section is configured to display a sectional image. One of the lens assemblies is optically coupled to each of the sections of each of the display devices to project the sectional image displayed on that section onto the screen. The multiple lens assemblies are configured to merge the projected sectional images to form a single tiled image. The projected sectional images may be merged on the screen by magnifying and shifting the images in an appropriate manner. The magnification and shifting of these images eliminates any visual effect on the tiled display that may result from dead-band regions defined between each pair of adjacent sections on each display device, and due to gaps between multiple display devices.

  19. High‑throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation.

    PubMed

    David, Vanessa; Terrat, Sébastien; Herzine, Khaled; Claisse, Olivier; Rousseaux, Sandrine; Tourdot-Maréchal, Raphaëlle; Masneuf-Pomarede, Isabelle; Ranjard, Lionel; Alexandre, Hervé

    2014-05-01

    We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITSRFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.

  20. Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae

    PubMed Central

    Lagkouvardos, Ilias; Weinmaier, Thomas; Lauro, Federico M; Cavicchioli, Ricardo; Rattei, Thomas; Horn, Matthias

    2014-01-01

    In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22 000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir. PMID:23949660

  1. Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

    PubMed

    Moralli, Daniela; Monaco, Zoia L

    2015-02-01

    De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

  2. Nitrogenase gene amplicons from global marine surface waters are dominated by genes of non-cyanobacteria.

    PubMed

    Farnelid, Hanna; Andersson, Anders F; Bertilsson, Stefan; Al-Soud, Waleed Abu; Hansen, Lars H; Sørensen, Søren; Steward, Grieg F; Hagström, Åke; Riemann, Lasse

    2011-04-29

    Cyanobacteria are thought to be the main N(2)-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N(2) fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

  3. Similar Diversity of Alphaproteobacteria and Nitrogenase Gene Amplicons on Two Related Sphagnum Mosses

    PubMed Central

    Bragina, Anastasia; Maier, Stefanie; Berg, Christian; Müller, Henry; Chobot, Vladimir; Hadacek, Franz; Berg, Gabriele

    2012-01-01

    Sphagnum mosses represent a main vegetation component in ombrotrophic wetlands. They harbor a specific and diverse microbial community with essential functions for the host. To understand the extend of host specificity and impact of environment, Sphagnum fallax and Sphagnum angustifolium, two phylogenetically closely related species, which show distinct habitat preference with respect to the nutrient level, were analyzed by a multifaceted approach. Microbial fingerprints obtained by PCR-single-strand conformation polymorphism of 16S rRNA and nitrogenase-encoding (nifH) genes were highly similar for both Sphagnum species. Similarity was confirmed for colonization patterns obtained by fluorescence in situ hybridization (FISH) coupled with confocal laser scanning microscopy (CLSM): Alphaproteobacteria were the main colonizers inside the hyaline cells of Sphagnum leaves. A deeper survey of Alphaproteobacteria by 16S rRNA gene amplicon sequencing reveals a high diversity with Acidocella, Acidisphaera, Rhodopila, and Phenylobacterium as major genera for both mosses. Nitrogen fixation is an important function of Sphagnum-associated bacteria, which is fulfilled by microbial communities of Sphagna in a similar way. NifH libraries of Sphagnum-associated microbial communities were characterized by high diversity and abundance of Alphaproteobacteria but contained also diverse amplicons of other taxa, e.g., Cyanobacteria and Deltaproteobacteria. Statistically significant differences between the microbial communities of both Sphagnum species could not be discovered in any of the experimental approach. Our results show that the same close relationship, which exists between the physical, morphological, and chemical characteristics of Sphagnum mosses and the ecology and function of bog ecosystems, also connects moss plantlets with their associated bacterial communities. PMID:22294982

  4. Avian host defense peptides.

    PubMed

    Cuperus, Tryntsje; Coorens, Maarten; van Dijk, Albert; Haagsman, Henk P

    2013-11-01

    Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense of many organisms. It is becoming increasingly clear that in the animal kingdom the functions of HDPs are not confined to direct antimicrobial actions. Research in mammals has indicated that HDPs have many immunomodulatory functions and are also involved in other physiological processes ranging from development to wound healing. During the past five years our knowledge about avian HDPs has increased considerably. This review addresses our current knowledge on the evolution, regulation and biological functions of HDPs of birds.

  5. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  6. Scalability of Robotic Displays: Display Size Investigation

    DTIC Science & Technology

    2008-05-01

    active matrix touch screen display (see figure 1). The screen is a super video graphics array 12.1 inches diagonal with 800x600-pixel resolution...ounces) super- video graphics display, high resolution (800x600) pictures with a 1.425-inch diagonal picture. The device used in this study was a...from a portable operator control unit that provides continuous data and video feedback for precise vehicle positioning. It was developed for the

  7. Probing Tumor Microenvironment with In Vivo Phage Display

    DTIC Science & Technology

    2013-07-01

    peptides may result in an efficient probe for breast tumor imaging and therapy . 15. SUBJECT TERMS Carcinoma-associated fibroblast; phage display...In Vivo Phage Display PRINCIPAL INVESTIGATOR: Dr. Erkki Ruoslahti CONTRACTING ORGANIZATION: Sanford Burnham Medical Research Institute...COVERED 01 July 2012 – 30 June 2013 4. TITLE AND SUBTITLE Probing Tumor Microenvironment with In Vivo Phage Display 5a. CONTRACT NUMBER W81XWH

  8. Probing Tumor Microenvironment with in Vivo Phage Display

    DTIC Science & Technology

    2013-07-01

    Vivo Phage Display PRINCIPAL INVESTIGATOR: Kazuki N. Sugahara, M.D., Ph.D...TITLE AND SUBTITLE 5a. CONTRACT NUMBER Probing Tumor Microenvironment with In Vivo Phage Display 5b. GRANT NUMBER W81XWH-12-1-0174 5c...cells and the matrix. The goal of our group is to make technical improvements in our phage display system, and find peptides that target carcinoma

  9. Advances in synthetic peptides reagent discovery

    NASA Astrophysics Data System (ADS)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Stratis-Cullum, Dimitra N.

    2013-05-01

    Bacterial display technology offers a number of advantages over competing display technologies (e.g, phage) for the rapid discovery and development of peptides with interaction targeted to materials ranging from biological hazards through inorganic metals. We have previously shown that discovery of synthetic peptide reagents utilizing bacterial display technology is relatively simple and rapid to make laboratory automation possible. This included extensive study of the protective antigen system of Bacillus anthracis, including development of discovery, characterization, and computational biology capabilities for in-silico optimization. Although the benefits towards CBD goals are evident, the impact is far-reaching due to our ability to understand and harness peptide interactions that are ultimately extendable to the hybrid biomaterials of the future. In this paper, we describe advances in peptide discovery including, new target systems (e.g. non-biological materials), advanced library development and clone analysis including integrated reporting.

  10. EMU helmet mounted display

    NASA Technical Reports Server (NTRS)

    Marmolejo, Jose (Inventor); Smith, Stephen (Inventor); Plough, Alan (Inventor); Clarke, Robert (Inventor); Mclean, William (Inventor); Fournier, Joseph (Inventor)

    1990-01-01

    A helmet mounted display device is disclosed for projecting a display on a flat combiner surface located above the line of sight where the display is produced by two independent optical channels with independent LCD image generators. The display has a fully overlapped field of view on the combiner surface and the focus can be adjusted from a near field of four feet to infinity.

  11. Digital video display system

    NASA Technical Reports Server (NTRS)

    Zygielbaum, A. I.; Martin, W. L.; Engle, A.

    1973-01-01

    System displays image data in real time on 120,000-element raster scan with 2, 4, or 8 shades of grey. Designed for displaying planetary range Doppler data, system can be used for X-Y plotting, displaying alphanumerics, and providing image animation.

  12. XVD Image Display Program

    NASA Technical Reports Server (NTRS)

    Deen, Robert G.; Andres, Paul M.; Mortensen, Helen B.; Parizher, Vadim; McAuley, Myche; Bartholomew, Paul

    2009-01-01

    The XVD [X-Windows VICAR (video image communication and retrieval) Display] computer program offers an interactive display of VICAR and PDS (planetary data systems) images. It is designed to efficiently display multiple-GB images and runs on Solaris, Linux, or Mac OS X systems using X-Windows.

  13. Screens and Displays.

    ERIC Educational Resources Information Center

    Edstrom, Malin

    1987-01-01

    Discusses the characteristics of different computer screen technologies including the possible harmful effects on health of cathode ray tube (CRT) terminals. CRT's are compared to other technologies including liquid crystal displays, plasma displays, electroluminiscence displays, and light emitting diodes. A chart comparing the different…

  14. Herpes simplex virus type 1-based amplicon vectors for fundamental research in neurosciences and gene therapy of neurological diseases.

    PubMed

    Jerusalinsky, Diana; Baez, María Verónica; Epstein, Alberto Luis

    2012-01-01

    Somatic manipulation of the nervous system without the involvement of the germinal line appears as a powerful counterpart of the transgenic strategy. The use of viral vectors to produce specific, transient and localized knockout, knockdown, ectopic expression or overexpression of a gene, leads to the possibility of analyzing both in vitro and in vivo molecular basis of neural function. In this approach, viral particles engineered to carry transgenic sequences are delivered into discrete brain regions, to transduce cells that will express the transgenic products. Amplicons are replication-incompetent helper-dependent vectors derived from herpes simplex virus type 1 (HSV-1), with several advantages that potentiate their use in neurosciences: (1) minimal toxicity: amplicons do not encode any virus proteins, are neither toxic for the infected cells nor pathogenic for the inoculated animals and elicit low levels of adaptive immune responses; (2) extensive transgene capacity to carry up to 150-kb of foreign DNA; i.e., entire genes with regulatory sequences could be delivered; (3) widespread cellular tropism: amplicons can experimentally infect several cell types including glial cells, though naturally the virus infects mainly neurons and epithelial cells; (4) since the viral genome does not integrate into cellular chromosomes there is low probability to induce insertional mutagenesis. Recent investigations on gene transfer into the brain using these vectors, have focused on gene therapy of inherited genetic diseases affecting the nervous system, such as ataxias, or on neurodegenerative disorders using experimental models of Parkinson's or Alzheimer's disease. Another group of studies used amplicons to investigate complex neural functions such as neuroplasticity, anxiety, learning and memory. In this short review, we summarize recent data supporting the potential of HSV-1 based amplicon vector model for gene delivery and modulation of gene expression in primary cultures

  15. The HER2 amplicon includes several genes required for the growth and survival of HER2 positive breast cancer cells.

    PubMed

    Sahlberg, Kristine Kleivi; Hongisto, Vesa; Edgren, Henrik; Mäkelä, Rami; Hellström, Kirsi; Due, Eldri U; Moen Vollan, Hans Kristian; Sahlberg, Niko; Wolf, Maija; Børresen-Dale, Anne-Lise; Perälä, Merja; Kallioniemi, Olli

    2013-06-01

    About 20% of breast cancers are characterized by amplification and overexpression of the HER2 oncogene. Although significant progress has been achieved for treating such patients with HER2 inhibitor trastuzumab, more than half of the patients respond poorly or become resistant to the treatment. Since the HER2 amplicon at 17q12 contains multiple genes, we have systematically explored the role of the HER2 co-amplified genes in breast cancer cell growth and their relation to trastuzumab resistance. We integrated aCGH data of the HER2 amplicon from 71 HER2 positive breast tumors and 10 cell lines with systematic functional RNA interference analysis of 23 core amplicon genes with several phenotypic endpoints in a panel of trastuzumab responding and non-responding HER2 positive breast cancer cells. Silencing of HER2 caused a greater growth arrest and apoptosis in the responding compared to the non-responding cell lines, indicating that the resistant cells are inherently less dependent on the HER2 pathway. Several other genes in the amplicon also showed a more pronounced effect when silenced; indicating that expression of HER2 co-amplified genes may be needed to sustain the growth of breast cancer cells. Importantly, co-silencing of STARD3, GRB7, PSMD3 and PERLD1 together with HER2 led to an additive inhibition of cell viability as well as induced apoptosis. These studies indicate that breast cancer cells may become addicted to the amplification of several genes that reside in the HER2 amplicon. The simultaneous targeting of these genes may increase the efficacy of the anti-HER2 therapies and possibly also counteract trastuzumab resistance. The observed additive effects seem to culminate to both apoptosis and cell proliferation pathways indicating that these pathways may be interesting targets for combinatorial treatment of HER2+ breast cancers.

  16. Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant region of hepatitis B core antigen: Broad protective efficacy of particles carrying four copies of M2e.

    PubMed

    Tsybalova, Liudmila M; Stepanova, Liudmila A; Kuprianov, Victor V; Blokhina, Elena A; Potapchuk, Marina V; Korotkov, Alexander V; Gorshkov, Andrey N; Kasyanenko, Marina A; Ravin, Nikolai V; Kiselev, Oleg I

    2015-06-26

    A long-term objective when designing influenza vaccines is to create one with broad cross-reactivity that will provide effective control over influenza, no matter which strain has caused the disease. Here we summarize the results from an investigation into the immunogenic and protective capacities inherent in variations of a recombinant protein, HBc/4M2e. This protein contains four copies of the ectodomain from the influenza virus protein M2 (M2e) fused within the immunodominant loop of the hepatitis B virus core antigen (HBc). Variations of this basic design include preparations containing M2e from the consensus human influenza virus; the M2e from the highly pathogenic avian A/H5N1 virus and a combination of two copies from human and two copies from avian influenza viruses. Intramuscular delivery in mice with preparations containing four identical copies of M2e induced high IgG titers in blood sera and bronchoalveolar lavages. It also provoked the formation of memory T-cells and antibodies were retained in the blood sera for a significant period of time post immunization. Furthermore, these preparations prevented the death of 75-100% of animals, which were challenged with lethal doses of virus. This resulted in a 1.2-3.5 log10 decrease in viral replication within the lungs. Moreover, HBc particles carrying only "human" or "avian" M2e displayed cross-reactivity in relation to human (A/H1N1, A/H2N2 and A/H3N2) or A/H5N1 and A(H1N1)pdm09 viruses, respectively; however, with the particles carrying both "human" and "avian" M2e this effect was much weaker, especially in relation to influenza virus A/H5N1. It is apparent from this work that to quickly produce vaccine for a pandemic it would be necessary to have several variations of a recombinant protein, containing four copies of M2e (each one against a group of likely influenza virus strains) with these relevant constructs housed within a comprehensive collection Escherichia coli-producers and maintained ready for use.

  17. Identification of peptide motif that binds to the surface of zirconia.

    PubMed

    Hashimoto, Kazuhiko; Yoshinari, Masao; Matsuzaka, Kenichi; Shiba, Kiyotaka; Inoue, Takashi

    2011-01-01

    A zirconia-binding peptide motif was identified using a peptide phage display system. Yttria stabilized zirconia beads and discs were used as the target. Quartz crystal microbalance was used to monitor the binding of phages to zirconia. Starting from a library of phages displaying random sequences of 12-mer peptides, we repeated cycles of biopanning against zirconia beads. After four cycles of biopanning, we isolated a phage clone Φ#17. DNA sequencing of the corresponding portion of Φ#17 unexpectedly revealed that it displayed a 58-mer peptide (amino acid sequence: WMPSDVDINDPQGGGSRPNLHQPKPAAEAASKKKSENRKVPFYSHSWY-SSMSEDKRGW). We found that Φ#17 had a 300-fold, significantly higher binding affinity for zirconia discs than phages displaying no peptide. In quartz crystal microbalance assay, a rapid increase in energy dissipation was observed from Φ#17 but not from the control phages, indicating that Φ#17 binds to the surface of zirconia via its displayed peptide. We successfully identified a peptide motif that binds zirconia.

  18. Virtual acoustics displays

    NASA Technical Reports Server (NTRS)

    Wenzel, Elizabeth M.; Fisher, Scott S.; Stone, Philip K.; Foster, Scott H.

    1991-01-01

    The real time acoustic display capabilities are described which were developed for the Virtual Environment Workstation (VIEW) Project at NASA-Ames. The acoustic display is capable of generating localized acoustic cues in real time over headphones. An auditory symbology, a related collection of representational auditory 'objects' or 'icons', can be designed using ACE (Auditory Cue Editor), which links both discrete and continuously varying acoustic parameters with information or events in the display. During a given display scenario, the symbology can be dynamically coordinated in real time with 3-D visual objects, speech, and gestural displays. The types of displays feasible with the system range from simple warnings and alarms to the acoustic representation of multidimensional data or events.

  19. Polyplanar optic display

    SciTech Connect

    Veligdan, J.; Biscardi, C.; Brewster, C.; DeSanto, L.; Beiser, L.

    1997-07-01

    The Polyplanar Optical Display (POD) is a unique display screen which can be used with any projection source. This display screen is 2 inches thick and has a matte black face which allows for high contrast images. The prototype being developed is a form, fit and functional replacement display for the B-52 aircraft which uses a monochrome ten-inch display. The new display uses a 100 milliwatt green solid state laser (532 nm) as its optical source. In order to produce real-time video, the laser light is being modulated by a Digital Light Processing (DLP{trademark}) chip manufactured by Texas Instruments, Inc. A variable astigmatic focusing system is used to produce a stigmatic image on the viewing face of the POD. In addition to the optical design, the authors discuss the electronic interfacing to the DLP{trademark} chip, the opto-mechanical design and viewing angle characteristics.

  20. Virtual acoustics displays

    NASA Astrophysics Data System (ADS)

    Wenzel, Elizabeth M.; Fisher, Scott S.; Stone, Philip K.; Foster, Scott H.

    1991-03-01

    The real time acoustic display capabilities are described which were developed for the Virtual Environment Workstation (VIEW) Project at NASA-Ames. The acoustic display is capable of generating localized acoustic cues in real time over headphones. An auditory symbology, a related collection of representational auditory 'objects' or 'icons', can be designed using ACE (Auditory Cue Editor), which links both discrete and continuously varying acoustic parameters with information or events in the display. During a given display scenario, the symbology can be dynamically coordinated in real time with 3-D visual objects, speech, and gestural displays. The types of displays feasible with the system range from simple warnings and alarms to the acoustic representation of multidimensional data or events.

  1. Multimission helicopter cockpit displays

    NASA Astrophysics Data System (ADS)

    Terry, William S.; Terry, Jody K.; Lovelace, Nancy D.

    1996-05-01

    A new operator display subsystem is being incorporated as part of the next generation United States Navy (USN) helicopter avionics system to be integrated into the multi-mission helicopter (MMH) that replaces both the SH-60B and the SH-60F in 2001. This subsystem exploits state-of-the-art technology for the display hardware, the display driver hardware, information presentation methodologies, and software architecture. Both of the existing SH-60 helicopter display systems are based on monochrome CRT technology; a key feature of the MMH cockpit is the integration of color AMLCD multifunction displays. The MMH program is one of the first military programs to use modified commercial AMLCD elements in a tactical aircraft. This paper presents the general configuration of the MMH cockpit and multifunction display subsystem and discusses the approach taken for presenting helicopter flight information to the pilots as well as presentation of mission sensor data for use by the copilot.

  2. Differential amplicons (ΔAmp)—a new molecular method to assess RNA integrity

    PubMed Central

    Björkman, J.; Švec, D.; Lott, E.; Kubista, M.; Sjöback, R.

    2015-01-01

    Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow. PMID:27077042

  3. Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing.

    PubMed

    Darby, Brian J; Erickson, Shay F; Hervey, Samuel D; Ellis-Felege, Susan N

    2016-07-01

    High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data.

  4. Shedding light on the microbial community of the macropod foregut using 454-amplicon pyrosequencing.

    PubMed

    Gulino, Lisa-Maree; Ouwerkerk, Diane; Kang, Alicia Y H; Maguire, Anita J; Kienzle, Marco; Klieve, Athol V

    2013-01-01

    Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

  5. Imaging herpes simplex virus type 1 amplicon vector-mediated gene expression in human glioma spheroids.

    PubMed

    Kaestle, Christine; Winkeler, Alexandra; Richter, Raphaela; Sauer, Heinrich; Hescheler, Jürgen; Fraefel, Cornel; Wartenberg, Maria; Jacobs, Andreas H

    2011-06-01

    Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector-mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP). After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector-mediated gene expression was investigated via laser scanning microscopy. Infection of spheroids with HSV-GFP demonstrated a maximal depth of vector-mediated GFP expression at 70 to 80 μm. A > 80% transduction efficiency was reached only in small spheroids with a diameter of < 150 μm. Guided vector injection into the spheroids showed transduction efficiencies ranging between < 10 and > 90%. The results demonstrated that vector-mediated gene expression in glioma spheroids was strongly dependent on the mode of vector application-injection pressure and injection time being the most important parameters. The assessment of these vector application parameters in tissue models will contribute to the development of safe and efficient gene therapy protocols for clinical application.

  6. Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

    NASA Astrophysics Data System (ADS)

    Thornhill, D. J.; Kemp, D. W.; Sampayo, E. M.; Schmidt, G. W.

    2010-03-01

    Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR-DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

  7. Accuracy of the high-throughput amplicon sequencing to identify species within the genus Aspergillus.

    PubMed

    Lee, Seungeun; Yamamoto, Naomichi

    2015-12-01

    This study characterized the accuracy of high-throughput amplicon sequencing to identify species within the genus Aspergillus. To this end, we sequenced the internal transcribed spacer 1 (ITS1), β-tubulin (BenA), and calmodulin (CaM) gene encoding sequences as DNA markers from eight reference Aspergillus strains with known identities using 300-bp sequencing on the Illumina MiSeq platform, and compared them with the BLASTn outputs. The identifications with the sequences longer than 250 bp were accurate at the section rank, with some ambiguities observed at the species rank due to mostly cross detection of sibling species. Additionally, in silico analysis was performed to predict the identification accuracy for all species in the genus Aspergillus, where 107, 210, and 187 species were predicted to be identifiable down to the species rank based on ITS1, BenA, and CaM, respectively. Finally, air filter samples were analysed to quantify the relative abundances of Aspergillus species in outdoor air. The results were reproducible across biological duplicates both at the species and section ranks, but not strongly correlated between ITS1 and BenA, suggesting the Aspergillus detection can be taxonomically biased depending on the selection of the DNA markers and/or primers.

  8. Flow cytometry community fingerprinting and amplicon sequencing for the assessment of landfill leachate cellulolytic bioaugmentation.

