Secretion of Thioredoxin Peroxidase Protein of Cat Liver Fluke Opisthorchis felineus during Modeling of Experimental Opisthorchiasis.

Mechanisms of thioredoxin peroxidase secretion by Opisthorchis felineus were studied in vivo and in vitro. Specific antibodies were obtained and used for western blotting and immunohistochemical detection in Syrian hamster model of opisthorchiasis. Secreted thioredoxin peroxidase protein was accumulated in the worm incubation medium under conditions of oxidative stress and in bile duct cells of hamsters with chronic opisthorchiasis.

Double-targeted polymersomes and liposomes for multiple barrier crossing.

In order to treat metastasis in the brain, drug delivery systems must overcome multiple physical barriers between the point of administration and the target, such as the Blood-brain barrier, that hinder their free access across them. Multiple targeting approaches arise as a promising alternative to this barrier and target certain tissues inside the brain at a time. Herein, two surface modification methods are presented to obtain dual-targeted vesicle-like carriers functionalized with an MCF-7-specific phage protein and a BBB-specific peptide, providing the system the ability to cross a BBB model, target breast cancer cells and deliver its payload. The aim of this study was to compare new designed polymersomes with liposomes, a well-established delivery vehicle, in terms of drug loading, targeting, release and tumor cell killing. The bilayer structure of both systems allowed the conjugation with different ligands both by insertion and covalent binding. Different behaviour was observed in release, uptake and tumor cell killing corresponding to differences in membrane permeability of both vehicles and type of targeting and ligands' combination. Preliminary results showed that both formulations were able to cross the BBB monolayer without harming it, showing cytotoxic activity in the abluminal compartment.

[The reagents kit to detect Metyorchis biis, Opisthorchis viverrini and Clonorchis sinensis, Opisthorchis felineus--agents of opisthorchiasis using technique of polymerase chain reaction in real-time].

The helminths Opisthorchis felineus, Opisthorchis viverrini, Clonorchis sinensis, Metorchis bilis are the agents of opisthorchiasis. The actual diagnostic of parasitic diseases based on microscope analysis of samples of human feces to detect presence of ova of parasites suffers of many shortcomings, in particular low sensitivity especially at earlier stages. The purpose of this study was to compare results of detection of parasites using both classical technique and technique of specific differentiation based on extraction of nucleic acids from samples of human feces and implementation of reaction of amplification of the chosen fragment of DNA with detection of products of polymerase chain reaction in the real time. The study detected 150 out of 165 positive samples and also 6 out of 37 negative samples both validated by coproovoscopy.

Phage protein-targeted cancer nanomedicines.

Nanoencapsulation of anticancer drugs improves their therapeutic indices by virtue of the enhanced permeation and retention effect which achieves passive targeting of nanoparticles in tumors. This effect can be significantly enhanced by active targeting of nanovehicles to tumors. Numerous ligands have been proposed and used in various studies with peptides being considered attractive alternatives to antibodies. This is further reinforced by the availability of peptide phage display libraries which offer an unlimited reservoir of target-specific probes. In particular landscape phages with multivalent display of target-specific peptides which enable the phage particle itself to become a nanoplatform creates a paradigm for high throughput selection of nanoprobes setting the stage for personalized cancer management. Despite its promise, this conjugate of combinatorial chemistry and nanotechnology has not made a significant clinical impact in cancer management due to a lack of using robust processes that facilitate scale-up and manufacturing. To this end we proposed the use of phage fusion protein as the navigating modules of novel targeted nanomedicine platforms which are described in this review.

Landscape phage ligands for PC3 prostate carcinoma cells.

Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display technology with its high throughput capacity for the analysis of targeting ligands possessing specific binding properties represents a very attractive tool in the quest for molecular ligands. Also, current phage nanobiotechnology concepts allow the use of intact phage particles and isolated phage coat proteins per se as components of nanomedicines. Herein, we describe the use of two landscape phage libraries to obtain phage ligands against PC3 prostate carcinoma cells. Following a very stringent selection scheme, we were able to identify three phage ligands, bearing the fusion peptides, DTDSHVNL, DTPYDLTG and DVVYALSDD that demonstrated specificity and selectivity to PC3 cells based on target-association assays, microscopy and flow cytometry. The phage ligands and their fusion coat proteins can be used as navigating modules in both therapeutic and diagnostic approaches to prostate carcinoma.

Mutations in fd phage major coat protein modulate affinity of the displayed peptide.