    PubMed

    Kinet, R; Dzaomuho, P; Baert, J; Taminiau, B; Daube, G; Nezer, C; Brostaux, Y; Nguyen, F; Dumont, G; Thonart, P; Delvigne, F

    2016-08-01

    Flow cytometry (FCM) is a high throughput single cell technology that is actually becoming widely used for studying phenotypic and genotypic diversity among microbial communities. This technology is considered in this work for the assessment of a bioaugmentation treatment in order to enhance cellulolytic potential of landfill leachate. The experimental results reveal the relevant increase of leachate cellulolytic potential due to bioaugmentation. Cytometric monitoring of microbial dynamics along these assays is then realized. The flow FP package is used to establish microbial samples fingerprint from initial 2D cytometry histograms. This procedure allows highlighting microbial communities' variation along the assays. Cytometric and 16S rRNA gene sequencing fingerprinting methods are then compared. The two approaches give same evidence about microbial dynamics throughout digestion assay. There are however a lack of significant correlation between cytometric and amplicon sequencing fingerprint at genus or species level. Same phenotypical profiles of microbiota during assays matched to several 16S rRNA gene sequencing ones. Flow cytometry fingerprinting can thus be considered as a promising routine on-site method suitable for the detection of stability/variation/disturbance of complex microbial communities involved in bioprocesses.

  9. Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

    PubMed Central

    Gulino, Lisa-Maree; Ouwerkerk, Diane; Kang, Alicia Y. H.; Maguire, Anita J.; Kienzle, Marco; Klieve, Athol V.

    2013-01-01

    Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as ‘shared’ OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry. PMID:23626688

  10. Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus

    PubMed Central

    Lu, Yang; Xu, Yanjuan; Di Bisceglie, Adrian M.; Fan, Xiaofeng

    2013-01-01

    The instability of recombinant clones accommodating large or full-length viral genomes is frequently a technical challenge in RNA virus research. In an attempt to establish a rapid plasmid-based reverse genetics system that utilizes long RT-PCR technique (LRP), similar difficulty was encountered in the cloning of 9022-bp LRP amplicon. All HCV genotype 1a strains used for LRP cloning showed a remarkable difference in terms of cloning stability. Subsequent analysis revealed the predictive value of phylogenetic positions in determining the cloning stability. Putative E. coli promoters on the HCV genome might be responsible for such cloning difference. An exhaustive exploration, testing nearly one hundred cloning protocols, did not reveal a general approach that can achieve stable cloning for all HCV 1a strains. The selection of appropriate strains, guided by phylogenetic analysis, appears to be necessary prior to the construction of infectious HCV 1a clones. These observations are not only valuable for potentially establishing an HCV 1a cell culture model but also have general implications for other RNA viruses due to concern about cloning instability. PMID:23602804

  11. Swarm v2: highly-scalable and high-resolution amplicon clustering

    PubMed Central

    Quince, Christopher; de Vargas, Colomban; Dunthorn, Micah

    2015-01-01

    Previously we presented Swarm v1, a novel and open source amplicon clustering program that produced fine-scale molecular operational taxonomic units (OTUs), free of arbitrary global clustering thresholds and input-order dependency. Swarm v1 worked with an initial phase that used iterative single-linkage with a local clustering threshold (d), followed by a phase that used the internal abundance structures of clusters to break chained OTUs. Here we present Swarm v2, which has two important novel features: (1) a new algorithm for d = 1 that allows the computation time of the program to scale linearly with increasing amounts of data; and (2) the new fastidious option that reduces under-grouping by grafting low abundant OTUs (e.g., singletons and doubletons) onto larger ones. Swarm v2 also directly integrates the clustering and breaking phases, dereplicates sequencing reads with d = 0, outputs OTU representatives in fasta format, and plots individual OTUs as two-dimensional networks. PMID:26713226

  12. Targeted Amplicon Sequencing (TAS): A Scalable Next-Gen Approach to Multilocus, Multitaxa Phylogenetics

    PubMed Central

    Bybee, Seth M.; Bracken-Grissom, Heather; Haynes, Benjamin D.; Hermansen, Russell A.; Byers, Robert L.; Clement, Mark J.; Udall, Joshua A.; Wilcox, Edward R.; Crandall, Keith A.

    2011-01-01

    Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach. PMID:22002916

  13. Paperlike thermochromic display

    NASA Astrophysics Data System (ADS)

    Liu, Liyu; Peng, Suili; Wen, Weijia; Sheng, Ping

    2007-05-01

    The authors report the design and implementation of a paperlike, thermally activated display fabricated from thermochromic composite and embedded conductive wiring patterns, shaped from mixture of metallic nanoparticles in polydimethylsioxane using soft lithography. The display exhibits good image quality and ease of control. Use of electric heating pulses is shown to reduce energy consumption while improving image quality control. The display has excellent mechanical bending flexibility.

  14. Display innovations through glass

    NASA Astrophysics Data System (ADS)

    Hamilton, Lori L.

    2016-03-01

    Prevailing trends in thin, lightweight, high-resolution, and added functionality, such as touch sensing, continue to drive innovation in the display market. While display volumes grow, so do consumers’ need for portability, enhanced optical performance, and mechanical reliability. Technical advancements in glass design and process have enabled display innovations in these areas while supporting industry growth. Opportunities for further innovation remain open for glass manufacturers to drive new applications, enhanced functionality, and increased demand.

  15. JAVA Stereo Display Toolkit

    NASA Technical Reports Server (NTRS)

    Edmonds, Karina

    2008-01-01

    This toolkit provides a common interface for displaying graphical user interface (GUI) components in stereo using either specialized stereo display hardware (e.g., liquid crystal shutter or polarized glasses) or anaglyph display (red/blue glasses) on standard workstation displays. An application using this toolkit will work without modification in either environment, allowing stereo software to reach a wider audience without sacrificing high-quality display on dedicated hardware. The toolkit is written in Java for use with the Swing GUI Toolkit and has cross-platform compatibility. It hooks into the graphics system, allowing any standard Swing component to be displayed in stereo. It uses the OpenGL graphics library to control the stereo hardware and to perform the rendering. It also supports anaglyph and special stereo hardware using the same API (application-program interface), and has the ability to simulate color stereo in anaglyph mode by combining the red band of the left image with the green/blue bands of the right image. This is a low-level toolkit that accomplishes simply the display of components (including the JadeDisplay image display component). It does not include higher-level functions such as disparity adjustment, 3D cursor, or overlays all of which can be built using this toolkit.

  16. Displaying Data As Movies

    NASA Technical Reports Server (NTRS)

    Moore, Judith G.

    1992-01-01

    NMSB Movie computer program displays large sets of data (more than million individual values). Presentation dynamic, rapidly displaying sequential image "frames" in main "movie" window. Any sequence of two-dimensional sets of data scaled between 0 and 255 (1-byte resolution) displayed as movie. Time- or slice-wise progression of data illustrated. Originally written to present data from three-dimensional ultrasonic scans of damaged aerospace composite materials, illustrates data acquired by thermal-analysis systems measuring rates of heating and cooling of various materials. Developed on Macintosh IIx computer with 8-bit color display adapter and 8 megabytes of memory using Symantec Corporation's Think C, version 4.0.

  17. Structure-function relationships of antimicrobial peptides.

    PubMed

    Hwang, P M; Vogel, H J

    1998-01-01

    Antimicrobial peptides are ubiquitously produced throughout nature. Many of these relatively short peptides (6-50 residues) are lethal towards bacteria and fungi, yet they display minimal toxicity towards mammalian cells. All of the peptides are highly cationic and hydrophobic. It is widely believed that they act through nonspecific binding to biological membranes, even though the exact nature of these interactions is presently unclear. High-resolution nuclear magnetic resonance (NMR) has contributed greatly to knowledge in this field, providing insight about peptide structure in aqueous solution, in organic cosolvents, and in micellar systems. Solid-state NMR can provide additional information about peptide-membrane binding. Here we review our current knowledge about the structure of antimicrobial peptides. We also discuss studies pertaining to the mechanism of action. Despite the different three-dimensional structural motifs of the various classes, they all have similar amphiphilic surfaces that are well-suited for membrane binding. Many antimicrobial peptides bind in a membrane-parallel orientation, interacting only with one face of the bilayer. This may be sufficient for antimicrobial action. At higher concentrations, peptides and phospholipids translocate to form multimeric transmembrane channels that seem to contribute to the peptide's hemolytic activity. An understanding of the key features of the secondary and tertiary structures of the antimicrobial peptides and their effects on bactericidal and hemolytic activity can aid the rational design of improved analogs for clinical use.

  18. Bioprospecting open reading frames for peptide effectors.

    PubMed

    Xiong, Ling; Scott, Charles

    2014-01-01

    Recent successes in the development of small-molecule antagonists of protein-protein interactions designed based on co-crystal structures of peptides bound to their biological targets confirm that short peptides derived from interacting proteins can be high-value ligands for pharmacologic validation of targets and for identification of druggable sites. Evolved sequence space is likely to be enriched for interacting peptides, but identifying minimal peptide effectors within genomic sequence can be labor intensive. Here we describe the use of incremental truncation to diversify genetic material on the scale of open reading frames into comprehensive libraries of constituent peptides. The approach is capable of generating peptides derived from both continuous and discontinuous sequence elements, and is compatible with the expression of free linear or backbone cyclic peptides, with peptides tethered to amino- or carboxyl-terminal fusion partners or with the expression of peptides displayed within protein scaffolds (peptide aptamers). Incremental truncation affords a valuable source of molecular diversity to interrogate the druggable genome or evaluate the therapeutic potential of candidate genes.

  19. Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants

    PubMed Central

    2004-01-01

    Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed. PMID:15560754

  20. Stabilization of exosome-targeting peptides via engineered glycosylation.

    PubMed

    Hung, Michelle E; Leonard, Joshua N

    2015-03-27

    Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics.

  1. Split image optical display

    DOEpatents

    Veligdan, James T.

    2005-05-31

    A video image is displayed from an optical panel by splitting the image into a plurality of image components, and then projecting the image components through corresponding portions of the panel to collectively form the image. Depth of the display is correspondingly reduced.

  2. Split image optical display

    DOEpatents

    Veligdan, James T.

    2007-05-29

    A video image is displayed from an optical panel by splitting the image into a plurality of image components, and then projecting the image components through corresponding portions of the panel to collectively form the image. Depth of the display is correspondingly reduced.

  3. Effective Monitor Display Design.

    ERIC Educational Resources Information Center

    Harrell, William

    1999-01-01

    Describes some of the factors that affect computer monitor display design and provides suggestions and insights into how screen displays can be designed more effectively. Topics include color, font choices, organizational structure of text, space outline, and general principles. (Author/LRW)

  4. Displays in space.

    PubMed

    Colford, Nicholas

    2002-04-01

    This chapter describes the human and environmental factors that dictate the way that displays must be designed for, and used in space. A brief history of the evolution of such display systems covers developments from the Mercury rockets to the International Space Station.

  5. Display and Presentation Boards.

    ERIC Educational Resources Information Center

    Midgley, Thomas Keith

    The use of display and presentation boards as tools to help teachers/trainers convey messages more clearly is briefly discussed, and 24 different types of display and presentation boards are described and illustrated; i.e., chalk, paste-up, hook-n-loop, electric, flannel, scroll, communication planning, acetate pocket, slot, pin-tack, preview,…

  6. Polyplanar optical display electronics

    SciTech Connect

    DeSanto, L.; Biscardi, C.

    1997-07-01

    The Polyplanar Optical Display (POD) is a unique display screen which can be used with any projection source. The prototype ten inch display is two inches thick and has a matte black face which allows for high contrast images. The prototype being developed is a form, fit and functional replacement display for the B-52 aircraft which uses a monochrome ten-inch display. In order to achieve a long lifetime, the new display uses a 100 milliwatt green solid-state laser (10,000 hr. life) at 532 nm as its light source. To produce real-time video, the laser light is being modulated by a Digital Light Processing (DLP{trademark}) chip manufactured by Texas Instruments. In order to use the solid-state laser as the light source and also fit within the constraints of the B-52 display, the Digital Micromirror Device (DMD{trademark}) circuit board is removed from the Texas Instruments DLP light engine assembly. Due to the compact architecture of the projection system within the display chassis, the DMD{trademark} chip is operated remotely from the Texas Instruments circuit board. The authors discuss the operation of the DMD{trademark} divorced from the light engine and the interfacing of the DMD{trademark} board with various video formats (CVBS, Y/C or S-video and RGB) including the format specific to the B-52 aircraft. A brief discussion of the electronics required to drive the laser is also presented.

  7. System status display evaluation

    NASA Technical Reports Server (NTRS)

    Summers, Leland G.

    1988-01-01

    The System Status Display is an electronic display system which provides the crew with an enhanced capability for monitoring and managing the aircraft systems. A flight simulation in a fixed base cockpit simulator was used to evaluate alternative design concepts for this display system. The alternative concepts included pictorial versus alphanumeric text formats, multifunction versus dedicated controls, and integration of the procedures with the system status information versus paper checklists. Twelve pilots manually flew approach patterns with the different concepts. System malfunctions occurred which required the pilots to respond to the alert by reconfiguring the system. The pictorial display, the multifunction control interfaces collocated with the system display, and the procedures integrated with the status information all had shorter event processing times and lower subjective workloads.

  8. Defense display market assessment

    NASA Astrophysics Data System (ADS)

    Desjardins, Daniel D.; Hopper, Darrel G.

    1998-09-01

    This paper addresses the number, function and size of principal military displays and establishes a basis to determine the opportunities for technology insertion in the immediate future and into the next millennium. Principal military displays are defined as those occupying appreciable crewstation real-estate and/or those without which the platform could not carry out its intended mission. DoD 'office' applications are excluded from this study. The military displays market is specified by such parameters as active area and footprint size, and other characteristics such as luminance, gray scale, resolution, angle, color, video capability, and night vision imaging system (NVIS) compatibility. Funded, future acquisitions, planned and predicted crewstation modification kits, and form-fit upgrades are taken into account. This paper provides an overview of the DoD niche market, allowing both government and industry a necessary reference by which to meet DoD requirements for military displays in a timely and cost-effective manner. The aggregate DoD market for direct-view and large-area military displays is presently estimated to be in excess of 242,000. Miniature displays are those which must be magnified to be viewed, involve a significantly different manufacturing paradigm and are used in helmet mounted displays and thermal weapon sight applications. Some 114,000 miniature displays are presently included within Service weapon system acquisition plans. For vendor production planning purposes it is noted that foreign military sales could substantially increase these quantities. The vanishing vendor syndrome (VVS) for older display technologies continues to be a growing, pervasive problem throughout DoD, which consequently must leverage the more modern display technologies being developed for civil- commercial markets.

  9. Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor.

    PubMed

    Ang, Geik Yong; Yu, Choo Yee; Yean, Chan Yean

    2012-01-01

    In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22-25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3 ng and 1 pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis.

  10. Prostate-Specific and Tumor-Specific Targeting of an Oncolytic HSV-1 Amplicon/Helper Virus for Prostate Cancer Treatment

    DTIC Science & Technology

    2009-11-01

    Targeting of an Oncolytic HSV - 1 Amplicon/Helper Virus for Prostate Cancer Treatment PRINCIPAL INVESTIGATOR: Cleo Lee CONTRACTING...5a. CONTRACT NUMBER Prostate-Specific and Tumor-Specific Targeting of an Oncolytic HSV - 1 Amplicon/Helper Virus for Prostate Cancer Treatment...untranslated region (3’UTR) of a herpes simplex virus- 1 ( HSV - 1 ) essential viral gene, ICP4, to create CMV-ICP4-143T and CMV-ICP4-145T amplicon viruses. Our

  11. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

    PubMed Central

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  12. Recent Advances Towards The Discovery Of Drug-Like Peptides De Novo

    NASA Astrophysics Data System (ADS)

    Goldflam, Michael; Ullman, Christopher

    2015-12-01

    Peptides are important natural molecules that possess functions as diverse as antibiotics, toxins, venoms and hormones, for example. However, whilst these peptides have useful properties, there are many targets and pathways that are not addressed through the activities of natural peptidic compounds. In these circumstances, directed evolution techniques, such as phage display, have been developed to sample the diverse chemical and structural repertoire of small peptides for useful means. In this review, we consider recent concepts that relate peptide structure to drug-like attributes and how these are incorporated within display technologies to deliver peptides de novo with valuable pharmaceutical properties.

  13. Recent Advances Toward the Discovery of Drug-Like Peptides De novo

    PubMed Central

    Goldflam, Michael; Ullman, Christopher G.

    2015-01-01

    Peptides are important natural molecules that possess functions as diverse as antibiotics, toxins, venoms and hormones, for example. However, whilst these peptides have useful properties, there are many targets and pathways that are not addressed through the activities of natural peptidic compounds. In these circumstances, directed evolution techniques, such as phage display, have been developed to sample the diverse chemical and structural repertoire of small peptides for useful means. In this review, we consider recent concepts that relate peptide structure to drug-like attributes and how these are incorporated within display technologies to deliver peptides de novo with valuable pharmaceutical properties. PMID:26734602

  14. Voltammetric detection of sequence-selective DNA hybridization related to Toxoplasma gondii in PCR amplicons.

    PubMed

    Gokce, Gultekin; Erdem, Arzum; Ceylan, Cagdas; Akgöz, Muslum

    2016-01-01

    This work describes the single-use electrochemical DNA biosensor technology developed for voltammetric detection of sequence selective DNA hybridization related to important human and veterinary pathogen; Toxoplasma gondii. In the principle of electrochemical label-free detection assay, the duplex of DNA hybrid formation was detected by measuring guanine oxidation signal occured in the presence of DNA hybridization. The biosensor design consisted of the immobilization of an inosine-modified (guanine-free) probe onto the surface of pencil graphite electrode (PGE), and the detection of the duplex formation in connection with the differential pulse voltammetry(DPV) by measuring the guanine signal. Toxoplasma gondii capture probe was firstly immobilized onto the surface of the activated PGE by wet adsorption. The extent of hybridization at PGE surface between the probe and the target was then determined by measuring the guanine signal observed at +1.0V. The electrochemical monitoring of optimum DNA hybridization has been performed in the target concentration of 40µg/mL in 50min of hybridization time. The specificity of the electrochemical biosensor was then tested using non-complementary, or mismatch short DNA sequences. Under the optimum conditions, the guanine oxidation signal indicating full hybridization was measured in various target concentration from 0.5 to 25µg/mL and a detection limit was found to be 1.78µg/mL. This single-use biosensor platform was successfully applied for the voltammetric detection of DNA hybridization related to Toxoplasma gondii in PCR amplicons.

  15. Antimicrobial Peptides

    PubMed Central

    Bahar, Ali Adem; Ren, Dacheng

    2013-01-01

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics). PMID:24287494

  16. Development of Bacterial Display Peptides for use in Biosensing Applications

    DTIC Science & Technology

    2012-09-01

    molded polypropylene (Pinnacle Polymers PP 5135C). The 200 µm deep fluidic channels are defined by two injected parts, which are laser- welded...providing predictive capability. The current computational suite combines a python -based application programming interface developed in-house, combined with

  17. Synthesis of stabilized alpha-helical peptides.

    PubMed

    Bernal, Federico; Katz, Samuel G

    2014-01-01

    Stabilized alpha-helical (SAH) peptides are valuable laboratory tools to explore important protein-protein interactions. Whereas most peptides lose their secondary structure when isolated from the host protein, stapled peptides incorporate an all-hydrocarbon "staple" that reinforces their natural alpha-helical structure. Thus, stapled peptides retain their functional ability to bind their native protein targets and serve multiple experimental uses. First, they are useful for structural studies such as NMR or crystal structures that map and better define binding sites. Second, they can be used to identify small molecules that specifically target that interaction site. Third, stapled peptides can be used to test the importance of specific amino acid residues or posttranslational modifications to the binding. Fourth, they can serve as structurally competent bait to identify novel binding partners to specific alpha-helical motifs. In addition to markedly improved alpha-helicity, stapled peptides also display resistance to protease cleavage and enhanced cell permeability. Most importantly, they are useful for intracellular experiments that explore the functional consequences of blocking particular protein interactions. Because of their remarkable stability, stapled peptides can be applied to whole-animal, in vivo studies. Here we describe a protocol for the synthesis of a peptide that incorporates an all-hydrocarbon "staple" employing a ring-closing olefin metathesis reaction. With proper optimization, stapled peptides can be a fundamental, accurate laboratory tool in the modern chemical biologist's armory.

  18. Molecular mechanism of selective binding of peptides to silicon surface.

    PubMed

    Ramakrishnan, Sathish Kumar; Martin, Marta; Cloitre, Thierry; Firlej, Lucyna; Gergely, Csilla

    2014-07-28

    Despite extensive recent research efforts on material-specific peptides, the fundamental problem to be explored yet is the molecular interactions between peptides and inorganic surfaces. Here we used computer simulations (density functional theory and classical molecular dynamics) to investigate the adsorption mechanism of silicon-binding peptides and the role of individual amino acids in the affinity of peptides for an n-type silicon (n(+)-Si) semiconductor. Three silicon binding 12-mer peptides previously elaborated using phage display technology have been studied. The peptides' conformations close to the surface have been determined and the best-binding amino acids have been identified. Adsorption energy calculations explain the experimentally observed different degrees of affinity of the peptides for n(+)-Si. Our residual scanning analysis demonstrates that the binding affinity relies on both the identity of the amino acid and its location in the peptide sequence.

  19. Map display design

    NASA Technical Reports Server (NTRS)

    Aretz, Anthony J.

    1990-01-01

    This paper presents a cognitive model of a pilot's navigation task and describes an experiment comparing a visual momentum map display to the traditional track-up and north-up approaches. The data show the advantage to a track-up map is its congruence with the ego-centered forward view; however, the development of survey knowledge is hindered by the inconsistency of the rotating display. The stable alignment of a north-up map aids the acquisition of survey knowledge, but there is a cost associated with the mental rotation of the display to a track-up alignment for ego-centered tasks. The results also show that visual momentum can be used to reduce the mental rotation costs of a north-up display.

  20. Gardens on Display.

    ERIC Educational Resources Information Center

    Steinheimer, Margaret

    1998-01-01

    Discusses display gardens and their development by students. Presents guidelines for construction and size consideration and describes details of an outdoor garden, volcanic garden, and shoe box dioramas. (DDR)

  1. Chapter 14. Biosynthesis of nonribosomal peptide precursors.

    PubMed

    Wilkinson, Barrie; Micklefield, Jason

    2009-01-01

    Nonribosomal peptides are natural products typically of bacterial and fungal origin. These highly complex molecules display a broad spectrum of biological activities, and have been exploited for the development of immunosuppressant, antibiotic, anticancer, and other therapeutic agents. The nonribosomal peptides are assembled by nonribosomal peptide synthetase (NRPS) enzymes comprising repeating modules that are responsible for the sequential selection, activation, and condensation of precursor amino acids. In addition to this, fatty acids, alpha-keto acids and alpha-hydroxy acids, as well as polyketide derived units, can also be utilized by NRPS assembly lines. Final tailoring-steps, including glycosylation and prenylation, serve to further decorate the nonribosomal peptides produced. The wide range of experimental methods that are employed in the elucidation of nonribosomal peptide precursor biosynthesis will be discussed, with particularly emphasis on genomics based approaches which have become wide spread over the last 5 years.