Multibillion-clone libraries of phages displaying guest peptides fused to the major coat protein pVIII (landscape libraries) are a rich source of probes for proteinaceous and non-proteinaceous targets. As opposed to the pIII-type fusion phages, which display peptides as independent structural domains, the guest peptides in the pVIII-fusion phages can be structurally and functionally influenced by contiguous subunits. To decipher the impact of the locale of a guest peptide on its affinity characteristics, we constructed a library of phages carrying beta-galactosidase-binding peptide ADTFAKSMQ at the N-terminus of the pVIII protein surrounded by random amino acids. It was found that mutagenesis of amino acids 12-19 (domain C) has polar effects on target binding affinity of the displayed peptide. The phages with highest affinity are characterized by: (i) a net electrostatic charge around -1 of domain C of the mutated phages at pH 7.0; (ii) a lower radius of cylinder coaxial to alpha-helix formed by domain C; (iii) a lower higher occupied molecular orbital (HOMO) of domain C leading to a decreased formation of hydrogen bonds and (iv) positively charged surface and torsion energy of domain C, which may require a conformational transition of N-terminal peptide ADTFAKSMQ for its binding with beta-galactosidase. Influence of the guest peptide on the diversity of mutations in the neighboring landscape area was also observed.

Diversity and censoring of landscape phage libraries.

Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd-tet (landscape libraries). They were obtained by replacement of amino acids 2-4 and 2-5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space.

Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors.

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1mg/ml BSA (blocking agent) onto the magnetoelastic resonator's surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.

The effect of salt and phage concentrations on the binding sensitivity of magnetoelastic biosensors for Bacillus anthracis detection.

This article presents an investigation of the effect of salt and phage concentrations on the binding affinity of magnetoelastic (ME) biosensors. The sensors were fabricated by immobilizing filamentous phage on the ME platform surface for the detection of Bacillus anthracis spores. In response to the binding of spores to the phage on the ME biosensor, a corresponding decrease occurs in resonance frequency. Transmission electron microscopy (TEM) was used to verify the structure of phage under different combinations of salt/phage concentration. The chemistry of the phage solution alters phage bundling characteristics and, hence, influences both the sensitivity and detection limit of the ME biosensors. The frequency responses of the sensors were measured to determine the effects of salt concentration on the sensors' performance. Scanning electron microscopy (SEM) was used to confirm and quantify the binding of spores to the sensor surface. This showed that 420 mM salt at a phage concentration of 1 x 10(11) vir/mL results in an optimal distribution of immobilized phages on the sensor surface, consequently promoting better binding of spores to the biosensor's surface. Additionally, the sensors immobilized with phage under this condition were exposed to B. anthracis spores in different concentrations ranging from 5 x 10(1) to 5 x 10(8) cfu/mL in a flowing system. The results showed that the sensitivity of this ME biosensor was 202 Hz/decade.

Alpha-helically constrained phage display library.

The library described here is a collection of phages with six degenerate codons in gene VIII, specifying amino acids 12, 13, 15-17 and 19 of the major coat protein. The randomized positions are surface exposed in the wild-type protein and thus might be expected to tolerate a great diversity of side chains without compromising phage viability. In agreement with this supposition, the new library showed great diversity of amino acids at the randomized positions and diversity did not diminish noticeably during repeated subculture. Despite their diversity, however, the randomized positions should be strongly constrained conformationally because they lie in an extended alpha-helical portion of the protein, stabilized by numerous inter- and intra-subunit contacts--a presupposition corroborated by circular dichroism spectroscopy of many library members. To reflect this conformational homogeneity and the fact that random amino acids subtend a major fraction of the surface 'landscape' of the particle, we call the new construct an alpha landscape library. It can be used as a source of alpha-helical ligands and substitute antibodies.

Phage display selection of peptides that affect prostate carcinoma cells attachment and invasion.

BACKGROUND: Prostate cancer-specific proteins must be identified to serve as diagnostic and prognostic markers. Cell surface proteins are especially important, because they have potential utility as diagnostic markers and therapeutic targets. Identification of ligands for these proteins will allow use of these ligands as diagnostic and therapeutic tools and permit the investigation of receptor function. We performed a search for peptide ligands to prostate cancer cell-specific receptors. METHODS: Peptide phage display library was used to isolate specific ligands to LNCaP prostate carcinoma cells receptors. Selected phage and cognate peptides were investigated for their cancer-related functions, such as the ability to interfere with cell adhesion, spreading, motility, and invasion. RESULTS: Phage designated pg35, blocked spreading of LNCaP cells and their derivatives C4-2 and C4-2b. Cognate peptide did not inhibit spreading, but incubation of C4-2 and C4-2b cells with cognate peptide increased their affinity for endothelial cells and invasiveness. In addition, the peptide activates matrix metalloproteinase (MMP)-2 and 9 in C4-2 and C4-2b cells. CONCLUSIONS: These results indicate that identified ligands may play a role in tumorigenicity and metastatic transformation of prostate cancer. To our knowledge, this is the first identification of a functional cancer-specific peptide ligand using the phage display approach.

Identifying diagnostic peptides for lyme disease through epitope discovery.