  2. Military display performance parameters

    NASA Astrophysics Data System (ADS)

    Desjardins, Daniel D.; Meyer, Frederick

    2012-06-01

    The military display market is analyzed in terms of four of its segments: avionics, vetronics, dismounted soldier, and command and control. Requirements are summarized for a number of technology-driving parameters, to include luminance, night vision imaging system compatibility, gray levels, resolution, dimming range, viewing angle, video capability, altitude, temperature, shock and vibration, etc., for direct-view and virtual-view displays in cockpits and crew stations. Technical specifications are discussed for selected programs.

  3. Liquid Crystal Airborne Display

    DTIC Science & Technology

    1977-08-01

    81/2X 11- 10 -9 .8 display using a large advertising alphanimeric ( TCI ) has been added to the front of the optical box used in the F-4 aircraft for HUD...properties over a wide range of tempera - tures, including normal room temperature. What are Liquid Crystals? Liquid crystals have been classified in three...natic fanctions and to present data needed for the semi- automatic and manual control of system functions. Existing aircraft using CRT display

  4. Peptide consensus sequence determination for the enhancement of the antimicrobial activity and selectivity of antimicrobial peptides

    PubMed Central

    Almaaytah, Ammar; Ajingi, Ya’u; Abualhaijaa, Ahmad; Tarazi, Shadi; Alshar’i, Nizar; Al-Balas, Qosay

    2017-01-01

    The rise of multidrug-resistant bacteria is causing a serious threat to the world’s human population. Recent reports have identified bacterial strains displaying pan drug resistance against antibiotics and generating fears among medical health specialists that humanity is on the dawn of entering a post-antibiotics era. Global research is currently focused on expanding the lifetime of current antibiotics and the development of new antimicrobial agents to tackle the problem of antimicrobial resistance. In the present study, we designed a novel consensus peptide named “Pepcon” through peptide consensus sequence determination among members of a highly homologous group of scorpion antimicrobial peptides. Members of this group were found to possess moderate antimicrobial activity with significant toxicity against mammalian cells. The aim of our design method was to generate a novel peptide with an enhanced antimicrobial potency and selectivity against microbial rather than mammalian cells. The results of our study revealed that the consensus peptide displayed potent antibacterial activities against a broad range of Gram-positive and Gram-negative bacteria. Our membrane permeation studies displayed that the peptide efficiently induced membrane damage and consequently led to cell death through the process of cell lysis. The microbial DNA binding assay of the peptide was found to be very weak suggesting that the peptide is not targeting the microbial DNA. Pepcon induced minimal cytotoxicity at the antimicrobial concentrations as the hemolytic activity was found to be zero at the minimal inhibitory concentrations (MICs). The results of our study demonstrate that the consensus peptide design strategy is efficient in generating peptides. PMID:28096686

  5. Dichroic Liquid Crystal Displays

    NASA Astrophysics Data System (ADS)

    Bahadur, Birendra

    The following sections are included: * INTRODUCTION * DICHROIC DYES * Chemical Structure * Chemical and Photochemical Stability * THEORETICAL MODELLING * DEFECTS CAUSED BY PROLONGED LIGHT IRRADIATION * CHEMICAL STRUCTURE AND PHOTOSTABILITY * OTHER PARAMETERS AFFECTING PHOTOSTABILITY * CELL PREPARATION * DICHROIC PARAMETERS AND THEIR MEASUREMENTS * Order Parameter and Dichroic Ratio Of Dyes * Absorbance, Order Parameter and Dichroic Ratio Measurements * IMPACT OF DYE STRUCTURE AND LIQUID CRYSTAL HOST ON PHYSICAL PROPERTIES OF A DICHROIC MIXTURE * Order Parameter and Dichroic Ratio * EFFECT OF LENGTH OF DICHROIC DYES ON THE ORDER PARAMETER * EFFECT OF THE BREADTH OF DYE ON THE ORDER PARAMETER * EFFECT OF THE HOST ON THE ORDER PARAMETER * TEMPERATURE VARIATION OF THE ORDER PARAMETER OF DYES IN A LIQUID CRYSTAL HOST * IMPACT OF DYE CONCENTRATION ON THE ORDER PARAMETER * Temperature Range * Viscosity * Dielectric Constant and Anisotropy * Refractive Indices and Birefringence * solubility43,153-156 * Absorption Wavelength and Auxochromic Groups * Molecular Engineering of Dichroic Dyes * OPTICAL, ELECTRO-OPTICAL AND LIFE PARAMETERS * Colour And CIE Colour space120,160-166 * CIE 1931 COLOUR SPACE * CIE 1976 CHROMATICITY DIAGRAM * CIE UNIFORM COLOUR SPACES & COLOUR DIFFERENCE FORMULAE120,160-166 * Electro-Optical Parameters120 * LUMINANCE * CONTRAST AND CONTRAST RATIO * SWITCHING SPEED * Life Parameters and Failure Modes * DICHROIC MIXTURE FORMULATION * Monochrome Mixture * Black Mixture * ACHROMATIC BLACK MIXTURE FOR HEILMEIER DISPLAYS * Effect of Illuminant on Display Colour * Colour of the Field-On State * Effect of Dye Linewidth * Optimum Centroid Wavelengths * Effect of Dye Concentration * Mixture Formulation Using More Than Three Dyes * ACHROMATIC MIXTURE FOR WHITE-TAYLOR TYPE DISPLAYS * HEILMEIER DISPLAYS * Theoretical Modelling * Threshold Characteristic * Effects of Dye Concentration on Electro-optical Parameters * Effect of Cholesteric Doping * Effect of Alignment

  6. Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands

    PubMed Central

    Shou, Yulin; Schmidt, Emily N.; Paavola, Chad D.; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I.; Bradbury, Andrew R. M.; Martinez, Jennifer S.

    2016-01-01

    Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands. PMID:27626637

  7. C-Peptide Test

    MedlinePlus

    ... vital for the body to use its main energy source, glucose . Since C-peptide and insulin are produced ... these cases, C-peptide measurement is a useful alternative to testing for insulin. C-peptide measurements can ...

  8. Effect of anchoring 4-anilidopiperidines to opioid peptides.

    PubMed

    Petrov, Ravil R; Lee, Yeon Sun; Vardanyan, Ruben S; Liu, Lu; Ma, Shou-wu; Davis, Peg; Lai, Josephine; Porreca, Frank; Vanderah, Todd W; Hruby, Victor J

    2013-06-01

    We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides.

  9. Dynamic plasmonic colour display

    NASA Astrophysics Data System (ADS)

    Duan, Xiaoyang; Kamin, Simon; Liu, Na

    2017-02-01

    Plasmonic colour printing based on engineered metasurfaces has revolutionized colour display science due to its unprecedented subwavelength resolution and high-density optical data storage. However, advanced plasmonic displays with novel functionalities including dynamic multicolour printing, animations, and highly secure encryption have remained in their infancy. Here we demonstrate a dynamic plasmonic colour display technique that enables all the aforementioned functionalities using catalytic magnesium metasurfaces. Controlled hydrogenation and dehydrogenation of the constituent magnesium nanoparticles, which serve as dynamic pixels, allow for plasmonic colour printing, tuning, erasing and restoration of colour. Different dynamic pixels feature distinct colour transformation kinetics, enabling plasmonic animations. Through smart material processing, information encoded on selected pixels, which are indiscernible to both optical and scanning electron microscopies, can only be read out using hydrogen as a decoding key, suggesting a new generation of information encryption and anti-counterfeiting applications.

  10. Dynamic plasmonic colour display

    PubMed Central

    Duan, Xiaoyang; Kamin, Simon; Liu, Na

    2017-01-01

    Plasmonic colour printing based on engineered metasurfaces has revolutionized colour display science due to its unprecedented subwavelength resolution and high-density optical data storage. However, advanced plasmonic displays with novel functionalities including dynamic multicolour printing, animations, and highly secure encryption have remained in their infancy. Here we demonstrate a dynamic plasmonic colour display technique that enables all the aforementioned functionalities using catalytic magnesium metasurfaces. Controlled hydrogenation and dehydrogenation of the constituent magnesium nanoparticles, which serve as dynamic pixels, allow for plasmonic colour printing, tuning, erasing and restoration of colour. Different dynamic pixels feature distinct colour transformation kinetics, enabling plasmonic animations. Through smart material processing, information encoded on selected pixels, which are indiscernible to both optical and scanning electron microscopies, can only be read out using hydrogen as a decoding key, suggesting a new generation of information encryption and anti-counterfeiting applications. PMID:28232722

  11. Stereo Painting Display Devices

    NASA Astrophysics Data System (ADS)

    Shafer, David

    1982-06-01

    The Spanish Surrealist artist Salvador Dali has recently perfected the art of producing two paintings which are stereo pairs. Each painting is separately quite remarkable, presenting a subject with the vivid realism and clarity for which Dali is famous. Due to the surrealistic themes of Dali's art, however, the subjects preser.ted with such naturalism only exist in his imagination. Despite this considerable obstacle to producing stereo art, Dali has managed to paint stereo pairs that display subtle differences of coloring and lighting, in addition to the essential perspective differences. These stereo paintings require a display method that will allow the viewer to experience stereo fusion, but which will not degrade the high quality of the art work. This paper gives a review of several display methods that seem promising in terms of economy, size, adjustability, and image quality.

  12. Viewing angle changeable display

    NASA Astrophysics Data System (ADS)

    Leng, Jinbi; Huang, Ziqiang; Yang, Wenjun; Chen, Xiaoxi

    2010-10-01

    Viewing angle changeable display can change the display viewing angle as needed: In the public place the display could have a narrow viewing angle for privacy, while in the private place the displays could have a wide viewing angle for the convenience of the operation and better viewing experience. This article propose a novel adjustable optical transmission device to realize the viewing angle changes for LCD by using the principle of guest- host effect of liquid crystal. The major technology is to insert a special equipment between the backlight and the LCD, through which the backlight will display either parallel or scattered features to get an either narrow or wide viewing angle. The equipment is an adjustable transmission cell (ATC) which is actually a black G-H LC cell. This ATC is the main focus of our invention. The ATC consists of a polarizer sheet and a special guest-host liquid crystal device filled with the two-phase dye (called as GH-LC in this report), to achieve the viewing angle change in the LCD. When an electrical field charges to the ATC, only the so-called near-axis lights can pass through the ATC within a relatively small angle, while the other scattered lights are absorbed sequentially by GH-LC and the polarizer sheet. On the other hand, when there is no electrical charge to the ATC, the cell behaves like a normal polarizer; and the scattered light can pass through the cell and polarizer in a normal way. This paper describes the principle and structure of the device, applies the electric field on the sample to observe the electro-optical properties, combine the theoretical and experimental research, getting the viewing angle effects of the display.

  13. Thin display optical projector

    DOEpatents

    Veligdan, James T.

    1999-01-01

    An optical system (20) projects light into a planar optical display (10). The display includes laminated optical waveguides (12) defining an inlet face (14) at one end and an outlet screen (16) at an opposite end. A first mirror (26) collimates light from a light source (18) along a first axis, and distributes the light along a second axis. A second mirror (28) collimates the light from the first mirror along the second axis to illuminate the inlet face and produce an image on the screen.

  14. Integrated display scanner

    DOEpatents

    Veligdan, James T.

    2004-12-21

    A display scanner includes an optical panel having a plurality of stacked optical waveguides. The waveguides define an inlet face at one end and a screen at an opposite end, with each waveguide having a core laminated between cladding. A projector projects a scan beam of light into the panel inlet face for transmission from the screen as a scan line to scan a barcode. A light sensor at the inlet face detects a return beam reflected from the barcode into the screen. A decoder decodes the return beam detected by the sensor for reading the barcode. In an exemplary embodiment, the optical panel also displays a visual image thereon.

  15. Universal electronic stereoscopic display

    NASA Astrophysics Data System (ADS)

    Lipton, Lenny; Halnon, Jeff

    1996-04-01

    SimulEYES VRTM, a new product for mass consumer electro-stereoscopic displays, is described. The system uses a unique indexing approach to allow content providers latitude in choosing the display mode. Board and PC manufacturers may also take advantage of the elegance of the solution by building in the SimulEYES VR capability. Hardware components consist, in part, of two custom chips which may be integrated at the board level, or employed in a VGA port dongle and control box. The liquid crystal shuttering eyewear is of a unique ergonomic design which is comfortable for people of all ages and most facial types, even when wearing eyeglasses.

  16. Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing.

    PubMed

    Campbell, Nathan R; Harmon, Stephanie A; Narum, Shawn R

    2015-07-01

    Genotyping-in-Thousands by sequencing (GT-seq) is a method that uses next-generation sequencing of multiplexed PCR products to generate genotypes from relatively small panels (50-500) of targeted single-nucleotide polymorphisms (SNPs) for thousands of individuals in a single Illumina HiSeq lane. This method uses only unlabelled oligos and PCR master mix in two thermal cycling steps for amplification of targeted SNP loci. During this process, sequencing adapters and dual barcode sequence tags are incorporated into the amplicons enabling thousands of individuals to be pooled into a single sequencing library. Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences. Genotyping is performed with a simple perl script which counts amplicon-specific sequences for each allele, and allele ratios are used to determine the genotypes. We demonstrate this technique by genotyping 2068 individual steelhead trout (Oncorhynchus mykiss) samples with a set of 192 SNP markers in a single library sequenced in a single Illumina HiSeq lane. Genotype data were 99.9% concordant to previously collected TaqMan(™) genotypes at the same 192 loci, but call rates were slightly lower with GT-seq (96.4%) relative to Taqman (99.0%). Of the 192 SNPs, 187 were genotyped in ≥90% of the individual samples and only 3 SNPs were genotyped in <70% of samples. This study demonstrates amplicon sequencing with GT-seq greatly reduces the cost of genotyping hundreds of targeted SNPs relative to existing methods by utilizing a simple library preparation method and massive efficiency of scale.

  17. Gas Vesicle Nanoparticles for Antigen Display

    PubMed Central

    DasSarma, Shiladitya; DasSarma, Priya

    2015-01-01

    Microorganisms like the halophilic archaeon Halobacterium sp. NRC-1 produce gas-filled buoyant organelles, which are easily purified as protein nanoparticles (called gas vesicles or GVNPs). GVNPs are non-toxic, exceptionally stable, bioengineerable, and self-adjuvanting. A large gene cluster encoding more than a dozen proteins has been implicated in their biogenesis. One protein, GvpC, found on the exterior surface of the nanoparticles, can accommodate insertions near the C-terminal region and results in GVNPs displaying the inserted sequences on the surface of the nanoparticles. Here, we review the current state of knowledge on GVNP structure and biogenesis as well as available studies on immunogenicity of pathogenic viral, bacterial, and eukaryotic proteins and peptides displayed on the nanoparticles. Recent improvements in genetic tools for bioengineering of GVNPs are discussed, along with future opportunities and challenges for development of vaccines and other applications. PMID:26350601

  18. Virtual Auditory Displays

    DTIC Science & Technology

    2000-01-01

    timbre , intensity, distance, room modeling, radio communication Virtual Environments Handbook Chapter 4 Virtual Auditory Displays Russell D... musical note “A” as a pure sinusoid, there will be 440 condensations and rarefactions per second. The distance between two adjacent condensations or...and complexity are pitch, loudness, and timbre respectively. This distinction between physical and perceptual measures of sound properties is an

  19. Refreshing Refreshable Braille Displays.

    PubMed

    Russomanno, Alexander; O'Modhrain, Sile; Gillespie, R Brent; Rodger, Matthew W M

    2015-01-01

    The increased access to books afforded to blind people via e-publishing has given them long-sought independence for both recreational and educational reading. In most cases, blind readers access materials using speech output. For some content such as highly technical texts, music, and graphics, speech is not an appropriate access modality as it does not promote deep understanding. Therefore blind braille readers often prefer electronic braille displays. But, these are prohibitively expensive. The search is on, therefore, for a low-cost refreshable display that would go beyond current technologies and deliver graphical content as well as text. And many solutions have been proposed, some of which reduce costs by restricting the number of characters that can be displayed, even down to a single braille cell. In this paper, we demonstrate that restricting tactile cues during braille reading leads to poorer performance in a letter recognition task. In particular, we show that lack of sliding contact between the fingertip and the braille reading surface results in more errors and that the number of errors increases as a function of presentation speed. These findings suggest that single cell displays which do not incorporate sliding contact are likely to be less effective for braille reading.

  20. Ferroelectric liquid crystal display

    NASA Technical Reports Server (NTRS)

    York, Paul K. (Inventor)

    1977-01-01

    A ferroelectric liquid crystal display device employs capacitance spoiling layers to minimize unneeded capacitances created by crossovers of X and Y address lines and to accurately define desired capacitances. The spoiler layers comprise low dielectric constant layers which space electrodes from the ferroelectric at crossover points where capacitance is not needed for device operation.

  1. A Plasma Display Terminal.

    ERIC Educational Resources Information Center

    Stifle, Jack

    A graphics terminal designed for use as a remote computer input/output terminal is described. Although the terminal is intended for use in teaching applications, it has several features which make it useful in many other computer terminal applications. These features include: a 10-inch square plasma display panel, permanent storage of information…

  2. Color Display Design Guide

    DTIC Science & Technology

    1978-10-01

    22 . 20 - MEAN/ALL COLORS/*. .. %.’ 18 -.-. YELLOW u- 16 . . RED /- ........ WHITE ൖ /- MAGENTA -,f 12 - / / CYAN ’"’- 10 /GREEN BLUE C= Ś S• l I I...Hawaii Laboratory P.O. Box 997 Kailua, Hawaii 96734 Attn: Dr. Ross L. Pepper Department of Psychology Panel Displays Incorporated Vanderbilt University

  3. Digital Holography Display (2)

    NASA Astrophysics Data System (ADS)

    Lee, Cheok Peng; Asundi, A.; Yu, Yang; Xiao, Zhen Zhong

    This paper describes the extension work from the last Digital Holography Projector System. From the developed works shows that, some unforeseen factors have created the difficulties for the system alignment. Such factors are the DMD frame rate, light source and diffractive zero order. It is really the challenging development works to achieve the virtual 3D model display on the high speed rotation screen. The three most key factors are emphasizing: 1) The display device's frame rate; 2) The light source orientation angle; and 3) The zero order filtering optic. 1) This device's is the digital micro mirror, in short is DMD. It is the high speed switching device has developed by the most recent technology. The switching frame rate can go up as high as 291fps. At first, the 8 bits depth file must be digitalized and stored for DMD onboard Ram. The digitalized data are transmitting from the PC USB to DMD onboard Ram. Instead of the data are downloading directly from the PC to DVI or VGA during display, this downloading method cause slower down the display speed, which is the common frame rate of 30 Hz. Next, the onboard Ram data then transfer to the DMD mirror's for display, at the 8 bits 291 fps speed. At this frame rate, the display 2D image can almost cover for 10 of out of the 360 0 in 1 revolution. 2) This laser light source must be installed such that free for orientated in any arbitrary angle from 220 to 450. Which is normalized to the DMD mirrors and the brief sketch show on figure (a). The purpose of orientated the light source is ensure that multi diffractive order would be reflected straight from the mirrors. (This multi diffractive order is the phenomenon of the digital micro mirror's characteristic). This mean, the reconstruct images would be followed the DMD normalized direction reflected up to fibre conduit. Moreover, this orientated method install of the laser light source is making space for other optical lenses or device driver/controller. Because, all

  4. Preparation of peptides which mimic glycosphingolipids by using phage peptide library and their modulation on beta-galactosidase activity.

    PubMed

    Taki, T; Ishikawa, D; Hamasaki, H; Handa, S

    1997-11-24

    We describe the use of a phage-displayed random pentadecamer peptide library for searching glycosphingolipid mimicking peptides. Two phage clones (AD-1 and AD-2) were selected by biopanning using monoclonal antibody AD117m, directed to lactotetraosylceramide (Lc4Cer). The amino acid sequences of the selected clones showed high homology (VPPXFXXXY) in 9-mer. Three phage clones were selected by using monoclonal antibody H11, directed to neolactotetraosylceramide (nLc4Cer), the linkage isomer of Lc4Cer, and the displayed amino acid sequences were compared. One of these peptides showed the same amino acid sequence as that of AD-2 except for one amino acid substitution. Pentadecamer, 9-mer and point mutated 9-mer peptides were synthesized on the basis of the displayed amino acid sequences. Binding activity of the peptides to the monoclonal antibodies or Ricinus communis lectin showed that 9-mer peptides are enough to mimic the epitope carbohydrate structure. Furthermore, six of the synthesized peptides inhibited Jack bean beta-galactosidase activity towards nLc4Cer at a high concentration of the enzyme, whereas at lower enzyme concentrations some peptides showed potent activation of the enzyme activity. This is the first report of carbohydrate mimicking peptides which modulate glycosidase activity.

  5. Virtual acoustic displays

    NASA Technical Reports Server (NTRS)

    Wenzel, Elizabeth M.

    1991-01-01

    A 3D auditory display can potentially enhance information transfer by combining directional and iconic information in a quite naturalistic representation of dynamic objects in the interface. Another aspect of auditory spatial clues is that, in conjunction with other modalities, it can act as a potentiator of information in the display. For example, visual and auditory cues together can reinforce the information content of the display and provide a greater sense of presence or realism in a manner not readily achievable by either modality alone. This phenomenon will be particularly useful in telepresence applications, such as advanced teleconferencing environments, shared electronic workspaces, and monitoring telerobotic activities in remote or hazardous situations. Thus, the combination of direct spatial cues with good principles of iconic design could provide an extremely powerful and information-rich display which is also quite easy to use. An alternative approach, recently developed at ARC, generates externalized, 3D sound cues over headphones in realtime using digital signal processing. Here, the synthesis technique involves the digital generation of stimuli using Head-Related Transfer Functions (HRTF's) measured in the two ear-canals of individual subjects. Other similar approaches include an analog system developed by Loomis, et. al., (1990) and digital systems which make use of transforms derived from normative mannikins and simulations of room acoustics. Such an interface also requires the careful psychophysical evaluation of listener's ability to accurately localize the virtual or synthetic sound sources. From an applied standpoint, measurement of each potential listener's HRTF's may not be possible in practice. For experienced listeners, localization performance was only slightly degraded compared to a subject's inherent ability. Alternatively, even inexperienced listeners may be able to adapt to a particular set of HRTF's as long as they provide adequate

  6. Peptides homing to tumor vasculature: imaging and therapeutics for cancer.

    PubMed

    Liu, Zhiguo; Wu, Kaichun

    2008-11-01

    A major obstacle to advances in anti-vascular therapy is the lack of molecule candidates that are effective in selectively targeting cancer tissues while sparing normal ones. Phage display peptide library greatly eases the discovery of peptides with specific homing capacity. Many novel peptides homing to angiogenic vessels were isolated recently. Notably, many such peptides showed relatively specific affinity with particular tumor types. These peptides appear to be able to accumulate in the target vascular site of tumor, making them particularly efficient to deliver drugs or other therapeutic and imaging agents. Some homing peptides could not only target to the desired location, but also be internalized into targeted cells, or even induce destruction in desired cells all by the same peptide sequence itself. Accumulating evidence has shown that by tumor specific targeting delivery, improved local effect can be achieved with well tolerated side effects. In the current review, recent literatures and patents in this field have been summarized.

  7. Peptide Inhibitors of the amyloidogenesis of IAPP: verification of the hairpin-binding geometry hypothesis.

    PubMed

    Sivanesam, Kalkena; Shu, Irene; Huggins, Kelly N L; Tatarek-Nossol, Marianna; Kapurniotu, Aphrodite; Andersen, Niels H

    2016-08-01

    Versions of a previously discovered β-hairpin peptide inhibitor of IAPP aggregation that are stabilized in that conformation, or even forced to remain in the hairpin conformation by a backbone cyclization constraint, display superior activity as inhibitors. The cyclized hairpin, cyclo-WW2, displays inhibitory activity at substoichiometric concentrations relative to this amyloidogenic peptide. The hairpin-binding hypothesis stands confirmed.

  8. Library-based display technologies: where do we stand?

    PubMed

    Galán, Asier; Comor, Lubos; Horvatić, Anita; Kuleš, Josipa; Guillemin, Nicolas; Mrljak, Vladimir; Bhide, Mangesh

    2016-07-19

    Over the past two decades, library-based display technologies have been staggeringly optimized since their appearance in order to mimic the process of natural molecular evolution. Display technologies are essential for the isolation of specific high-affinity binding molecules (proteins, polypeptides, nucleic acids and others) for diagnostic and therapeutic applications in cancer, infectious diseases, autoimmune, neurodegenerative, inflammatory pathologies etc. Applications extend to other fields such as antibody and enzyme engineering, cell-free protein synthesis and the discovery of protein-protein interactions. Phage display technology is the most established of these methods but more recent fully in vitro alternatives, such as ribosome display, mRNA display, cis-activity based (CIS) display and covalent antibody display (CAD), as well as aptamer display and in vitro compartmentalization, offer advantages over phage in library size, speed and the display of unnatural amino acids and nucleotides. Altogether, they have produced several molecules currently approved or in diverse stages of clinical or preclinical testing and have provided researchers with tools to address some of the disadvantages of peptides and nucleotides such as their low affinity, low stability, high immunogenicity and difficulty to cross membranes. In this review we assess the fundamental technological features and point out some recent advances and applications of display technologies.

  9. Phage Display Breast Carcinoma cDNA Libraries: Isolation of Clones Which Specifically Bind to Membrane Glycoproteins, Mucins, and Endothelial Cell Surface

    DTIC Science & Technology

    1999-07-01

    Display of Peptides and Proteins: A Laboratory Manual, ed. B. Kay. 1996, San Diego: Academic Press. 10. Smith, G.P., Filamentous fusion phage : novel expression vectors that display cloned antigens on the virion surface . Science

  10. Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software.

    PubMed

    Poulet, Axel; Privat, Maud; Ponelle, Flora; Viala, Sandrine; Decousus, Stephanie; Perin, Axel; Lafarge, Laurence; Ollier, Marie; El Saghir, Nagi S; Uhrhammer, Nancy; Bignon, Yves-Jean; Bidet, Yannick

    Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.

  11. Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software

    PubMed Central

    Poulet, Axel; Privat, Maud; Viala, Sandrine; Decousus, Stephanie; Perin, Axel; Lafarge, Laurence; Ollier, Marie; El Saghir, Nagi S.

    2016-01-01

    Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts. PMID:27656653

  12. A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene

    PubMed Central

    Sestini, Roberta; Candita, Luisa; Capone, Gabriele Lorenzo; Barbetti, Lorenzo; Falconi, Serena; Frusconi, Sabrina; Giotti, Irene; Giuliani, Costanza; Torricelli, Francesca; Benelli, Matteo; Papi, Laura

    2015-01-01

    The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design. PMID:26066488

  13. RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

    PubMed Central

    Zhang, Yanming; Ji, Peifeng; Wang, Jinfeng; Zhao, Fangqing

    2016-01-01

    16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities. PMID:26984526

  14. Parallel tagged amplicon sequencing reveals major lineages and phylogenetic structure in the North American tiger salamander (Ambystoma tigrinum) species complex.

    PubMed

    O'Neill, Eric M; Schwartz, Rachel; Bullock, C Thomas; Williams, Joshua S; Shaffer, H Bradley; Aguilar-Miguel, X; Parra-Olea, Gabriela; Weisrock, David W

    2013-01-01

    Modern analytical methods for population genetics and phylogenetics are expected to provide more accurate results when data from multiple genome-wide loci are analysed. We present the results of an initial application of parallel tagged sequencing (PTS) on a next-generation platform to sequence thousands of barcoded PCR amplicons generated from 95 nuclear loci and 93 individuals sampled across the range of the tiger salamander (Ambystoma tigrinum) species complex. To manage the bioinformatic processing of this large data set (344 330 reads), we developed a pipeline that sorts PTS data by barcode and locus, identifies high-quality variable nucleotides and yields phased haplotype sequences for each individual at each locus. Our sequencing and bioinformatic strategy resulted in a genome-wide data set with relatively low levels of missing data and a wide range of nucleotide variation. structure analyses of these data in a genotypic format resulted in strongly supported assignments for the majority of individuals into nine geographically defined genetic clusters. Species tree analyses of the most variable loci using a multi-species coalescent model resulted in strong support for most branches in the species tree; however, analyses including more than 50 loci produced parameter sampling trends that indicated a lack of convergence on the posterior distribution. Overall, these results demonstrate the potential for amplicon-based PTS to rapidly generate large-scale data for population genetic and phylogenetic-based research.

  15. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India

    PubMed Central

    Rao, Pavitra N.; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K.; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P.; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C.

    2016-01-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy. PMID:27008882

  16. Multifunctional aerial display through use of polarization-processing display

    NASA Astrophysics Data System (ADS)

    Uchida, Keitaro; Ito, Shusei; Yamamoto, Hirotsugu

    2017-02-01

    We have realized a multifunctional aerial display. An aerial image of a polarization-processing display is formed through aerial imaging by retro-reflection. By changing the polarization modulation patterns, we can switch between a three-layered display and a secure display.

  17. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested.

  18. Targeted Amplicon Sequencing for Single-Nucleotide-Polymorphism Genotyping of Attaching and Effacing Escherichia coli O26:H11 Cattle Strains via a High-Throughput Library Preparation Technique

    PubMed Central

    Delannoy, Sabine; Bugarel, Marie; Nagaraja, Tiruvoor G.; Renter, David G.; den Bakker, Henk C.; Nightingale, Kendra K.; Fach, Patrick; Loneragan, Guy H.

    2015-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O26:H11, a serotype within Shiga toxin-producing E. coli (STEC) that causes severe human disease, has been considered to have evolved from attaching and effacing E. coli (AEEC) O26:H11 through the acquisition of a Shiga toxin-encoding gene. Targeted amplicon sequencing using next-generation sequencing technology of 48 phylogenetically informative single-nucleotide polymorphisms (SNPs) and three SNPs differentiating Shiga toxin-positive (stx-positive) strains from Shiga toxin-negative (stx-negative) strains were used to infer the phylogenetic relationships of 178 E. coli O26:H11 strains (6 stx-positive strains and 172 stx-negative AEEC strains) from cattle feces to 7 publically available genomes of human clinical strains. The AEEC cattle strains displayed synonymous SNP genotypes with stx2-positive sequence type 29 (ST29) human O26:H11 strains, while stx1 ST21 human and cattle strains clustered separately, demonstrating the close phylogenetic relatedness of these Shiga toxin-negative AEEC cattle strains and human clinical strains. With the exception of seven stx-negative strains, five of which contained espK, three stx-related SNPs differentiated the STEC strains from non-STEC strains, supporting the hypothesis that these AEEC cattle strains could serve as a potential reservoir for new or existing pathogenic human strains. Our results support the idea that targeted amplicon sequencing for SNP genotyping expedites strain identification and genetic characterization of E. coli O26:H11, which is important for food safety and public health. PMID:26567298

  19. Image Descriptors for Displays

    DTIC Science & Technology

    1977-02-01

    information. In Section V of the report, however, we have extended our descriptor for the total channel capacity of a display to include both chromi - nance and...frequency and for constant chromi - nance. The quantities nl(w) represent the number of perceivable colors for a given spatial frequancy and luminance value...the chromi - nance contribution to the total channel capacity, we shall utilize a linear model for thot distribution of perceived chrominance levels. We

  20. Text File Display Program

    NASA Technical Reports Server (NTRS)

    Vavrus, J. L.

    1986-01-01

    LOOK program permits user to examine text file in pseudorandom access manner. Program provides user with way of rapidly examining contents of ASCII text file. LOOK opens text file for input only and accesses it in blockwise fashion. Handles text formatting and displays text lines on screen. User moves forward or backward in file by any number of lines or blocks. Provides ability to "scroll" text at various speeds in forward or backward directions.

  1. Image Descriptors for Displays

    DTIC Science & Technology

    1975-03-01

    gain an insight into the detailed mechanisms of aliasing, but it does not predict how important aliasing is. Our statistical approach predicts the...undersampled limit has a maximum edge discrimination ability equivalent to an analog display with a flat pass- band and limiting resolution given by... discrimination ability of the observer is proportional to the statistical average of a quantity that is representative of the perceived information content

  2. Attention-Seeking Displays

    PubMed Central

    Számadó, Szabolcs

    2015-01-01

    Animal communication abounds with extravagant displays. These signals are usually interpreted as costly signals of quality. However, there is another important function for these signals: to call the attention of the receiver to the signaller. While there is abundant empirical evidence to show the importance of this stage, it is not yet incorporated into standard signalling theory. Here I investigate a general model of signalling - based on a basic action-response game - that incorporates this searching stage. I show that giving attention-seeking displays and searching for them can be an ESS. This is a very general result and holds regardless whether only the high quality signallers or both high and low types give them. These signals need not be costly at the equilibrium and they need not be honest signals of any quality, as their function is not to signal quality but simply to call the attention of the potential receivers. These kind of displays are probably more common than their current weight in the literature would suggest. PMID:26287489

  3. Engine monitoring display study

    NASA Technical Reports Server (NTRS)

    Hornsby, Mary E.

    1992-01-01

    The current study is part of a larger NASA effort to develop displays for an engine-monitoring system to enable the crew to monitor engine parameter trends more effectively. The objective was to evaluate the operational utility of adding three types of information to the basic Boeing Engine Indicating and Crew Alerting System (EICAS) display formats: alphanumeric alerting messages for engine parameters whose values exceed caution or warning limits; alphanumeric messages to monitor engine parameters that deviate from expected values; and a graphic depiction of the range of expected values for current conditions. Ten training and line pilots each flew 15 simulated flight scenarios with five variants of the basic EICAS format; these variants included different combinations of the added information. The pilots detected engine problems more quickly when engine alerting messages were included in the display; adding a graphic depiction of the range of expected values did not affect detection speed. The pilots rated both types of alphanumeric messages (alert and monitor parameter) as more useful and easier to interpret than the graphic depiction. Integrating engine parameter messages into the EICAS alerting system appears to be both useful and preferred.

  4. Microgap flat panel display

    DOEpatents

    Wuest, Craig R.

    1998-01-01

    A microgap flat panel display which includes a thin gas-filled display tube that utilizes switched X-Y "pixel" strips to trigger electron avalanches and activate a phosphor at a given location on a display screen. The panel utilizes the principal of electron multiplication in a gas subjected to a high electric field to provide sufficient electron current to activate standard luminescent phosphors located on an anode. The X-Y conductive strips of a few micron widths may for example, be deposited on opposite sides of a thin insulating substrate, or on one side of the adjacent substrates and function as a cathode. The X-Y strips are separated from the anode by a gap filled with a suitable gas. Electrical bias is selectively switched onto X and Y strips to activate a "pixel" in the region where these strips overlap. A small amount of a long-lived radioisotope is used to initiate an electron avalanche in the overlap region when bias is applied. The avalanche travels through the gas filled gap and activates a luminescent phosphor of a selected color. The bias is adjusted to give a proportional electron multiplication to control brightness for given pixel.

  5. Microgap flat panel display

    DOEpatents

    Wuest, C.R.

    1998-12-08

    A microgap flat panel display is disclosed which includes a thin gas-filled display tube that utilizes switched X-Y ``pixel`` strips to trigger electron avalanches and activate a phosphor at a given location on a display screen. The panel utilizes the principal of electron multiplication in a gas subjected to a high electric field to provide sufficient electron current to activate standard luminescent phosphors located on an anode. The X-Y conductive strips of a few micron widths may for example, be deposited on opposite sides of a thin insulating substrate, or on one side of the adjacent substrates and function as a cathode. The X-Y strips are separated from the anode by a gap filled with a suitable gas. Electrical bias is selectively switched onto X and Y strips to activate a ``pixel`` in the region where these strips overlap. A small amount of a long-lived radioisotope is used to initiate an electron avalanche in the overlap region when bias is applied. The avalanche travels through the gas filled gap and activates a luminescent phosphor of a selected color. The bias is adjusted to give a proportional electron multiplication to control brightness for given pixel. 6 figs.

  6. Stage Cylindrical Immersive Display

    NASA Technical Reports Server (NTRS)

    Abramyan, Lucy; Norris, Jeffrey S.; Powell, Mark W.; Mittman, David S.; Shams, Khawaja S.

    2011-01-01

    Panoramic images with a wide field of view intend to provide a better understanding of an environment by placing objects of the environment on one seamless image. However, understanding the sizes and relative positions of the objects in a panorama is not intuitive and prone to errors because the field of view is unnatural to human perception. Scientists are often faced with the difficult task of interpreting the sizes and relative positions of objects in an environment when viewing an image of the environment on computer monitors or prints. A panorama can display an object that appears to be to the right of the viewer when it is, in fact, behind the viewer. This misinterpretation can be very costly, especially when the environment is remote and/or only accessible by unmanned vehicles. A 270 cylindrical display has been developed that surrounds the viewer with carefully calibrated panoramic imagery that correctly engages their natural kinesthetic senses and provides a more accurate awareness of the environment. The cylindrical immersive display offers a more natural window to the environment than a standard cubic CAVE (Cave Automatic Virtual Environment), and the geometry allows multiple collocated users to simultaneously view data and share important decision-making tasks. A CAVE is an immersive virtual reality environment that allows one or more users to absorb themselves in a virtual environment. A common CAVE setup is a room-sized cube where the cube sides act as projection planes. By nature, all cubic CAVEs face a problem with edge matching at edges and corners of the display. Modern immersive displays have found ways to minimize seams by creating very tight edges, and rely on the user to ignore the seam. One significant deficiency of flat-walled CAVEs is that the sense of orientation and perspective within the scene is broken across adjacent walls. On any single wall, parallel lines properly converge at their vanishing point as they should, and the sense of

  7. Defense Display Strategy and Roadmaps

    DTIC Science & Technology

    2002-08-06

    ultra-resolution, true 3D , and intelligent displays (integration of computers and communication functions into screens). The new strategy is Service...led. Keywords: defense, electronic displays, high definition, micro-display, 25-megapixel, true 3D , novel and intelligent displays 1...megapixel and true 3D devices. The approved roadmap is illustrated in Figure 1. * Paper

  8. Landing Hazard Avoidance Display

    NASA Technical Reports Server (NTRS)

    Abernathy, Michael Franklin (Inventor); Hirsh, Robert L. (Inventor)

    2016-01-01

    Landing hazard avoidance displays can provide rapidly understood visual indications of where it is safe to land a vehicle and where it is unsafe to land a vehicle. Color coded maps can indicate zones in two dimensions relative to the vehicles position where it is safe to land. The map can be simply green (safe) and red (unsafe) areas with an indication of scale or can be a color coding of another map such as a surface map. The color coding can be determined in real time based on topological measurements and safety criteria to thereby adapt to dynamic, unknown, or partially known environments.

  9. Pictorial Format Display Evaluation

    DTIC Science & Technology

    1984-05-01

    the pathway moved out of the theoretical field of view of the HUDM It did not disappear from the HUD, however, instead it was pegged to the side of...the HUD in the direction of its current position. When the pathway was pegged to the side, a transitional flight director symbol (an inverted "T...of view,. the flashing tractor beam remained, and the "jewel light" was pegged to the side of the display at the end of the ’flashing tractor beam

  10. Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

    PubMed Central

    Rucevic, Marijana; Kourjian, Georgio; Boucau, Julie; Blatnik, Renata; Garcia Bertran, Wilfredo; Berberich, Matthew J.; Walker, Bruce D.; Riemer, Angelika B.

    2016-01-01

    ABSTRACT Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity. IMPORTANCE The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA

  11. Synthesis and screening of peptide libraries with free C-termini

    PubMed Central

    Wang, Yen-Chih; Distefano, Mark D.

    2016-01-01

    Peptide libraries are useful tools to investigate the relationship between structure and function of proteins. The creation of peptide libraries with free C-termini presents unique synthetic challenges. In this review, methods for creating peptide libraries using either solid-phase peptide synthesis or phage display are described. Methods for screening such libraries and their application in studying several important biological problems are also reported.

  12. Synthesis and screening of peptide libraries with free C-termini.

    PubMed

    Wang, Yen-Chih; Distefano, Mark D

    2014-01-01

    Peptide libraries are useful tools to investigate the relationship between structure and function of proteins. The creation of peptide libraries with free C-termini presents unique synthetic challenges. In this review, methods for creating peptide libraries using either solid-phase peptide synthesis or phage display are described. Methods for screening such libraries and their application in studying several important biological problems are also reported.

  13. Peptides specific for Mycobacterium avium subspecies paratuberculosis infection: diagnostic potential.

    PubMed

    Casey, J L; Sanalla, A M; Tamvakis, D; Thalmann, C; Carroll, E L; Parisi, K; Coley, A M; Stewart, D J; Vaughan, J A; Michalski, W P; Luke, R; Foley, M

    2011-08-01

    Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of Johne's disease (JD). Current serological diagnostic tests for JD are limited by their sensitivity when used in sub-clinical stages of the disease. Our objective was to identify peptides that mimic diagnostically important Map epitopes that might be incorporated into a new-generation JD diagnostic. Four peptides were isolated from a phage-displayed random peptide library by screening on antibodies derived from Map-infected goats. The peptides were recognised by antibodies from Map-infected goats but not by antibodies from uninfected goats. The peptides elicited immune responses in rabbits, which reacted strongly with bona fide Map antigens proving the peptides were true epitope mimics. To assess the diagnostic value a panel of goat sera was screened for reactivity's with peptides. The peptides were recognised by antibodies from a proportion of goats infected with Map compared with control animals with a diagnostic specificity of 100% and the sensitivity ranged from 50 to 75%. Combinations of any two peptides improved sensitivity 62.5-87.5% and 100% sensitivity was achieved with three of the four peptides in combination. These data suggest peptides representing diagnostically important Map epitopes could be incorporated into a sensitive diagnostic test.

  14. HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization

    PubMed Central

    Meisal, Roger; Rounge, Trine Ballestad; Christiansen, Irene Kraus; Eieland, Alexander Kirkeby; Worren, Merete Molton; Molden, Tor Faksvaag; Kommedal, Øyvind; Hovig, Eivind; Leegaard, Truls Michael

    2017-01-01

    Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution. PMID:28045981

  15. Phage display: applications, innovations, and issues in phage and host biology.

    PubMed

    Wilson, D R; Finlay, B B

    1998-04-01

    In the 7 years since the first publications describing phage-displayed peptide libraries, phage display has been successfully employed in a variety of research. Innovations in vector design and methods to identify target clones account for much of this success. At the same time, not all ventures have been entirely successful and it appears that phage and host biology play important roles in this. A key issue concerns the role played by a displayed peptide or protein in its successful expression and incorporation into virions. While few studies have examined these issues specifically in context of phage display, the literature as a whole provides insight. Accordingly, we review phage biology, relevant aspects of host biology, and phage display applications with the goals of illustrating (i) relevant aspects of the interplay between phage-host biology and successful phage display and (ii) the limitations and considerable potential of this important technology.

  16. Construction and packaging of herpes simplex virus/adeno-associated virus (HSV/AAV) Hybrid amplicon vectors.

    PubMed

    Saydam, Okay; Glauser, Daniel L; Fraefel, Cornel

    2012-03-01

    Herpes simplex virus type 1 (HSV-1)-based amplicon vectors conserve most properties of the parental virus: broad host range, the ability to transduce dividing and nondiving cells, and a large transgene capacity. This permits incorporation of genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell-specific expression, multiple transgene cassettes, or genetic elements from other viruses. Hybrid vectors use elements from HSV-1 that allow replication and packaging of large-vector DNA into highly infectious particles, and elements from other viruses that confer genetic stability to vector DNA in the transduced cell. For example, adeno-associated virus (AAV) has the unique ability to integrate its genome into a specific site on human chromosome 19. The viral rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. However, AAV-based vectors have a very small transgene capacity and do not conventionally contain the rep gene to support site-specific genomic integration. HSV/AAV hybrid vectors contain both HSV-1 replication and packaging functions and the AAV rep gene and a transgene cassette flanked by the AAV ITRs. This combines the large transgene capacity of HSV-1 with the capability of site-specific genomic transgene integration and long-term transgene expression of AAV. This protocol describes the preparation of HSV/AAV hybrid vectors using a replication-competent/conditional, packaging-defective HSV-1 genome cloned as a bacterial artificial chromosome (BAC) to provide helper functions for vector replication and packaging. The advantages and limitations of such vectors compared to standard HSV-1 amplicon vectors are also discussed.

  17. Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments.

    PubMed

    Miller, Christopher S; Handley, Kim M; Wrighton, Kelly C; Frischkorn, Kyle R; Thomas, Brian C; Banfield, Jillian F

    2013-01-01

    In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the "long tail" of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change.

  18. Black optic display

    DOEpatents

    Veligdan, James T.

    1997-01-01

    An optical display includes a plurality of stacked optical waveguides having first and second opposite ends collectively defining an image input face and an image screen, respectively, with the screen being oblique to the input face. Each of the waveguides includes a transparent core bound by a cladding layer having a lower index of refraction for effecting internal reflection of image light transmitted into the input face to project an image on the screen, with each of the cladding layers including a cladding cap integrally joined thereto at the waveguide second ends. Each of the cores is beveled at the waveguide second end so that the cladding cap is viewable through the transparent core. Each of the cladding caps is black for absorbing external ambient light incident upon the screen for improving contrast of the image projected internally on the screen.