Serum antibodies from patients with Lyme disease (LD) were used to affinity select peptide epitopes from 12 large random peptide libraries in phage display format. The selected peptides were surveyed for reactivity with a panel of positive sera (from LD patients) and negative sera (from subjects without LD), thus identifying 17 peptides with a diagnostically useful binding pattern: reactivity with at least three positive sera and no reactivity with any of the negative sera. The peptides define eight sequence motifs, none of which can be matched convincingly with segments of proteins from Borrelia burgdorferi, the LD pathogen; evidently, then, they are "mimotopes," mimicking natural pathogen epitopes without matching contiguous amino acids of pathogen proteins. Peptides like these could be the basis of a new diagnostic enzyme-linked immunosorbent assay for LD, with sufficient specificity and sensitivity to replace expensive immunoblotting tests that are currently required for definitive serological diagnosis. Moreover, the method used to discover these peptides did not require any knowledge of the pathogen and involved generic procedures that are applicable to almost any infectious disease, including emerging diseases for which no pathogen has yet been identified.

Phages from landscape libraries as substitute antibodies.

In 'landscape' phage, as in traditional phage-display constructs, foreign peptides or proteins are fused to coat proteins on the surface of a filamentous phage particle. Unlike conventional constructs, however, each virion displays thousands of copies of the peptide in a repeating pattern, subtending a major fraction of the viral surface. The phage body serves as an interacting scaffold to constrain the peptide into a particular conformation, creating a defined organic surface structure ('landscape') that varies from one phage clone to the next. By testing landscape libraries with three representative antigens (streptavidin from the bacterium Streptomyces avidinii, avidin from chicken egg white and beta-galactosidase from Escherichia coli) we have shown that landscape phages may be used as a new type of substitute antibodies-filaments that can bind protein and glycoprotein antigens with nanomolar affinities and high specificity. In many ways these substitute antibodies are more convenient than their natural immunoglobulin counterparts.

Cross-linked filamentous phage as an affinity matrix.

Filamentous phage can be cross-linked to make a hydrophilic aggregate that is pelleted by low-speed centrifugation. The aggregate is stable at near-neutral pHs, and withstands exposure to the acid buffers (pH down to 2.2) that are often used as eluents in immunoaffinity purification. If a peptide epitope is genetically fused to a coat protein on the virion surface, the aggregate serves as an effective affinity matrix for absorbing and affinity-purifying antibodies that bind the peptide. When the peptide epitope is first obtained in this form by selection from large phage display libraries, this ability to fashion an affinity matrix directly from the selected phage represents a significant streamlining of research and development.

A library of organic landscapes on filamentous phage.

A billion-clone library of filamentous phage with different surface structures ("landscapes') was generated by fusing random octapeptides to the N-terminus of all 4000 copies of the major coat protein. Such a "landscape library' might include clones exhibiting emergent properties that inhere in the entire surface architecture, not in the peptides by themselves. Because the diverse surface landscapes are displayed on viable phage, they can be surveyed for exceedingly rare functions using microbiological selection methods. Clones with several emergent properties of the sort envisioned were successfully selected, suggesting that landscape libraries have promise as a novel source of nanomaterials with exploitable surface properties.

[Ability of a recombinant protein containing fragment 114-122 of myelin basic protein, to cause allergic encephalomyelitis in guinea pigs]. / O sposobnosti rekombinantnogo belka, soderzhashchego fragment 114-122 osnovnogo belka mielina, vyzyvat' allergicheskii éntsefalomielit u morskikh svinok.

Four genetic constructions have been designed, capable of producing in E. coli the hybrid beta-galactosidases containing the encephalitogenic determinant 114-122 of myelin basic protein. The ability of chromatography-purified proteins to cause allergic encephalomyelitis in guinea pigs has been investigated. Only one out of four proteins carrying at least one complete replica of encephalitogenic determinant did induce allergic encephalomyelitis in animals. Effects of the structural context of the encephalitogenic determinant on its functional activity are discussed.

[Detection of bovine infectious rhinotracheitis virus by hybridization using nonradioactive DNA-probes]. / Vyiavlenie virusa infektsionnogo rinotrakheita krupnogo rogatogo skota gibridizatsiei s neradioaktivnymi DNK-zondami.

Biotin-labeled DNA probes for infectious bovine rhinotracheitis virus also known as bovine herpesvirus-1 (BHV-1) have been developed. The procedure is based on dot-blot hybridization using biotin-labeled bacteriophage M13 and plasmid probes containing cloned PstI and EcoRI-PstI restriction fragments of viral genome. The probes obtained were used to detect viral nucleic acids in specimens of bovine spermatic fluid or nasal swabs of calves. The method is simple and rapid, taking less than 24 h, and is highly specific and sensitive, this recommending it for practical veterinary.