  19. Three dimensional interactive display

    NASA Technical Reports Server (NTRS)

    Vranish, John M. (Inventor)

    2005-01-01

    A three-dimensional (3-D) interactive display and method of forming the same, includes a transparent capaciflector (TC) camera formed on a transparent shield layer on the screen surface. A first dielectric layer is formed on the shield layer. A first wire layer is formed on the first dielectric layer. A second dielectric layer is formed on the first wire layer. A second wire layer is formed on the second dielectric layer. Wires on the first wire layer and second wire layer are grouped into groups of parallel wires with a turnaround at one end of each group and a sensor pad at the opposite end. An operational amplifier is connected to each of the sensor pads and the shield pad biases the pads and receives a signal from connected sensor pads in response to intrusion of a probe. The signal is proportional to probe location with respect to the monitor screen.

  20. Versatile microbial surface-display for environmental remediation and biofuels production

    SciTech Connect

    Wu, Cindy H.; Mulchandani, Ashok; Chen, wilfred

    2008-02-14

    Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.

  1. Peptide Antimicrobial Agents

    PubMed Central

    Jenssen, Håvard; Hamill, Pamela; Hancock, Robert E. W.

    2006-01-01

    Antimicrobial host defense peptides are produced by all complex organisms as well as some microbes and have diverse and complex antimicrobial activities. Collectively these peptides demonstrate a broad range of antiviral and antibacterial activities and modes of action, and it is important to distinguish between direct microbicidal and indirect activities against such pathogens. The structural requirements of peptides for antiviral and antibacterial activities are evaluated in light of the diverse set of primary and secondary structures described for host defense peptides. Peptides with antifungal and antiparasitic activities are discussed in less detail, although the broad-spectrum activities of such peptides indicate that they are important host defense molecules. Knowledge regarding the relationship between peptide structure and function as well as their mechanism of action is being applied in the design of antimicrobial peptide variants as potential novel therapeutic agents. PMID:16847082

  2. Display Factors and Subjective Evaluation of Dynamic Text Display

    NASA Astrophysics Data System (ADS)

    So, Joey C. Y.; Chan, Alan H. S.

    2009-01-01

    Communications technology has exploded in past decades, leading to the question of which display method is the best to deliver electronic text messages. Many of these systems employ cathode ray tubes, liquid crystal displays, gas plasma displays, or light-emitting diodes as the output device. In order to overcome the limitations of screen size of the display units, numerous means of presenting dynamic display on screens have been invented. There are many factors that affect the readability of electronic text. This paper reviews some related empirical studies concerning the various display methods of dynamic text presentation, such as text display type, character type, text display direction, and text/background color combination, highlighting method and validity of highlighting. The subjective evaluation questionnaire is also discussed. According to the readability and preference ratings of the subjects given under different conditions, the best display method and color for comprehending the delivered messages were investigated. General recommendations of displaying dynamic information are made for the large display units which have been widely used for delivering important messages.

  3. Developing Intepretive Soil Education Displays.

    ERIC Educational Resources Information Center

    Hansmeyer, T. L.; Cooper, T. H.

    1993-01-01

    Describes several soil educational displays developed for park and nature center trails. Displays include full-scale soil monoliths displayed along the trails with explanations on why and how the soils are different, and micro-monoliths exhibiting the different soil types. (MDH)

  4. Phage display in the study of infectious diseases.

    PubMed

    Mullen, Lisa M; Nair, Sean P; Ward, John M; Rycroft, Andrew N; Henderson, Brian

    2006-03-01

    Microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection. Phage display is a simple functional genomic methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping, antibody engineering and screening for receptor agonists or antagonists. Phage display is also used widely in various forms, including the use of fragment libraries of whole microbial genomes, to identify peptide-ligand and protein-ligand interactions that are of importance in infection. In particular, this technique has proved successful in identifying microbial adhesins that are vital for colonization.

  5. Biomathematical Description of Synthetic Peptide Libraries

    PubMed Central

    Trepel, Martin

    2015-01-01

    Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org), allowing scientists to plan and analyse their peptide libraries. PMID:26042419

  6. Quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) using amplicon-fusion-site polymerase chain reaction (AFS-PCR).

    PubMed

    Weber, Axel; Taube, Sylvia; Zur Stadt, Udo; Horstmann, Martin; Krohn, Knut; Bradtke, Jutta; Teigler-Schlegel, Andrea; Leiblein, Sabine; Christiansen, Holger

    2012-11-09

    The amplification of putative oncogenes is a common finding within the genome of various cancer types. Identification and further targeting of specific junction sites within the sequence of genomic amplicons (amplicon fusion sites, AFS) by PCR (AFS-PCR) is suitable for quantification of minimal residual disease (MRD). This approach has recently been developed and described for MYCN amplified neuroblastomas. To compare AFS-PCR directly to routinely used MRD diagnostic strategies, we mapped the amplified genomic regions (ampGR) of an iAMP21-amplicon in high resolution of a patient with acute lymphoblastic leukemia (ALL). Successfully, we established AFS-PCR covering junction sites between ampGR within the iAMP21-amplicon. Quantification of MRD by AFS-PCR was directly comparable to IgH/TCR based real time quantitative PCR and fluorescence activated cell sorting (FACS) analysis in consecutive bone marrow (BM) specimens. Our data give an additional proof of concept of AFS-PCR for quantification of MRD. The method could be taken into account for ALL patients with genomic amplifications as alternative MRD diagnostic, if no or qualitatively poor Ig/TCR-PCRs are available.

  7. Metagenomic 16S rDNA Illumina tags are a powerful alternative to amplicon sequencing to explore diversity and structure of microbial communities.

    PubMed

    Logares, Ramiro; Sunagawa, Shinichi; Salazar, Guillem; Cornejo-Castillo, Francisco M; Ferrera, Isabel; Sarmento, Hugo; Hingamp, Pascal; Ogata, Hiroyuki; de Vargas, Colomban; Lima-Mendez, Gipsi; Raes, Jeroen; Poulain, Julie; Jaillon, Olivier; Wincker, Patrick; Kandels-Lewis, Stefanie; Karsenti, Eric; Bork, Peer; Acinas, Silvia G

    2014-09-01

    Sequencing of 16S rDNA polymerase chain reaction (PCR) amplicons is the most common approach for investigating environmental prokaryotic diversity, despite the known biases introduced during PCR. Here we show that 16S rDNA fragments derived from Illumina-sequenced environmental metagenomes (mi tags) are a powerful alternative to 16S rDNA amplicons for investigating the taxonomic diversity and structure of prokaryotic communities. As part of the Tara Oceans global expedition, marine plankton was sampled in three locations, resulting in 29 subsamples for which metagenomes were produced by shotgun Illumina sequencing (ca. 700 Gb). For comparative analyses, a subset of samples was also selected for Roche-454 sequencing using both shotgun (m454 tags; 13 metagenomes, ca. 2.4 Gb) and 16S rDNA amplicon (454 tags; ca. 0.075 Gb) approaches. Our results indicate that by overcoming PCR biases related to amplification and primer mismatch, mi tags may provide more realistic estimates of community richness and evenness than amplicon 454 tags. In addition, mi tags can capture expected beta diversity patterns. Using mi tags is now economically feasible given the dramatic reduction in high-throughput sequencing costs, having the advantage of retrieving simultaneously both taxonomic (Bacteria, Archaea and Eukarya) and functional information from the same microbial community.

  8. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  9. PH dependent adhesive peptides

    SciTech Connect

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  10. Colorimetry for CRT displays

    NASA Astrophysics Data System (ADS)

    Golz, Jürgen; MacLeod, Donald I. A.

    2003-05-01

    We analyze the sources of error in specifying color in CRT displays. These include errors inherent in the use of the color matching functions of the CIE 1931 standard observer when only colorimetric, not radiometric, calibrations are available. We provide transformation coefficients that prove to correct the deficiencies of this observer very well. We consider four different candidate sets of cone sensitivities. Some of these differ substantially; variation among candidate cone sensitivities exceeds the variation among phosphors. Finally, the effects of the recognized forms of observer variation on the visual responses (cone excitations or cone contrasts) generated by CRT stimuli are investigated and quantitatively specified. Cone pigment polymorphism gives rise to variation of a few per cent in relative excitation by the different phosphors-a variation larger than the errors ensuing from the adoption of the CIE standard observer, though smaller than the differences between some candidate cone sensitivities. Macular pigmentation has a larger influence, affecting mainly responses to the blue phosphor. The estimated combined effect of all sources of observer variation is comparable in magnitude with the largest differences between competing cone sensitivity estimates but is not enough to disrupt very seriously the relation between the L and M cone weights and the isoluminance settings of individual observers. It is also comparable with typical instrumental colorimetric errors, but we discuss these only briefly.

  11. Signal Processing, Analysis, & Display

    SciTech Connect

    Lager, Darrell; Azevado, Stephen

    1986-06-01

    SIG is a general-purpose signal processing, analysis, and display program. Its main purpose is to perform manipulations on time- and frequency-domain signals. However, it has been designed to ultimately accommodate other representations for data such as multiplexed signals and complex matrices. Two user interfaces are provided in SIG - a menu mode for the unfamiliar user and a command mode for more experienced users. In both modes errors are detected as early as possible and are indicated by friendly, meaningful messages. An on-line HELP package is also included. A variety of operations can be performed on time- and frequency-domain signals including operations on the samples of a signal, operations on the entire signal, and operations on two or more signals. Signal processing operations that can be performed are digital filtering (median, Bessel, Butterworth, and Chebychev), ensemble average, resample, auto and cross spectral density, transfer function and impulse response, trend removal, convolution, Fourier transform and inverse window functions (Hamming, Kaiser-Bessel), simulation (ramp, sine, pulsetrain, random), and read/write signals. User definable signal processing algorithms are also featured. SIG has many options including multiple commands per line, command files with arguments,commenting lines, defining commands, and automatic execution for each item in a repeat sequence. Graphical operations on signals and spectra include: x-y plots of time signals; real, imaginary, magnitude, and phase plots of spectra; scaling of spectra for continuous or discrete domain; cursor zoom; families of curves; and multiple viewports.

  12. Identification of peptides that bind to irradiated pancreatic tumor cells

    SciTech Connect

    Huang Canhui; Liu, Xiang Y.; Rehemtulla, Alnawaz; Lawrence, Theodore S. . E-mail: tsl@med.umich.edu

    2005-08-01

    Purpose: Peptides targeting tumor vascular cells or tumor cells themselves have the potential to be used as vectors for delivering either DNA in gene therapy or antitumor agents in chemotherapy. We wished to determine if peptides identified by phage display could be used to target irradiated pancreatic cancer cells. Methods and Materials: Irradiated Capan-2 cells were incubated with 5 x 10{sup 12} plaque-forming units of a phage display library. Internalized phage were recovered and absorbed against unirradiated cells. After five such cycles of enrichment, the recovered phage were subjected to DNA sequencing analysis and synthetic peptides made. The binding of both phage and synthetic peptides was evaluated by fluorescence staining and flow cytometry in vitro and in vivo. Results: We identified one 12-mer peptide (PA1) that binds to irradiated Capan-2 pancreatic adenocarcinoma cells but not to unirradiated cells. The binding of peptide was significant after 48 h incubation with cells. In vivo experiments with Capan-2 xenografts in nude mice demonstrated that these small peptides are able to penetrate tumor tissue after intravenous injections and bind specifically to irradiated tumor cells. Conclusion: These data suggest that peptides can be identified that target tumors with radiation-induced cell markers and may be clinically useful.

  13. Antibody repertoire profiling using bacterial display identifies reactivity signatures of celiac disease.

    PubMed

    Spatola, Bradley N; Murray, Joseph A; Kagnoff, Martin; Kaukinen, Katri; Daugherty, Patrick S

    2013-01-15

    A general strategy to identify serum antibody specificities associated with a given disease state and peptide reagents for their detection was developed using bacterial display peptide libraries and multiparameter flow cytometry (MPFC). Using sera from patients with celiac disease (CD) (n = 45) or healthy subjects (n = 40), bacterial display libraries were screened for peptides that react specifically with antibodies from CD patients and not with those from healthy patients. The libraries were screened for peptides that simultaneously cross-react with CD patient antibodies present in two separate patient groups labeled with spectrally distinct fluorophores but do not react with unlabeled non-CD antibodies, thus affording a quantitative separation. A panel of six unique peptide sequences yielded 85% sensitivity and 91% specificity (AUC = 0.91) on a set of 60 samples not used for discovery, using leave-one-out cross-validation. Individual peptides were dissimilar with known CD-specific antigens tissue transglutaminase (tTG) and deamidated gliadin, and the classifier accuracy was independent of anti-tTG antibody titer. These results demonstrate that bacterial display/MPFC provides a highly effective tool for the unbiased discovery of disease-associated antibody specificities and peptide reagents for their detection that may have broad utility for diagnostic development.

  14. Unique interactive projection display screen

    SciTech Connect

    Veligdan, J.T.

    1997-11-01

    Projection systems continue to be the best method to produce large (1 meter and larger) displays. However, in order to produce a large display, considerable volume is typically required. The Polyplanar Optic Display (POD) is a novel type of projection display screen, which for the first time, makes it possible to produce a large projection system that is self-contained and only inches thick. In addition, this display screen is matte black in appearance allowing it to be used in high ambient light conditions. This screen is also interactive and can be remotely controlled via an infrared optical pointer resulting in mouse-like control of the display. Furthermore, this display need not be flat since it can be made curved to wrap around a viewer as well as being flexible.

  15. LED instrument approach instruction display

    NASA Technical Reports Server (NTRS)

    Meredith, B. D.; Kelly, W. L., IV; Crouch, R. K.

    1979-01-01

    A display employing light emitting diodes (LED's) was developed to demonstrate the feasibility of such displays for presenting landing and navigation information to reduce the workload of general aviation pilots during IFR flight. The display consists of a paper tape reader, digital memory, control electronics, digital latches, and LED alphanumeric displays. A presentable digital countdown clock-timer is included as part of the system to provide a convenient means of monitoring time intervals for precise flight navigation. The system is a limited capability prototype assembled to test pilot reaction to such a device under simulated IFR operation. Pilot opinion indicates that the display is helpful in reducing the IFR pilots workload when used with a runway approach plate. However, the development of a compact, low power second generation display was recommended which could present several instructions simultaneously and provide information update capability. A microprocessor-based display could fulfill these requirements.

  16. Visualized detection of single-base difference in multiplexed loop-mediated isothermal amplification amplicons by invasive reaction coupled with oligonucleotide probe-modified gold nanoparticles.

    PubMed

    Lu, Yan; Ma, Xueping; Wang, Jianping; Sheng, Nan; Dong, Tianhui; Song, Qinxin; Rui, Jianzhong; Zou, Bingjie; Zhou, Guohua

    2017-04-15

    Loop-mediated isothermal amplification (LAMP) is a well-developed DNA amplification method with an ultra-high sensitivity, but it is difficult to recognize a single-base difference (like genotyping) in target-specific amplicons by conventional detection ways, such as the intercalation of dyes into dsDNA amplicons or the increase of solution turbidity along with the polymerization process. To allow genotyping based on LAMP suitable for POCT (point-of-care testing) or on-site testing, here we proposed a highly specific and cost-effective method for detecting a single-base difference in LAMP amplicons. The method includes three key steps, sequence amplifier to amplify multiple fragments containing the single nucleotide polymorphisms (SNPs) of interest, allele identifier to recognize a targeted base in the amplicons by invasive reaction, and signal generator to yield signals by hybridization-induced assembly of oligonucleotide probe-modified gold nanoparticles. Because the allele identifier is sensitive to one base difference, it is possible to use multiplexed LAMP (mLAMP) to generate amplicon mixtures for multiple SNP typing. Genotyping of 3 different SNPs (CYP2C19*2, CYP2C19*3 and MDR1-C3435T) for guiding the dosage of clopidogrel is successfully carried out in a 3-plex LAMP on real clinical samples. As our method relies on the naked-eye detection and constant-temperature reaction, no expensive instrument is required for both target amplification and sequence identification, thus much suitable for inexpensive gene-guided personalized medicine in source-limited regions.

  17. De Novo Design of Potent Antimicrobial Peptides

    PubMed Central

    Frecer, V.; Ho, B.; Ding, J. L.

    2004-01-01

    Lipopolysaccharide (LPS), shed by gram-negative bacteria during infection and antimicrobial therapy, may lead to lethal endotoxic shock syndrome. A rational design strategy based on the presumed mechanism of antibacterial effect was adopted to design cationic antimicrobial peptides capable of binding to LPS through tandemly repeated sequences of alternating cationic and nonpolar residues. The peptides were designed to achieve enhanced antimicrobial potency due to initial bacterial membrane binding with a reduced risk of endotoxic shock. The peptides designed displayed binding affinities to LPS and lipid A (LA) in the low micromolar range and by molecular modeling were predicted to form amphipathic β-hairpin-like structures when they bind to LPS or LA. They also exhibited strong effects against gram-negative bacteria, with MICs in the nanomolar range, and low cytotoxic and hemolytic activities at concentrations significantly exceeding their MICs. Quantitative structure-activity relationship (QSAR) analysis of peptide sequences and their antimicrobial, cytotoxic, and hemolytic activities revealed that site-directed substitutions of residues in the hydrophobic face of the amphipathic peptides with less lipophilic residues selectively decrease the hemolytic effect without significantly affecting the antimicrobial or cytotoxic activity. On the other hand, the antimicrobial effect can be enhanced by substitutions in the polar face with more polar residues, which increase the amphipathicity of the peptide. On the basis of the QSARs, new analogs that have strong antimicrobial effects but that lack hemolytic activity can be proposed. The findings highlight the importance of peptide amphipathicity and allow a rational method that can be used to dissociate the antimicrobial and hemolytic effects of cationic peptides, which have potent antimicrobial properties, to be proposed. PMID:15328096

  18. A switchable stapled peptide.

    PubMed

    Kalistratova, Aleksandra; Legrand, Baptiste; Verdié, Pascal; Naydenova, Emilia; Amblard, Muriel; Martinez, Jean; Subra, Gilles

    2016-03-01

    The O-N acyl transfer reaction has gained significant popularity in peptide and medicinal chemistry. This reaction has been successfully applied to the synthesis of difficult sequence-containing peptides, cyclic peptides, epimerization-free fragment coupling and more recently, to switchable peptide polymers. Herein, we describe a related strategy to facilitate the synthesis and purification of a hydrophobic stapled peptide. The staple consists of a serine linked through an amide bond formed from its carboxylic acid function and the side chain amino group of diaminopropionic acid and through an ester bond formed from its amino group and the side chain carboxylic acid function of aspartic acid. The α-amino group of serine was protonated during purification. Interestingly, when the peptide was placed at physiological pH, the free amino group initiated the O-N shift reducing the staple length by one atom, leading to a more hydrophobic stapled peptide.

  19. Coupling of carbon and peptide nanotubes.

    PubMed

    Montenegro, Javier; Vázquez-Vázquez, Carlos; Kalinin, Arseny; Geckeler, Kurt E; Granja, Juan R

    2014-02-12

    Two of the main types of nanotubular architectures are the single-walled carbon nanotubes (SWCNTs) and the self-assembling cyclic peptide nanotubes (SCPNs). We here report the preparation of the dual composite resulting from the ordered combination of both tubular motifs. In the resulting architecture, the SWCNTs can act as templates for the assembly of SCPNs that engage the carbon nanotubes noncovalently via pyrene "paddles", each member of the resulting hybrid stabilizing the other in aqueous solution. The particular hybrids obtained in the present study formed highly ordered oriented arrays and display complementary properties such as electrical conductivity. Furthermore, a self-sorting of the cyclic peptides toward semiconducting rather than metallic SWCNTs is also observed in the aqueous dispersions. It is envisaged that a broad range of exploitable properties may be achieved and/or controlled by varying the cyclic peptide components of similar SWCNT/SCPN hybrids.

  20. Augmenting digital displays with computation

    NASA Astrophysics Data System (ADS)

    Liu, Jing

    As we inevitably step deeper and deeper into a world connected via the Internet, more and more information will be exchanged digitally. Displays are the interface between digital information and each individual. Naturally, one fundamental goal of displays is to reproduce information as realistically as possible since humans still care a lot about what happens in the real world. Human eyes are the receiving end of such information exchange; therefore it is impossible to study displays without studying the human visual system. In fact, the design of displays is rather closely coupled with what human eyes are capable of perceiving. For example, we are less interested in building displays that emit light in the invisible spectrum. This dissertation explores how we can augment displays with computation, which takes both display hardware and the human visual system into consideration. Four novel projects on display technologies are included in this dissertation: First, we propose a software-based approach to driving multiview autostereoscopic displays. Our display algorithm can dynamically assign views to hardware display zones based on multiple observers' current head positions, substantially reducing crosstalk and stereo inversion. Second, we present a dense projector array that creates a seamless 3D viewing experience for multiple viewers. We smoothly interpolate the set of viewer heights and distances on a per-vertex basis across the arrays field of view, reducing image distortion, crosstalk, and artifacts from tracking errors. Third, we propose a method for high dynamic range display calibration that takes into account the variation of the chrominance error over luminance. We propose a data structure for enabling efficient representation and querying of the calibration function, which also allows user-guided balancing between memory consumption and the amount of computation. Fourth, we present user studies that demonstrate that the ˜ 60 Hz critical flicker fusion

  1. PACS displays: how to select the right display technology.

    PubMed

    Hirschorn, David S; Krupinski, Elizabeth A; Flynn, Michael J

    2014-12-01

    The medical imaging display is a precision instrument with many features not found in commercial-grade displays. The more one understands what these features are and their corresponding clinical value, the better one can make a purchase decision. None of these displays maintain themselves for 5 years or more without some degree of automatic or manual performance testing. Routine calibration conformance checks are beginning to be mandated by the departments of health of many states. Most manufacturers provide mechanisms to perform these checks and keep track of their results, some more easily than others. A consistent display brightness of about 400 cd/m(2) and close conformance to the DICOM curve are the key components of a successful check. Displays are typically characterized by the number of pixels they contain, usually 2, 3, or 5 megapixels, but this is the least useful determinant of image quality. What matters most is the size of the pixels and the size of the whole display, which should be selected on the basis of the typical viewing distance. The farther one's eyes are from the display, the larger the pixels and the overall display size can be while still feeding the eye as much information as it can see. Care should be taken to use the appropriate display in a given setting for the clinical purpose at hand.

  2. 6000 x 2000 display prototype

    NASA Astrophysics Data System (ADS)

    Masuishi, Tetsuya; Small, David; MacNeil, Ronald L.

    1992-07-01

    While electronic technology has evolved enormously, there are no displays which are both very large and of high resolution. This paper describes our 6 K X 2 K, 60 inch by 20 inch, display prototype which consists of three 2 K X 2 K CRT displays connected seamlessly. Using a custom frame and a half-silvered mirror, the three images are joined by reflecting the center display image from above and transmitting the two side display images directly. Two problems must be solved to achieve a truly seamless effect. First, viewers can still see seams between regular screen images even if the displays are strictly aligned. Second, each physical display has a different geometrical space, and the center display image must be drawn in reverse because it will be reflected by the mirror. We developed a seamless window system to solve these problems. The window system displays overlapping images with translucent borders to enable better blending of the three display screens. Custom application software treats the system as a single 6 K X 2 K area. A concept named ''virtual framebuffer architecture'' enables us to implement the two kinds of seamlessness easily. To evaluate the visual effects, we developed some application systems which include video in a window, stereo sound and a high speed channel to the Connection Machine II for image processing.

  3. Display Parameters and Requirements

    NASA Astrophysics Data System (ADS)

    Bahadur, Birendra

    The following sections are included: * INTRODUCTION * HUMAN FACTORS * Anthropometry * Sensory * Cognitive * Discussions * THE HUMAN VISUAL SYSTEM - CAPABILITIES AND LIMITATIONS * Cornea * Pupil and Iris * Lens * Vitreous Humor * Retina * RODS - NIGHT VISION * CONES - DAY VISION * RODS AND CONES - TWILIGHT VISION * VISUAL PIGMENTS * MACULA * BLOOD * CHOROID COAT * Visual Signal Processing * Pathways to the Brain * Spatial Vision * Temporal Vision * Colour Vision * Colour Blindness * DICHROMATISM * Protanopia * Deuteranopia * Tritanopia * ANOMALOUS TRICHROMATISM * Protanomaly * Deuteranomaly * Tritanomaly * CONE MONOCHROMATISM * ROD MONOCHROMATISM * Using Colour Effectively * COLOUR MIXTURES AND THE CHROMATICITY DIAGRAM * Colour Matching Functions and Chromaticity Co-ordinates * CIE 1931 Colour Space * CIE PRIMARIES * CIE COLOUR MATCHING FUNCTIONS AND CHROMATICITY CO-ORDINATES * METHODS FOR DETERMINING TRISTIMULUS VALUES AND COLOUR CO-ORDINATES * Spectral Power Distribution Method * Filter Method * CIE 1931 CHROMATICITY DIAGRAM * ADDITIVE COLOUR MIXTURE * CIE 1976 Chromaticity Diagram * CIE Uniform Colour Spaces and Colour Difference Formulae * CIELUV OR L*u*v* * CIELAB OR L*a*b* * CIE COLOUR DIFFERENCE FORMULAE * Colour Temperature and CIE Standard Illuminants and source * RADIOMETRIC AND PHOTOMETRIC QUANTITIES * Photopic (Vλ and Scotopic (Vλ') Luminous Efficiency Function * Photometric and Radiometric Flux * Luminous and Radiant Intensities * Incidence: Illuminance and Irradiance * Exitance or Emittance (M) * Luminance and Radiance * ERGONOMIC REQUIREMENTS OF DISPLAYS * ELECTRO-OPTICAL PARAMETERS AND REQUIREMENTS * Contrast and Contrast Ratio * Luminance and Brightness * Colour Contrast and Chromaticity * Glare * Other Aspects of Legibility * SHAPE AND SIZE OF CHARACTERS * DEFECTS AND BLEMISHES * FLICKER AND DISTORTION * ANGLE OF VIEW * Switching Speed * Threshold and Threshold Characteristic * Measurement Techniques For Electro-optical Parameters * RADIOMETRIC

  4. Antimicrobial Peptides with Differential Bacterial Binding Characteristics

    DTIC Science & Technology

    2013-03-01

    screened also displayed discriminatory binding to pathogenic E. coli O157:H7 relative to non -pathogenic E. coli ML35. The three fragments that were...screened for binding to pathogenic and non -pathogenic Escherichia coli (a Gram- negative bacterium) as well as Staphylococcus aureus (a Gram-positive...strain-specific (pathogenic vs. non -pathogenic E. coli). Several of the peptide fragments demonstrated the ability to discriminate between

  5. Antimicrobial peptides: a new class of antimalarial drugs?

    PubMed Central

    Vale, Nuno; Aguiar, Luísa; Gomes, Paula

    2014-01-01

    A range of antimicrobial peptides (AMP) exhibit activity on malaria parasites, Plasmodium spp., in their blood or mosquito stages, or both. These peptides include a diverse array of both natural and synthetic molecules varying greatly in size, charge, hydrophobicity, and secondary structure features. Along with an overview of relevant literature reports regarding AMP that display antiplasmodial activity, this review makes a few considerations about those molecules as a potential new class of antimalarial drugs. PMID:25566072

  6. Laser illuminated flat panel display

    SciTech Connect

    Veligdan, J.T.

    1995-12-31

    A 10 inch laser illuminated flat panel Planar Optic Display (POD) screen has been constructed and tested. This POD screen technology is an entirely new concept in display technology. Although the initial display is flat and made of glass, this technology lends itself to applications where a plastic display might be wrapped around the viewer. The display screen is comprised of hundreds of planar optical waveguides where each glass waveguide represents a vertical line of resolution. A black cladding layer, having a lower index of refraction, is placed between each waveguide layer. Since the cladding makes the screen surface black, the contrast is high. The prototype display is 9 inches wide by 5 inches high and approximately I inch thick. A 3 milliwatt HeNe laser is used as the illumination source and a vector scanning technique is employed.

  7. Flat panel planar optic display

    SciTech Connect

    Veligdan, J.T.

    1994-11-01

    A prototype 10 inch flat panel Planar Optic Display, (POD), screen has been constructed and tested. This display screen is comprised of hundreds of planar optic class sheets bonded together with a cladding layer between each sheet where each glass sheet represents a vertical line of resolution. The display is 9 inches wide by 5 inches high and approximately 1 inch thick. A 3 milliwatt HeNe laser is used as the illumination source and a vector scanning technique is employed.

  8. Prototyping user displays using CLIPS

    NASA Technical Reports Server (NTRS)

    Kosta, Charles P.; Miller, Ross; Krolak, Patrick; Vesty, Matt

    1990-01-01

    CLIPS is being used as an integral module of a rapid prototyping system. The prototyping system consists of a display manager for object browsing, a graph program for displaying line and bar charts, and a communications server for routing messages between modules. A CLIPS simulation of a physical model provides dynamic control of the user's display. Currently, a project is well underway to prototype the Advanced Automation System (AAS) for the Federal Aviation Administration.

  9. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions.

  10. High-resolution melting analysis using unlabeled probe and amplicon scanning simultaneously detects several lactase persistence variants.

    PubMed

    Janukonyté, Jurgita; Vestergaard, Else M; Ladefoged, Søren A; Nissen, Peter H

    2010-12-01

    Lactase persistence and thereby tolerance to lactose is a common trait in people of Northern European descent. It is linked to the LCT -13910C>T variant located in intron 13 of the MCM6 gene 13.9 kb upstream of the lactase (LCT) gene. In people of African and Middle Eastern descent, lactase persistence can be associated with other variants nearby the -13910C>T variant, limiting the use of the -13910C>T-based SNP analysis, e.g. TaqMan assays for the diagnosis of lactose intolerance. Using high-resolution melting analysis, we identified five samples that were heterozygous for the -13915T>G variant among 78 patients genotyped as -13910C/C by a TaqMan assay. All samples originated from patients of probable Middle Eastern descent. In order to detect the -13910 and -13915 variants simultaneously, we developed a new high-resolution melting (HRM) analysis assay based on unlabeled probe genotyping and simultaneous amplicon scanning analysis. By using this assay we were able to distinguish the -13910 and -13915 genotypes clearly. Furthermore, we identified two rare variants, the -13907C>G and -13913T>C. With this method, based on an inexpensive unlabeled probe, it is possible to simultaneously detect the -13910C>T and -13915T>G variants in addition to rarer variants surrounding the -13910 site. This new method may contribute to improve the diagnostic performance of the genetic analysis for lactose intolerance.

  11. A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

    PubMed

    Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

    2016-12-15

    The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.

  12. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    PubMed

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  13. Molecular Epidemiology of Plasmodium falciparum kelch13 Mutations in Senegal Determined by Using Targeted Amplicon Deep Sequencing.

    PubMed

    Talundzic, Eldin; Ndiaye, Yaye D; Deme, Awa B; Olsen, Christian; Patel, Dhruviben S; Biliya, Shweta; Daniels, Rachel; Vannberg, Fredrik O; Volkman, Sarah K; Udhayakumar, Venkatachalam; Ndiaye, Daouda

    2017-03-01

    The emergence of Plasmodium falciparum resistance to artemisinin in Southeast Asia threatens malaria control and elimination activities worldwide. Multiple polymorphisms in the P. falciparum kelch gene found in chromosome 13 (Pfk13) have been associated with artemisinin resistance. Surveillance of potential drug resistance loci within a population that may emerge under increasing drug pressure is an important public health activity. In this context, P. falciparum infections from an observational surveillance study in Senegal were genotyped using targeted amplicon deep sequencing (TADS) for Pfk13 polymorphisms. The results were compared to previously reported Pfk13 polymorphisms from around the world. A total of 22 Pfk13 propeller domain polymorphisms were identified in this study, of which 12 have previously not been reported. Interestingly, of the 10 polymorphisms identified in the present study that were also previously reported, all had a different amino acid substitution at these codon positions. Most of the polymorphisms were present at low frequencies and were confined to single isolates, suggesting they are likely transient polymorphisms that are part of naturally evolving parasite populations. The results of this study underscore the need to identify potential drug resistance loci existing within a population, which may emerge under increasing drug pressure.

  14. Amplicon-Based Pyrosequencing Reveals High Diversity of Protistan Parasites in Ships' Ballast Water: Implications for Biogeography and Infectious Diseases.

    PubMed

    Pagenkopp Lohan, K M; Fleischer, R C; Carney, K J; Holzer, K K; Ruiz, G M

    2016-04-01

    Ships' ballast water (BW) commonly moves macroorganisms and microorganisms across the world's oceans and along coasts; however, the majority of these microbial transfers have gone undetected. We applied high-throughput sequencing methods to identify microbial eukaryotes, specifically emphasizing the protistan parasites, in ships' BW collected from vessels calling to the Chesapeake Bay (Virginia and Maryland, USA) from European and Eastern Canadian ports. We utilized tagged-amplicon 454 pyrosequencing with two general primer sets, amplifying either the V4 or V9 domain of the small subunit (SSU) of the ribosomal RNA (rRNA) gene complex, from total DNA extracted from water samples collected from the ballast tanks of bulk cargo vessels. We detected a diverse group of protistan taxa, with some known to contain important parasites in marine systems, including Apicomplexa (unidentified apicomplexans, unidentified gregarines, Cryptosporidium spp.), Dinophyta (Blastodinium spp., Euduboscquella sp., unidentified syndinids, Karlodinium spp., Syndinium spp.), Perkinsea (Parvilucifera sp.), Opisthokonta (Ichthyosporea sp., Pseudoperkinsidae, unidentified ichthyosporeans), and Stramenopiles (Labyrinthulomycetes). Further characterization of groups with parasitic taxa, consisting of phylogenetic analyses for four taxa (Cryptosporidium spp., Parvilucifera spp., Labyrinthulomycetes, and Ichthyosporea), revealed that sequences were obtained from both known and novel lineages. This study demonstrates that high-throughput sequencing is a viable and sensitive method for detecting parasitic protists when present and transported in the ballast water of ships. These data also underscore the potential importance of human-aided dispersal in the biogeography of these microbes and emerging diseases in the world's oceans.

  15. Amplicon-pyrosequencing-based detection of compositional shifts in bryophyte-associated fungal communities along an elevation gradient.

    PubMed

    Davey, Marie L; Heegaard, Einar; Halvorsen, Rune; Kauserud, Håvard; Ohlson, Mikael

    2013-01-01

    Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low-alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.

  16. Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

    PubMed Central

    Chaitanya, Lakshmi; Ralf, Arwin; van Oven, Mannis; Kupiec, Tomasz; Chang, Joseph; Lagacé, Robert

    2015-01-01

    ABSTRACT Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long‐range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications. PMID:26387877

  17. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    PubMed Central

    Fischer, Sandra; Kumar, Neeraj

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea. PMID:28097056

  18. High-throughput amplicon sequencing and stream benthic bacteria: identifying the best taxonomic level for multiple-stressor research

    PubMed Central

    Salis, R. K.; Bruder, A.; Piggott, J. J.; Summerfield, T. C.; Matthaei, C. D.

    2017-01-01

    Disentangling the individual and interactive effects of multiple stressors on microbial communities is a key challenge to our understanding and management of ecosystems. Advances in molecular techniques allow studying microbial communities in situ and with high taxonomic resolution. However, the taxonomic level which provides the best trade-off between our ability to detect multiple-stressor effects versus the goal of studying entire communities remains unknown. We used outdoor mesocosms simulating small streams to investigate the effects of four agricultural stressors (nutrient enrichment, the nitrification inhibitor dicyandiamide (DCD), fine sediment and flow velocity reduction) on stream bacteria (phyla, orders, genera, and species represented by Operational Taxonomic Units with 97% sequence similarity). Community composition was assessed using amplicon sequencing (16S rRNA gene, V3-V4 region). DCD was the most pervasive stressor, affecting evenness and most abundant taxa, followed by sediment and flow velocity. Stressor pervasiveness was similar across taxonomic levels and lower levels did not perform better in detecting stressor effects. Community coverage decreased from 96% of all sequences for abundant phyla to 28% for species. Order-level responses were generally representative of responses of corresponding genera and species, suggesting that this level may represent the best compromise between stressor sensitivity and coverage of bacterial communities. PMID:28327636

  19. Fungi Sailing the Arctic Ocean: Speciose Communities in North Atlantic Driftwood as Revealed by High-Throughput Amplicon Sequencing.

    PubMed

    Rämä, Teppo; Davey, Marie L; Nordén, Jenni; Halvorsen, Rune; Blaalid, Rakel; Mathiassen, Geir H; Alsos, Inger G; Kauserud, Håvard

    2016-08-01

    High amounts of driftwood sail across the oceans and provide habitat for organisms tolerating the rough and saline environment. Fungi have adapted to the extremely cold and saline conditions which driftwood faces in the high north. For the first time, we applied high-throughput sequencing to fungi residing in driftwood to reveal their taxonomic richness, community composition, and ecology in the North Atlantic. Using pyrosequencing of ITS2 amplicons obtained from 49 marine logs, we found 807 fungal operational taxonomic units (OTUs) based on clustering at 97 % sequence similarity cut-off level. The phylum Ascomycota comprised 74 % of the OTUs and 20 % belonged to Basidiomycota. The richness of basidiomycetes decreased with prolonged submersion in the sea, supporting the general view of ascomycetes being more extremotolerant. However, more than one fourth of the fungal OTUs remained unassigned to any fungal class, emphasising the need for better DNA reference data from the marine habitat. Different fungal communities were detected in coniferous and deciduous logs. Our results highlight that driftwood hosts a considerably higher fungal diversity than currently known. The driftwood fungal community is not a terrestrial relic but a speciose assemblage of fungi adapted to the stressful marine environment and different kinds of wooden substrates found in it.

  20. Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring

    PubMed Central

    Vierheilig, J.; Savio, D.; Ley, R. E.; Mach, R. L.; Farnleitner, A. H.

    2016-01-01

    The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multicompartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems. PMID:26606090

  1. Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring.

    PubMed

    Vierheilig, J; Savio, D; Ley, R E; Mach, R L; Farnleitner, A H; Reischer, G H

    2015-01-01

    The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multi-compartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems.

  2. Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.

    PubMed

    Chaitanya, Lakshmi; Ralf, Arwin; van Oven, Mannis; Kupiec, Tomasz; Chang, Joseph; Lagacé, Robert; Kayser, Manfred

    2015-12-01

    Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long-range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications.

  3. High-throughput amplicon sequencing and stream benthic bacteria: identifying the best taxonomic level for multiple-stressor research

    NASA Astrophysics Data System (ADS)

    Salis, R. K.; Bruder, A.; Piggott, J. J.; Summerfield, T. C.; Matthaei, C. D.

    2017-03-01

    Disentangling the individual and interactive effects of multiple stressors on microbial communities is a key challenge to our understanding and management of ecosystems. Advances in molecular techniques allow studying microbial communities in situ and with high taxonomic resolution. However, the taxonomic level which provides the best trade-off between our ability to detect multiple-stressor effects versus the goal of studying entire communities remains unknown. We used outdoor mesocosms simulating small streams to investigate the effects of four agricultural stressors (nutrient enrichment, the nitrification inhibitor dicyandiamide (DCD), fine sediment and flow velocity reduction) on stream bacteria (phyla, orders, genera, and species represented by Operational Taxonomic Units with 97% sequence similarity). Community composition was assessed using amplicon sequencing (16S rRNA gene, V3-V4 region). DCD was the most pervasive stressor, affecting evenness and most abundant taxa, followed by sediment and flow velocity. Stressor pervasiveness was similar across taxonomic levels and lower levels did not perform better in detecting stressor effects. Community coverage decreased from 96% of all sequences for abundant phyla to 28% for species. Order-level responses were generally representative of responses of corresponding genera and species, suggesting that this level may represent the best compromise between stressor sensitivity and coverage of bacterial communities.

  4. High-throughput identification and quantification of Candida species using high resolution derivative melt analysis of panfungal amplicons.

    PubMed

    Mandviwala, Tasneem; Shinde, Rupali; Kalra, Apoorv; Sobel, Jack D; Akins, Robert A

    2010-01-01

    Fungal infections pose unique challenges to molecular diagnostics; fungal molecular diagnostics consequently lags behind bacterial and viral counterparts. Nevertheless, fungal infections are often life-threatening, and early detection and identification of species is crucial to successful intervention. A high throughput PCR-based method is needed that is independent of culture, is sensitive to the level of one fungal cell per milliliter of blood or other tissue types, and is capable of detecting species and resistance mutations. We introduce the use of high resolution melt analysis, in combination with more sensitive, inclusive, and appropriately positioned panfungal primers, to address these needs. PCR-based amplification of the variable internal transcribed regions of the rDNA genes generates an amplicon whose sequence melts with a shape that is characteristic and therefore diagnostic of the species. Simple analysis of the differences between test and reference melt curves generates a single number that calls the species. Early indications suggest that high resolution melt analysis can distinguish all eight major species of Candida of clinical significance without interference from excess human DNA. Candida species, including mixed and novel species, can be identified directly in vaginal samples. This tool can potentially detect, count, and identify fungi in hundreds of samples per day without further manipulation, costs, or delays, offering a major step forward in fungal molecular diagnostics.

  5. Program For A Pushbutton Display

    NASA Technical Reports Server (NTRS)

    Busquets, Anthony M.; Luck, William S., Jr.

    1989-01-01

    Programmable Display Pushbutton (PDP) is pushbutton device available from Micro Switch having programmable 16X35 matrix of light-emitting diodes on pushbutton surface. Any desired legends display on PDP's, producing user-friendly applications reducing need for dedicated manual controls. Interacts with operator, calls for correct response before transmitting next message. Both simple manual control and sophisticated programmable link between operator and host system. Programmable Display Pushbutton Legend Editor (PDPE) computer program used to create light-emitting-diode (LED) displays for pushbuttons. Written in FORTRAN.

  6. Liquid crystal Fresnel lens display

    NASA Astrophysics Data System (ADS)

    Wang, Xiao-Qian; Abhishek Kumar, Srivastava; Alwin Tam, Ming-Wai; Zheng, Zhi-Gang; Shen, Dong; Vladimir, Chigrinov G.; Kwok, Hoi-Sing

    2016-09-01

    A novel see-through display with a liquid crystal lens array was proposed. A liquid crystal Fresnel lens display (LCFLD) with a holographic screen was demonstrated. The proposed display system has high efficiency, simple fabrication, and low manufacturing cost due to the absence of a polarizer and color filter. Project supported by Partner State Key Laboratory on Advanced Displays and Optoelectronics Technologies HKUST, China, the National Natural Science Foundation of China (Grant Nos. 61435008 and 61575063), and the Fundamental Research Funds for the Central Universities, China (Grant No. WM1514036).

  7. The display of tactile information

    NASA Technical Reports Server (NTRS)

    Sherrick, Carl E.

    1991-01-01

    There are a number of examples of natural tactile displays that can five us some insights about the solid geometry of touch, and recent experimental work on the subject has extended our thinking considerably. The concern of here is, however, more with synthetic or artificial displays for the production of a virtual environment. Features of synthetic displays that have enjoyed some success in one of the following two enterprises are discussed: the study of the spatio-temporal dimensions of stimuli that afford accurate and rapid processing of environmental information, or the use of displays in the design of sensory aids for disabled persons.

  8. Miniature information displays: primary applications

    NASA Astrophysics Data System (ADS)

    Alvelda, Phillip; Lewis, Nancy D.

    1998-04-01

    Positioned to replace current liquid crystal display technology in many applications, miniature information displays have evolved to provide several truly portable platforms for the world's growing personal computing and communication needs. The technology and functionality of handheld computer and communicator systems has finally surpassed many of the standards that were originally established for desktop systems. In these new consumer electronics, performance, display size, packaging, power consumption, and cost have always been limiting factors for fabricating genuinely portable devices. The rapidly growing miniature information display manufacturing industry is making it possible to bring a wide range of highly anticipated new products to new markets.

  9. Maintenance Procedure Display: Head Mounted Display (HMD) Evaluations

    NASA Technical Reports Server (NTRS)

    Whitmore, Milrian; Litaker, Harry L., Jr.; Solem, Jody A.; Holden, Kritina L.; Hoffman, Ronald R.

    2007-01-01

    A viewgraph presentation describing maintenance procedures for head mounted displays is shown. The topics include: 1) Study Goals; 2) Near Eye Displays (HMDs); 3) Design; 4) Phase I-Evaluation Methods; 5) Phase 1 Results; 6) Improved HMD Mounting; 7) Phase 2 -Evaluation Methods; 8) Phase 2 Preliminary Results; and 9) Next Steps.

  10. Comparative Analysis of the Antimicrobial Activities of Plant Defensin-Like and Ultrashort Peptides against Food-Spoiling Bacteria

    PubMed Central

    Kraszewska, Joanna; Beckett, Michael C.; James, Tharappel C.

    2016-01-01

    ABSTRACT Antimicrobial peptides offer potential as novel therapeutics to combat food spoilage and poisoning caused by pathogenic and nonpathogenic bacteria. Our previous studies identified the peptide human beta-defensin 3 (HBD3) as a potent antimicrobial agent against a wide range of beer-spoiling bacteria. Thus, HBD3 is an excellent candidate for development as an additive to prevent food and beverage spoilage. To expand the repertoire of peptides with antimicrobial activity against bacteria associated with food spoilage and/or food poisoning, we carried out an in silico discovery pipeline to identify peptides with structure and activity similar to those of HBD3, focusing on peptides of plant origin. Using a standardized assay, we compared the antimicrobial activities of nine defensin-like plant peptides to the activity of HBD3. Only two of the peptides, fabatin-2 and Cp-thionin-2, displayed antimicrobial activity; however, the peptides differed from HBD3 in being sensitive to salt and were thermostable. We also compared the activities of several ultrashort peptides to that of HBD3. One of the peptides, the synthetic tetrapeptide O3TR, displayed biphasic antimicrobial activity but had a narrower host range than HBD3. Finally, to determine if the peptides might act in concert to improve antimicrobial activity, we compared the activities of the peptides in pairwise combinations. The plant defensin-like peptides fabatin-2 and Cp-thionin-2 displayed a synergistic effect with HBD3, while O3TR was antagonistic. Thus, some plant defensin-like peptides are effective antimicrobials and may act in concert with HBD3 to control bacteria associated with food spoilage and food poisoning. IMPORTANCE Food spoilage and food poisoning caused by bacteria can have major health and economic implications for human society. With the rise in resistance to conventional antibiotics, there is a need to identify new antimicrobials to combat these outbreaks in our food supply. Here we

  11. Development of a Small Peptide Tag for Covalent Labeling of Proteins

    PubMed Central

    Tanaka, Fujie; Fuller, Roberta; Asawapornmongkol, Lily; Warsinke, Axel; Gobuty, Sarah; Barbas, Carlos F.

    2008-01-01

    A 21-mer peptide that can be used to covalently introduce synthetic molecules into proteins has been developed. Phage displayed peptide libraries were subjected to reaction-based selection with 1,3-diketones. The peptide was further evolved by addition of a randomized region and reselection for improved binding. The resulting 21-mer peptide had a reactive amino group that formed an enaminone with 1,3-diketone and was used as a tag for labeling of maltose binding protein. Using this peptide tag and 1,3-diketone derivatives, a variety of molecules such as reporter probes and functionalities may be covalently introduced into proteins of interest. PMID:17602682

  12. Peptides for functionalization of InP semiconductors.

    PubMed

    Estephan, Elias; Saab, Marie-belle; Larroque, Christian; Martin, Marta; Olsson, Fredrik; Lourdudoss, Sebastian; Gergely, Csilla

    2009-09-15

    The challenge is to achieve high specificity in molecular sensing by proper functionalization of micro/nano-structured semiconductors by peptides that reveal specific recognition for these structures. Here we report on surface modification of the InP semiconductors by adhesion peptides produced by the phage display technique. An M13 bacteriophage library has been used to screen 10(10) different peptides against the InP(001) and the InP(111) surfaces to finally isolate specific peptides for each orientation of the InP. MALDI-TOF/TOF mass spectrometry has been employed to study real affinity of the peptide towards the InP surfaces. The peptides serve for controlled placement of biotin onto InP to bind then streptavidin. Our Atomic Force Microscopy study revealed a total surface coverage of molecules when the InP surface was functionalized by its specific biotinylated peptide (YAIKGPSHFRPS). Finally, fluorescence microscopy has been employed to demonstrate the preferential attachment of the peptide onto a micro-patterned InP surface. Use of substrate specific peptides could present an alternative solution for the problems encountered in the actually existing sensing methods and molecular self-assembly due to the unwanted unspecific interactions.

  13. Perspective of Use of Antiviral Peptides against Influenza Virus

    PubMed Central

    Skalickova, Sylvie; Heger, Zbynek; Krejcova, Ludmila; Pekarik, Vladimir; Bastl, Karel; Janda, Jozef; Kostolansky, Frantisek; Vareckova, Eva; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

    2015-01-01

    The threat of a worldwide influenza pandemic has greatly increased over the past decade with the emergence of highly virulent avian influenza strains. The increased frequency of drug-resistant influenza strains against currently available antiviral drugs requires urgent development of new strategies for antiviral therapy, too. The research in the field of therapeutic peptides began to develop extensively in the second half of the 20th century. Since then, the mechanisms of action for several peptides and their antiviral prospect received large attention due to the global threat posed by viruses. Here, we discussed the therapeutic properties of peptides used in influenza treatment. Peptides with antiviral activity against influenza can be divided into three main groups. First, entry blocker peptides such as a Flupep that interact with influenza hemagglutinin, block its binding to host cells and prevent viral fusion. Second, several peptides display virucidal activity, disrupting viral envelopes, e.g., Melittin. Finally, a third set of peptides interacts with the viral polymerase complex and act as viral replication inhibitors such as PB1 derived peptides. Here, we present a review of the current literature describing the antiviral activity, mechanism and future therapeutic potential of these influenza antiviral peptides. PMID:26492266

  14. Macrocyclic peptides self-assemble into robust vesicles with molecular recognition capabilities.

    PubMed

    Jeong, Woo-jin; Lim, Yong-beom

    2014-11-19

    In this study, we developed macrocyclic peptide building blocks that formed self-assembled peptide vesicles with molecular recognition capabilities. Macrocyclic peptides were significantly different from conventional amphiphiles, in that they could self-assemble into vesicles at very high hydrophilic-to-total mass ratios. The flexibility of the hydrophobic self-assembly segment was critical for vesicle formation. The unique features of this peptide vesicle system include a homogeneous size distribution, unusually small size, and robust structural and thermal stability. The peptide vesicles successfully entrapped a hydrophilic model drug, released the payload very slowly, and were internalized by cells in a highly efficient manner. Moreover, the peptide vesicles exhibited molecular recognition capabilities, in that they selectively bound to target RNA through surface-displayed peptides. This study demonstrates that self-assembled peptide vesicles can be used as strong intracellular delivery vehicles that recognize specific biomacromolecular targets.

  15. Structure of a peptide adsorbed on graphene and graphite.

    PubMed

    Katoch, Jyoti; Kim, Sang Nyon; Kuang, Zhifeng; Farmer, Barry L; Naik, Rajesh R; Tatulian, Suren A; Ishigami, Masa

    2012-05-09

    Noncovalent functionalization of graphene using peptides is a promising method for producing novel sensors with high sensitivity and selectivity. Here we perform atomic force microscopy, Raman spectroscopy, infrared spectroscopy, and molecular dynamics simulations to investigate peptide-binding behavior to graphene and graphite. We studied a dodecamer peptide identified with phage display to possess affinity for graphite. Optical spectroscopy reveals that the peptide forms secondary structures both in powder form and in an aqueous medium. The dominant structure in the powder form is α-helix, which undergoes a transition to a distorted helical structure in aqueous solution. The peptide forms a complex reticular structure upon adsorption on graphene and graphite, having a helical conformation different from α-helix due to its interaction with the surface. Our observation is consistent with our molecular dynamics calculations, and our study paves the way for rational functionalization of graphene using biomolecules with defined structures and, therefore, functionalities.

  16. Antimicrobial Dendrimeric Peptides: Structure, Activity and New Therapeutic Applications.

    PubMed

    Scorciapino, Mariano A; Serra, Ilaria; Manzo, Giorgia; Rinaldi, Andrea C

    2017-03-03

    Microbial resistance to conventional antibiotics is one of the most outstanding medical and scientific challenges of our times. Despite the recognised need for new anti-infective agents, however, very few new drugs have been brought to the market and to the clinic in the last three decades. This review highlights the properties of a new class of antibiotics, namely dendrimeric peptides. These intriguing novel compounds, generally made of multiple peptidic sequences linked to an inner branched core, display an array of antibacterial, antiviral and antifungal activities, usually coupled to low haemolytic activity. In addition, several peptides synthesized in oligobranched form proved to be promising tools for the selective treatment of cancer cells.

  17. Hydroxyapatite-binding peptides for bone growth and inhibition

    DOEpatents

    Bertozzi, Carolyn R.; Song, Jie; Lee, Seung-Wuk

    2011-09-20

    Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

  18. Roles of Hydrophobicity and Charge Distribution of Cationic Antimicrobial Peptides in Peptide-Membrane Interactions*

    PubMed Central

    Yin, Lois M.; Edwards, Michelle A.; Li, Jessica; Yip, Christopher M.; Deber, Charles M.

    2012-01-01

    Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms, including humans, and offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In this work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in-tandem atomic force microscopy, attenuated total reflection-FTIR, CD spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane-disruptive abilities and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase in aggregated β-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKKALFALWLAFLA-NH2) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKKAAFAAWAAFAA-NH2). Redistribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, whereas those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 μm, but the four-Leu peptides induced 40–80% hemolysis at the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (minimum inhibitory concentrations of 4–32 μm). The overall findings suggest optimum routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes. PMID:22253439

  19. Updated defense display market assessment

    NASA Astrophysics Data System (ADS)

    Desjardins, Daniel D.; Hopper, Darrel G.

    1999-08-01

    This paper addresses the number, function and size of principal military displays and establishes a basis to determine the opportunities for technology insertion in the immediate future and into the next millennium. Principal military displays are defined as those occupying appreciable crewstation real-estate and/or those without which the platform could not carry out its intended mission. DoD 'office' applications are excluded from this study. The military displays market is specified by such parameters as active area and footprint size, and other characteristics such as luminance, gray scale, resolution, angle, color, video capability, and night vision imaging system compatibility. Funded, future acquisitions, planned and predicted crewstation modification kits, and form-fit upgrades are taken into account. This paper provides an overview of the DoD niche market, allowing both government and industry a necessary reference by which to meet DoD requirements for military displays in a timely and cost-effective manner. The aggregate DoD installed base for direct-view and large-area military displays is presently estimated to be in excess of 313,000. Miniature displays are those which must be magnified to be viewed, involve a significantly different manufacturing paradigm and are used in helmet mounted displays and thermal weapon sight applications. Some 114,000 miniature displays are presently included within future weapon system acquisition plans. For vendor production planning purposes it is noted that foreign military sales could substantially increase these quantities. The vanishing vendor syndrome (VVS) for older display technologies continues to be a growing, pervasive problem throughout DoD, which consequently must leverage the more modern, especially flat panel, display technologies being developed to replace older, especially cathode ray tube, technology for civil-commercial markets. Total DoD display needs (FPD, HMD) are some 427,000.

  20. Antigen discovery using whole-genome phage display libraries.

    PubMed

    Beghetto, Elisa; Gargano, Nicola

    2013-01-01

    In the last two decades phage display technology has been used for investigating complex biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical cloning and expression system, has also been exploited for generating display libraries of small peptides and protein domains. More recently, large cDNA and whole-genome lambda-display libraries of human pathogens have been generated for the discovery of new antigens for biomedical applications. Here, we describe the construction of a whole-genome library of a common pathogen-Streptococcus pneumoniae-and the use of this library for the molecular dissection of the human B-cell response against bacterial infection and colonization.

  1. Peptide Based Radiopharmaceuticals: Specific Construct Approach

    SciTech Connect

    Som, P; Rhodes, B A; Sharma, S S

    1997-10-21

    The objective of this project was to develop receptor based peptides for diagnostic imaging and therapy. A series of peptides related to cell adhesion molecules (CAM) and immune regulation were designed for radiolabeling with 99mTc and evaluated in animal models as potential diagnostic imaging agents for various disease conditions such as thrombus (clot), acute kidney failure, and inflection/inflammation imaging. The peptides for this project were designed by the industrial partner, Palatin Technologies, (formerly Rhomed, Inc.) using various peptide design approaches including a newly developed rational computer assisted drug design (CADD) approach termed MIDAS (Metal ion Induced Distinctive Array of Structures). In this approach, the biological function domain and the 99mTc complexing domain are fused together so that structurally these domains are indistinguishable. This approach allows construction of conformationally rigid metallo-peptide molecules (similar to cyclic peptides) that are metabolically stable in-vivo. All the newly designed peptides were screened in various in vitro receptor binding and functional assays to identify a lead compound. The lead compounds were formulated in a one-step 99mTc labeling kit form which were studied by BNL for detailed in-vivo imaging using various animals models of human disease. Two main peptides usingMIDAS approach evolved and were investigated: RGD peptide for acute renal failure and an immunomodulatory peptide derived from tuftsin (RMT-1) for infection/inflammation imaging. Various RGD based metallopeptides were designed, synthesized and assayed for their efficacy in inhibiting ADP-induced human platelet aggregation. Most of these peptides displayed biological activity in the 1-100 µM range. Based on previous work by others, RGD-I and RGD-II were evaluated in animal models of acute renal failure. These earlier studies showed that after acute ischemic injury the renal cortex displays

  2. Development of peptides as potential drugs for cancer therapy.

    PubMed

    Li, Zhi Jie; Cho, Chi Hin

    2010-01-01

    The development of more selective agents focused on targeted delivery of imaging probes and drugs to different tumor sites is the current trend in cancer diagnosis and therapies. Peptides are small amino acid sequences that can be isolated to bind to a predetermined target and are potentially capable of interfering with its function. These specific peptides isolated can inhibit individual signaling components, which are essential in cancer development and progression. Phage display is a powerful technology for selecting and cloning peptides displayed on the surface of bacteriophage. Billionclone-peptide libraries can be rapidly and simultaneously selected by phage biopanning, leading to large numbers of hits. Although peptides account for only a small part of current therapeutic agents, their potential is being improved by new technologies affecting their modification, delivery, stability and their application in preclinical settings. This review will highlight how to isolate peptides that target pivotal molecules in cancer development and progression through phage library biopanning and how to modify these peptides to enhance their anticancer efficacy.

  3. Reassociation with beta 2-microglobulin is necessary for Kb class I major histocompatibility complex binding of exogenous peptides.

    PubMed Central

    Rock, K L; Rothstein, L E; Gamble, S R; Benacerraf, B

    1990-01-01

    T lymphocytes recognize endogenously produced antigenic peptides in association with major histocompatibility complex (MHC)-encoded molecules. Peptides from the extracellular fluid can be displayed in association with class I and class II MHC molecules. Here we report that mature Kb class I MHC molecules bind peptides upon dissociation and reassociation of their light chain. Intact Kb heterodimers, unlike class II MHC molecules, are relatively unreceptive to binding peptides. This property may maintain segregation of class I and class II MHC-restricted peptides and has implications for the use of peptides as vaccines. Images PMID:2217182

  4. You Be the Judge: Display.

    ERIC Educational Resources Information Center

    Koeninger, Jimmy G.

    The instructional package was developed to provide the distributive education teacher-coordinator with visual materials that can be used to supplement existing textbook offerings in the area of display (visual merchandising). Designed for use with 35mm slides of retail store displays, the package allows the student to view the slides of displays…

  5. Flexible Bistable Cholesteric Reflective Displays

    NASA Astrophysics Data System (ADS)

    Yang, Deng-Ke

    2006-03-01

    Cholesteric liquid crystals (ChLCs) exhibit two stable states at zero field condition-the reflecting planar state and the nonreflecting focal conic state. ChLCs are an excellent candidate for inexpensive and rugged electronic books and papers. This paper will review the display cell structure,materials and drive schemes for flexible bistable cholesteric (Ch) reflective displays.

  6. Displays: Entering a New Dimension

    ERIC Educational Resources Information Center

    Starkman, Neal

    2007-01-01

    As display technologies prepare to welcome 3-D, the 21st-century classroom will soon bear little resemblance to anything students and teachers have ever seen. In this article, the author presents the latest innovations in the world of digital display technology. These include: (1) Touchlight, an interactive touch screen program that takes a normal…

  7. Discovery of a 9-mer Cationic Peptide (LTX-315) as a Potential First in Class Oncolytic Peptide.

    PubMed

    Haug, Bengt Erik; Camilio, Ketil André; Eliassen, Liv Tone; Stensen, Wenche; Svendsen, John Sigurd; Berg, Kristel; Mortensen, Bjarte; Serin, Guillaume; Mirjolet, Jean-Francois; Bichat, Francis; Rekdal, Øystein

    2016-04-14

    Oncolytic immunotherapies represent a new promising strategy in the treatment of cancer. In our efforts to develop oncolytic peptides, we identified a series of chemically modified 9-mer cationic peptides that were highly effective against both drug-resistant and drug-sensitive cancer cells and with lower toxicity toward normal cells. Among these peptides, LTX-315 displayed superior anticancer activity and was selected as a lead candidate. This peptide showed relative high plasma protein binding abilities and a human plasma half-life of 160 min, resulting in formation of nontoxic metabolites. In addition, the lead candidate demonstrated relatively low ability to inhibit CYP450 enzymes. Collectively these data indicated that this peptide has potential to be developed as a new anticancer agent for intratumoral administration and is currently being evaluated in a phase I/IIa study.

  8. Peptide binding properties of the three PDZ domains of Bazooka (Drosophila Par-3).

    PubMed

    Yu, Cao Guo; Tonikian, Raffi; Felsensteiner, Corinna; Jhingree, Jacquelyn R; Desveaux, Darrell; Sidhu, Sachdev S; Harris, Tony J C

    2014-01-01

    The Par complex is a conserved cell polarity regulator. Bazooka/Par-3 is scaffold for the complex and contains three PDZ domains in tandem. PDZ domains can act singly or synergistically to bind the C-termini of interacting proteins. Sequence comparisons among Drosophila Baz and its human and C. elegans Par-3 counterparts indicate a divergence of the peptide binding pocket of PDZ1 and greater conservation for the pockets of PDZ2 and PDZ3. However, it is unclear whether the domains from different species share peptide binding preferences, or if their tandem organization affects their peptide binding properties. To investigate these questions, we first used phage display screens to identify unique peptide binding profiles for each single PDZ domain of Baz. Comparisons with published phage display screens indicate that Baz and C. elegans PDZ2 bind to similar peptides, and that the peptide binding preferences of Baz PDZ3 are more similar to C. elegans versus human PDZ3. Next we quantified the peptide binding preferences of each Baz PDZ domain using single identified peptides in surface plasmon resonance assays. In these direct binding studies, each peptide had a binding preference for a single PDZ domain (although the peptide binding of PDZ2 was weakest and the least specific). PDZ1 and PDZ3 bound their peptides with dissociation constants in the nM range, whereas PDZ2-peptide binding was in the µM range. To test whether tandem PDZ domain organization affects peptide binding, we examined a fusion protein containing all three PDZ domains and their normal linker regions. The binding strengths of the PDZ-specific peptides to single PDZ domains and to the PDZ domain tandem were indistinguishable. Thus, the peptide binding pockets of each PDZ domain in Baz are not obviously affected by the presence of neighbouring PDZ domains, but act as isolated modules with specific in vitro peptide binding preferences.

  9. Antimicrobial Peptides in Reptiles

    PubMed Central

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  10. The natriuretic peptides.

    PubMed

    Baxter, Gary F

    2004-03-01

    The natriuretic peptides are a family of widely distributed, but evolutionarily conserved, polypeptide mediators that exert a range of actions throughout the body. In cardiovascular homeostasis, the endocrine roles of the cardiac-derived atrial and B-type natriuretic peptide (ANP and BNP) in regulating central fluid volume and blood pressure have been recognised for two decades. However, there is a growing realisation that natriuretic peptide actions go far beyond their volume regulating effects. These pleiotropic actions include local (autocrine/paracrine) regulatory actions of ANP and BNP within the heart, and of another natriuretic peptide, CNP, within the vessel wall. Effects on function and growth of the local tissue environment are likely to be of great importance, especially in disease states where tissue and circulating levels of ANP and BNP rise markedly. At present, the relevance of other natriuretic peptides (notably uroguanylin and DNP) to human physiology and pathology remain uncertain. Other articles in this issue of Basic Research in Cardiology review the molecular physiology of natriuretic peptide signalling, with a particular emphasis on the lessons from genetically targetted mice; the vascular activity of natriuretic peptides; the regulation and roles of natriuretic peptides in ischaemic myocardium; and the diagnostic, prognostic and therapeutic roles of natriuretic peptides in heart failure.

  11. Three-dimensional display technologies.

    PubMed

    Geng, Jason

    2013-01-01

    The physical world around us is three-dimensional (3D), yet traditional display devices can show only two-dimensional (2D) flat images that lack depth (i.e., the third dimension) information. This fundamental restriction greatly limits our ability to perceive and to understand the complexity of real-world objects. Nearly 50% of the capability of the human brain is devoted to processing visual information [Human Anatomy & Physiology (Pearson, 2012)]. Flat images and 2D displays do not harness the brain's power effectively. With rapid advances in the electronics, optics, laser, and photonics fields, true 3D display technologies are making their way into the marketplace. 3D movies, 3D TV, 3D mobile devices, and 3D games have increasingly demanded true 3D display with no eyeglasses (autostereoscopic). Therefore, it would be very beneficial to readers of this journal to have a systematic review of state-of-the-art 3D display technologies.

  12. INFORMATION DISPLAY: CONSIDERATIONS FOR DESIGNING COMPUTER-BASED DISPLAY SYSTEMS.

    SciTech Connect

    O'HARA,J.M.; PIRUS,D.; BELTRATCCHI,L.

    2004-09-19

    This paper discussed the presentation of information in computer-based control rooms. Issues associated with the typical displays currently in use are discussed. It is concluded that these displays should be augmented with new displays designed to better meet the information needs of plant personnel and to minimize the need for interface management tasks (the activities personnel have to do to access and organize the information they need). Several approaches to information design are discussed, specifically addressing: (1) monitoring, detection, and situation assessment; (2) routine task performance; and (3) teamwork, crew coordination, collaborative work.

  13. Flat panel display Impurity doping technology for flat panel displays

    NASA Astrophysics Data System (ADS)

    Suzuki, Toshiharu

    2005-08-01

    Features of the flat panel displays (FPDs) such as liquid crystal display (LCD) and organic light emitting diode (OLED) display, etc. using low temperature poly-Si (LTPS) thin film transistors (TFTs) are briefly reviewed comparing with other FPDs. The requirements for fabricating TFTs used for high performance FPDs and system on glass (SoG) are addressed. This paper focuses on the impurity doping technology, which is one of the key technologies together with crystallization by laser annealing, formation of high quality gate insulator and gate-insulator/poly-Si interface. The issues to be solved in impurity doping technology for state of the art and future TFTs are clarified.

  14. Phage display: development of nanocarriers for targeted drug delivery to the brain

    PubMed Central

    Bakhshinejad, Babak; Karimi, Marzieh; Khalaj-Kondori, Mohammad

    2015-01-01

    The blood brain barrier represents a formidable obstacle for the transport of most systematically administered neurodiagnostics and neurotherapeutics to the brain. Phage display is a high throughput screening strategy that can be used for the construction of nanomaterial peptide libraries. These libraries can be screened for finding brain targeting peptide ligands. Surface functionalization of a variety of nanocarriers with these brain homing peptides is a sophisticated way to develop nanobiotechnology-based drug delivery platforms that are able to cross the blood brain barrier. These efficient drug delivery systems raise our hopes for the diagnosis and treatment of various brain disorders in the future. PMID:26199590

  15. Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing

    PubMed Central

    Mehetre, Gajanan T.; Paranjpe, Aditi; Dastager, Syed G.

    2016-01-01

    Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche. PMID:26950332

  16. Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

    PubMed

    Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N

    2015-02-03

    The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

  17. Applications of Circular Dichroism for Structural Analysis of Gelatin and Antimicrobial Peptides

    PubMed Central

    Gopal, Ramamourthy; Park, Jin Soon; Seo, Chang Ho; Park, Yoonkyung

    2012-01-01

    Circular dichroism (CD) is a useful technique for monitoring changes in the conformation of antimicrobial peptides or gelatin. In this study, interactions between cationic peptides and gelatin were observed without affecting the triple helical content of the gelatin, which was more strongly affected by anionic surfactant. The peptides did not adopt a secondary structure in the presence of aqueous solution or Tween 80, but a peptide secondary structure formed upon the addition of sodium dodecyl sulfate (SDS). The peptides bound to the phosphate group of lipopolysaccharide (LPS) and displayed an alpha-helical conformation while (KW)4 adopted a folded conformation. Further, the peptides did not specifically interact with the fungal cell wall components of mannan or laminarin. Tryptophan blue shift assay indicated that these peptides interacted with SDS, LPS, and gelatin but not with Tween 80, mannan, or laminarin. The peptides also displayed antibacterial activity against P. aeruginosa without cytotoxicity against HaCaT cells at MIC, except for HPA3NT3-analog peptide. In this study, we used a CD spectroscopic method to demonstrate the feasibility of peptide characterization in numerous environments. The CD method can thus be used as a screening method of gelatin-peptide interactions for use in wound healing applications. PMID:22489150

  18. Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display

    PubMed Central

    Guo, Nini; Duan, Hongying; Kachko, Alla; Krause, Benjamin W.; Major, Marian E.; Krause, Philip R.

    2015-01-01

    Vaccine reverse engineering is emerging as an important approach to vaccine antigen identification, recently focusing mainly on structural characterization of interactions between neutralizing monoclonal antibodies (mAbs) and antigens. Using mAbs that bind unknown antigen structures, we sought to probe the intrinsic features of antibody antigen-binding sites with a high complexity peptide library, aiming to identify conformationally optimized mimotope antigens that capture mAb-specific epitopes. Using a high throughput sequencing-enhanced messenger ribonucleic acid (mRNA) display approach, we identified high affinity binding peptides for a hepatitis C virus neutralizing mAb. Immunization with the selected peptides induced neutralizing activity similar to that of the original mAb. Antibodies elicited by the most commonly selected peptides were predominantly against specific epitopes. Thus, using mRNA display to interrogate mAbs permits high resolution identification of functional peptide antigens that direct targeted immune responses, supporting its use in vaccine reverse engineering for pathogens against which potent neutralizing mAbs are available. Research in Context We used a large number of randomly produced small proteins (“peptides”) to identify peptides containing specific protein sequences that bind efficiently to an antibody that can prevent hepatitis C virus infection in cell culture. After the identified peptides were injected into mice, the mice produced their own antibodies with characteristics similar to the original antibody. This approach can provide previously unavailable information about antibody binding and could also be useful in developing new vaccines. PMID:26425692

  19. High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley.

    PubMed

    Hofinger, Bernhard J; Jing, Hai-Chun; Hammond-Kosack, Kim E; Kanyuka, Kostya

    2009-09-01

    An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species.

  20. Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer.

    PubMed

    Etemadmoghadam, Dariush; George, Joshy; Cowin, Prue A; Cullinane, Carleen; Kansara, Maya; Gorringe, Kylie L; Smyth, Gordon K; Bowtell, David D L

    2010-11-12

    Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.

  1. Amplicon-Dependent CCNE1 Expression Is Critical for Clonogenic Survival after Cisplatin Treatment and Is Correlated with 20q11 Gain in Ovarian Cancer

    PubMed Central

    Etemadmoghadam, Dariush; George, Joshy; Cowin, Prue A.; Cullinane, Carleen; Kansara, Maya; Gorringe, Kylie L.; Smyth, Gordon K.; Bowtell, David D. L.

    2010-01-01

    Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers. PMID:21103391

  2. Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing.

    PubMed

    Delforno, Tiago Palladino; Lacerda Júnior, Gileno Vieira; Noronha, Melline F; Sakamoto, Isabel K; Varesche, Maria Bernadete A; Oliveira, Valéria M

    2017-02-23

    The 16S rRNA gene amplicon and whole-genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide-spectrum profiles of microbial composition and metabolic diversity from a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one-carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin).

  3. Amplicon-based profiling of bacteria in raw and secondary treated wastewater from treatment plants across Australia.

    PubMed

    Ahmed, Warish; Staley, Christopher; Sidhu, Jatinder; Sadowsky, Michael; Toze, Simon

    2017-02-01

    In this study, we investigated the use of Illumina high-throughput sequencing of 16S ribosomal RNA (rRNA) amplicons to explore microbial diversity and community structure in raw and secondary treated wastewater (WW) samples from four municipal wastewater treatment plants (WWTPs A-D) across Australia. Sequence reads were analyzed to determine the abundance and diversity of bacterial communities in raw and secondary treated WW samples across the four WWTPs. In addition, sequence reads were also characterized to phenotypic features and to estimate the abundance of potential pathogenic bacterial genera and antibiotic-resistant genes in total bacterial communities. The mean coverage, Shannon diversity index, observed richness (S obs), and abundance-based coverage estimate (ACE) of richness for raw and secondary treated WW samples did not differ significantly (P > 0.05) among the four WWTPs examined. Generally, raw and secondary treated WW samples were dominated by members of the genera Pseudomonas, Arcobacter, and Bacteroides. Evaluation of source contributions to secondary treated WW, done using SourceTracker, revealed that 8.80-61.4% of the bacterial communities in secondary treated WW samples were attributed to raw WW. Twenty-five bacterial genera were classified as containing potential bacterial pathogens. The abundance of potentially pathogenic genera in raw WW samples was higher than that found in secondary treated WW samples. Among the pathogenic genera identified, Pseudomonas and Arcobacter had the greatest percentage of the sequence reads. The abundances of antibiotic resistance genes were generally low (<0.5%), except for genes encoding ABC transporters, which accounted for approximately 3% of inferred genes. These findings provided a comprehensive profile of bacterial communities, including potential bacterial pathogens and antibiotic-resistant genes, in raw and secondary treated WW samples from four WWTPs across Australia and demonstrated that Illumina

  4. Use of propidium monoazide and increased amplicon length reduce false-positive signals in quantitative PCR for bioburden analysis.

    PubMed

    Schnetzinger, Franz; Pan, Youwen; Nocker, Andreas

    2013-03-01

    Rapid microbiological methods (RMMs) as an alternative to conventional cultivation-based bioburden analysis are receiving increasing attention although no single technology is currently able to satisfy the needs of the health care industry. Among the RMMs, quantitative PCR (qPCR) seems particularly suited. Its implementation is, however, hampered by false-positive signals originating from free DNA in PCR reagents or from dead cells in the samples to be analysed. In this study, we assessed the capability of propidium monoazide (PMA) to inactivate exogenous DNA in PCR reagents and thus to minimise its impact in bioburden analysis. PMA is a membrane-impermeant dye that intercalates into DNA and covalently binds to it upon photoactivation leading to strong inhibition of PCR amplification. PMA is currently used mainly for treatment of microbiological samples to exclude signals from membrane-compromised cells, but is also very useful for suppression of exogenous DNA signals. In addition to testing the effect of different PMA concentrations on non-template controls and target DNA, we demonstrate the effect of amplicon length on the exclusion of background amplification. Targeting a 1,108-bp 16S rRNA gene fragment using universal bacterial primers and PCR reagents treated with 5 μM PMA resulted in complete suppression of signals from exogenous DNA within 50 cycles of amplification, while a limit of detection of 10 copies of Escherichia coli genomic DNA per PCR reaction was achieved. A combined PMA treatment of sample and PCR reagents furthermore improved the selective detection of live cells making this method appear a highly attractive RMM.

  5. Nuclear species-diagnostic SNP markers mined from 454 amplicon sequencing reveal admixture genomic structure of modern citrus varieties.

    PubMed

    Curk, Franck; Ancillo, Gema; Ollitrault, Frédérique; Perrier, Xavier; Jacquemoud-Collet, Jean-Pierre; Garcia-Lor, Andres; Navarro, Luis; Ollitrault, Patrick

    2015-01-01

    Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha) with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP) markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105) were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species-diagnostic SNP

  6. Uncultured bacterial diversity in a seawater recirculating aquaculture system revealed by 16S rRNA gene amplicon sequencing.

    PubMed

    Lee, Da-Eun; Lee, Jinhwan; Kim, Young-Mog; Myeong, Jeong-In; Kim, Kyoung-Ho

    2016-04-01

    Bacterial diversity in a seawater recirculating aquaculture system (RAS) was investigated using 16S rRNA amplicon sequencing to understand the roles of bacterial communities in the system. The RAS was operated at nine different combinations of temperature (15°C, 20°C, and 25°C) and salinity (20‰, 25‰, and 32.5‰). Samples were collected from five or six RAS tanks (biofilters) for each condition. Fifty samples were analyzed. Proteobacteria and Bacteroidetes were most common (sum of both phyla: 67.2% to 99.4%) and were inversely proportional to each other. Bacteria that were present at an average of ≥ 1% included Actinobacteria (2.9%) Planctomycetes (2.0%), Nitrospirae (1.5%), and Acidobacteria (1.0%); they were preferentially present in packed bed biofilters, mesh biofilters, and maturation biofilters. The three biofilters showed higher diversity than other RAS tanks (aerated biofilters, floating bed biofilters, and fish tanks) from phylum to operational taxonomic unit (OTU) level. Samples were clustered into several groups based on the bacterial communities. Major taxonomic groups related to family Rhodobacteraceae and Flavobacteriaceae were distributed widely in the samples. Several taxonomic groups like [Saprospiraceae], Cytophagaceae, Octadecabacter, and Marivita showed a cluster-oriented distribution. Phaeobacter and Sediminicola-related reads were detected frequently and abundantly at low temperature. Nitrifying bacteria were detected frequently and abundantly in the three biofilters. Phylogenetic analysis of the nitrifying bacteria showed several similar OTUs were observed widely through the biofilters. The diverse bacterial communities and the minor taxonomic groups, except for Proteobacteria and Bacteroidetes, seemed to play important roles and seemed necessary for nitrifying activity in the RAS, especially in packed bed biofilters, mesh biofilters, and maturation biofilters.

  7. Nuclear Species-Diagnostic SNP Markers Mined from 454 Amplicon Sequencing Reveal Admixture Genomic Structure of Modern Citrus Varieties

    PubMed Central

    Curk, Franck; Ancillo, Gema; Ollitrault, Frédérique; Perrier, Xavier; Jacquemoud-Collet, Jean-Pierre; Garcia-Lor, Andres; Navarro, Luis; Ollitrault, Patrick

    2015-01-01

    Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha) with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP) markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105) were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species-diagnostic SNP

  8. Amplicon-based taxonomic characterization of bacteria in urban and peri-urban roof-harvested rainwater stored in tanks.

    PubMed

    Ahmed, W; Staley, C; Hamilton, K A; Beale, D J; Sadowsky, M J; Toze, S; Haas, C N

    2017-01-15

    Overall, 26% of Australian households use rainwater tanks as a source of potable and nonpotable water. Limited information is available on the total bacterial communities in tank water. Therefore, identification of dominant bacterial communities, diversity, and their distribution is important in understanding the microbial quality of tank water. In this study, the abundance and diversity of bacterial communities in 88 tank water samples collected from the urban areas of Brisbane (n=44) and the peri-urban center of Currumbin (n=44) in Southeast Queensland, Australia were determined using amplicon-based Illumina next-generation sequencing. In addition, the SourceTracker program was used to identify the sources of fecal contamination in tank water samples. Sequence reads were also analyzed to detect potential bacterial pathogenic genera in the tank water samples collected. Differences in sample coverage, alpha diversity, and richness did not differ significantly between the Brisbane and Currumbin tank water samples. Comamonadaceae and Planctomycetaceae were the most abundant families in all tank water samples. Curvibacter was the most abundant genus in all tank water samples. SourceTracker revealed that around 34% (Brisbane) and 43% (Currumbin) of tank water samples had a signature for bird fecal contamination. The potential opportunistic pathogenic genera including Burkholderia, Chromobacterium, Clostridium, Legionella, Mycobacterium, Nocardia, and Pseudomonas were most prevalent in tank water samples. Next-generation sequencing can be used as an initial screening tool to identify a wide array of potential pathogenic genera in tank water samples followed by quantifying specific pathogen(s) of interest using more sensitive molecular assays such as quantitative PCR (qPCR).

  9. Responses of soil microeukaryotic communities to short-term fumigation-incubation revealed by MiSeq amplicon sequencing

    PubMed Central

    Chen, Lin; Xu, Jianming; Feng, Youzhi; Wang, Juntao; Yu, Yongjie; Brookes, Philip C.

    2015-01-01

    In soil microbiology, there is a “paradox” of soil organic carbon (SOC) mineralization, which is that even though chloroform fumigation destroys majority of the soil microbial biomass, SOC mineralization continues at the same rate as in the non-fumigated soil during the incubation period. Soil microeukaryotes as important SOC decomposers, however, their community-level responses to chloroform fumigation are not well understood. Using the 18S rRNA gene amplicon sequencing, we analyzed the composition, diversity, and C-metabolic functions of a grassland soil and an arable soil microeukaryotic community in response to fumigation followed by a 30-day incubation. The grassland and arable soil microeukaryotic communities were dominated by the fungal Ascomycota (80.5–93.1% of the fungal sequences), followed by the protistan Cercozoa and Apicomplexa. In the arable soil fungal community, the predominance of the class Sordariomycetes was replaced by the class Eurotiomycetes after fumigation at days 7 and 30 of the incubation. Fumigation changed the microeukaryotic α-diversity in the grassland soil at days 0 and 7, and β-diversity in the arable soil at days 7 and 30. Network analysis indicated that after fumigation fungi were important groups closely related to other taxa. Most phylotypes (especially Sordariomycetes, Dothideomycetes, Coccidia, and uncultured Chytridiomycota) were inhibited, and only a few were positively stimulated by fumigation. Despite the inhibited Sordariomycetes, the fumigated communities mainly consisted of Eurotiomycetes and Sordariomycetes (21.9 and 36.5% relative frequency, respectively), which are able to produce hydrolytic enzymes associated with SOC mineralization. Our study suggests that fumigation not only decreases biomass size, but modulates the composition and diversity of the soil microeukaryotic communities, which are capable of driving SOC mineralization by release of hydrolytic enzymes during short-term fumigation-incubation. PMID

  10. Probing ADAMTS13 substrate specificity using phage display.

    PubMed

    Desch, Karl C; Kretz, Colin; Yee, Andrew; Gildersleeve, Robert; Metzger, Kristin; Agrawal, Nidhi; Cheng, Jane; Ginsburg, David

    2015-01-01

    Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2' and P11', for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13-VWF exosite interactions outside of VWF73.

  11. Tone compatibility between HDR displays

    NASA Astrophysics Data System (ADS)

    Bist, Cambodge; Cozot, Rémi; Madec, Gérard; Ducloux, Xavier

    2016-09-01

    High Dynamic Range (HDR) is the latest trend in television technology and we expect an in ux of HDR capable consumer TVs in the market. Initial HDR consumer displays will operate on a peak brightness of about 500-1000 nits while in the coming years display peak brightness is expected to go beyond 1000 nits. However, professionally graded HDR content can range from 1000 to 4000 nits. As with Standard Dynamic Range (SDR) content, we can expect HDR content to be available in variety of lighting styles such as low key, medium key and high key video. This raises concerns over tone-compatibility between HDR displays especially when adapting to various lighting styles. It is expected that dynamic range adaptation between HDR displays uses similar techniques as found with tone mapping and tone expansion operators. In this paper, we survey simple tone mapping methods of 4000 nits color-graded HDR content for 1000 nits HDR displays. We also investigate tone expansion strategies when HDR content graded in 1000 nits is displayed on 4000 nits HDR monitors. We conclude that the best tone reproduction technique between HDR displays strongly depends on the lighting style of the content.

  12. Lizard threat display handicaps endurance.

    PubMed Central

    Brandt, Y

    2003-01-01

    Honest-signalling theory asserts that threat displays reliably advertise attributes that influence fighting success. Endurance, as measured by treadmill performance, predicts the outcome of agonistic interactions among lizards. If threat displays in lizards function to advertise endurance capacity then variation in threat displays should correlate with endurance. I tested this prediction for the duration of threat posturing in male side-blotched lizards (Uta stansburiana) and examined whether threat displays act as quality handicaps, reliable signals that expend the attribute that is advertised. Individual variation in the duration of threat posturing correlated with endurance, while an experimental reduction of endurance diminished the duration of threat posturing. As expected of a quality handicap, endurance fell below baseline after display production. A restriction of aerobic metabolism can account for this effect. In threat posturing, lateral compression of the thorax may interfere with respiration or with circulation, limiting aerobic metabolism and causing a compensatory increase in anaerobic metabolism, thereby generating lactate and diminishing locomotor capacity. Concentrations of lactate measured after display production were higher than baseline, consistent with the proposed mechanism. By restricting aerobic metabolism, the threat posture can act as a quality handicap, simultaneously advertising and expending the endurance capacity of displaying lizards. PMID:12803896

  13. Texture-Based Correspondence Display

    NASA Technical Reports Server (NTRS)

    Gerald-Yamasaki, Michael

    2004-01-01

    Texture-based correspondence display is a methodology to display corresponding data elements in visual representations of complex multidimensional, multivariate data. Texture is utilized as a persistent medium to contain a visual representation model and as a means to create multiple renditions of data where color is used to identify correspondence. Corresponding data elements are displayed over a variety of visual metaphors in a normal rendering process without adding extraneous linking metadata creation and maintenance. The effectiveness of visual representation for understanding data is extended to the expression of the visual representation model in texture.

  14. Circular displays: control/display arrangements and stereotype strength with eight different display locations.

    PubMed

    Chan, Alan H S; Hoffmann, Errol R

    2015-01-01

    Two experiments are reported that were designed to investigate control/display arrangements having high stereotype strengths when using circular displays. Eight display locations relative to the operator and control were tested with rotational and translational controls situated on different planes according to the Frame of Reference Transformation Tool (FORT) model of Wickens et al. (2010). (Left. No, Right! Development of the Frame of Reference Transformation Tool (FORT), Proceedings of the Human Factors and Ergonomics Society 54th Annual Meeting, 54: 1022-1026). In many cases, there was little effect of display locations, indicating the importance of the Worringham and Beringer (1998. Directional stimulus-response compatibility: a test of three alternative principles. Ergonomics, 41(6), 864-880) Visual Field principle and an extension of this principle for rotary controls (Hoffmann and Chan (2013). The Worringham and Beringer 'visual field' principle for rotary controls. Ergonomics, 56(10), 1620-1624). The initial indicator position (12, 3, 6 and 9 o'clock) had a major effect on control/display stereotype strength for many of the six controls tested. Best display/control arrangements are listed for each of the different control types (rotational and translational) and for the planes on which they are mounted. Data have application where a circular display is used due to limited display panel space and applies to space-craft, robotics operators, hospital equipment and home appliances. Practitioner Summary: Circular displays are often used when there is limited space available on a control panel. Display/control arrangements having high stereotype strength are listed for four initial indicator positions. These arrangements are best for design purposes.

  15. Transmissible gastroenteritis virus; identification of M protein-binding peptide ligands with antiviral and diagnostic potential

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and ...

  16. Peptide bioregulators inhibit apoptosis.

    PubMed

    Khavinson, V K; Kvetnoii, I M

    2000-12-01

    The effects of peptide bioregulators epithalon and vilon on the dynamics of irradiation-induced apoptotic death of spleen lymphocytes in rats indicate that these agents inhibit physiologically programmed cell death. The antiapoptotic effect of vilon was more pronounced, which corroborates the concept on tissue-specific effect of peptide bioregulators.

  17. Bacteriocin Inducer Peptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  18. Antimicrobial Peptides from Fish

    PubMed Central

    Masso-Silva, Jorge A.; Diamond, Gill

    2014-01-01

    Antimicrobial peptides (AMPs) are found widely distributed through Nature, and participate in the innate host defense of each species. Fish are a great source of these peptides, as they express all of the major classes of AMPs, including defensins, cathelicidins, hepcidins, histone-derived peptides, and a fish-specific class of the cecropin family, called piscidins. As with other species, the fish peptides exhibit broad-spectrum antimicrobial activity, killing both fish and human pathogens. They are also immunomodulatory, and their genes are highly responsive to microbes and innate immuno-stimulatory molecules. Recent research has demonstrated that some of the unique properties of fish peptides, including their ability to act even in very high salt concentrations, make them good potential targets for development as therapeutic antimicrobials. Further, the stimulation of their gene expression by exogenous factors could be useful in preventing pathogenic microbes in aquaculture. PMID:24594555

  19. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

    PubMed Central

    Conway, J E; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1997-01-01

    Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate

  20. Phage Selection of Chemically Stabilized α-Helical Peptide Ligands.

    PubMed

    Diderich, Philippe; Bertoldo, Davide; Dessen, Pierre; Khan, Maola M; Pizzitola, Irene; Held, Werner; Huelsken, Joerg; Heinis, Christian

    2016-05-20

    Short α-helical peptides stabilized by linkages between constituent amino acids offer an attractive format for ligand development. In recent years, a range of excellent ligands based on stabilized α-helices were generated by rational design using α-helical peptides of natural proteins as templates. Herein, we developed a method to engineer chemically stabilized α-helical ligands in a combinatorial fashion. In brief, peptides containing cysteines in position i and i + 4 are genetically encoded by phage display, the cysteines are modified with chemical bridges to impose α-helical conformations, and binders are isolated by affinity selection. We applied the strategy to affinity mature an α-helical peptide binding β-catenin. We succeeded in developing ligands with Kd's as low as 5.2 nM, having >200-fold improved affinity. The strategy is generally applicable for affinity maturation of any α-helical peptide. Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, as no unnatural amino acids are required and the cyclization reaction is more eff