Generation and characterization of phage-GnRH chemical conjugates for potential use in cat and dog immunocontraception.

Overpopulation of cats and dogs is a serious worldwide problem that demands novel, safe and cost-effective solutions. The objective of this study was to generate and characterize phage-peptide conjugates with gonadotropin-releasing hormone (GnRH) for potential use as an immunocontraceptive. A filamentous phage vector f5-8 with wild-type phage coat proteins was used as a carrier for construction of chemical conjugates with GnRH, a peptide that acts as a master reproductive hormone. In such conjugates, the phage body plays the role of a carrier protein, while multiple copies of GnRH peptide stimulate production of neutralizing anti-GnRH antibodies potentially leading to contraceptive effects. To generate the constructs, four different GnRH-based peptides were synthesized and conjugated to phage particles in a two-step procedure: (i) peptides were reacted with phage to form a conjugate using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride chemistry (EDC) and (ii) the conjugates were separated from remaining free peptides by dialysis. Formation and specificity of phage-GnRH conjugates were confirmed by three independent methods: spectrophotometry, electron microscopy and ELISA. When the conjugates were tested for interaction with sera collected from cats and dogs immunized with GnRH-based vaccines in independent studies, strong specific ELISA signals were obtained, suggesting the potential use of the conjugates for cat and dog immunosterilization. The ability of the conjugates to stimulate production of anti-GnRH antibodies in vivo was evaluated in mice. While optimization of dose, immunization route and adjuvant still requires investigation, our preliminary results demonstrated the presence of anti-GnRH antibodies in sera of mice immunized with such conjugates. Fertility trials in cats and dogs will be needed to evaluate contraceptive potentials of the phage-GnRH peptide chemical conjugates.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 5. SkQ1 prolongs lifespan and prevents development of traits of senescence.

Very low (nano- and subnanomolar) concentrations of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) were found to prolong lifespan of a fungus (Podospora anserina), a crustacean (Ceriodaphnia affinis), an insect (Drosophila melanogaster), and a mammal (mouse). In the latter case, median lifespan is doubled if animals live in a non-sterile vivarium. The lifespan increase is accompanied by rectangularization of the survival curves (an increase in survival is much larger at early than at late ages) and disappearance of typical traits of senescence or retardation of their development. Data summarized here and in the preceding papers of this series suggest that mitochondria-targeted antioxidant SkQ1 is competent in slowing down execution of an aging program responsible for development of age-related senescence.

Peptide phage display: opportunities for development of personalized anti-cancer strategies.

Personalized medicine is critical for cancer patients, because (1) cancer is a highly heterogeneous disease with major molecular differences in the expression and distribution of tumor cell surface markers among patients with the same type and grade of cancer, (2) cellular mutations tend to accumulate as cancer progresses, further increasing tumor heterogeneity, and (3) currently used cancer therapies often are toxic to normal cells, causing severe side effects rarely seen in other diseases. To prevent side effects and to improve effectiveness, cytotoxic therapies should be targeted and each patient should be profiled for the presence of cancer targets before the therapy is administered. Phage display technology utilizes combinatorial libraries of proteins expressed on phage particles that can be selected for specific binding to cancer cells. Such cancer-specific molecules can be used in a variety of applications, including identification of cell-specific targeting molecules; identification of cell surface biomarkers; profiling of specimens obtained from individual cancer patients, and the design of peptide-based anti-cancer therapeutics for personalized treatments. This review is focused on peptide phage display strategies that target cell surfaces because many biomarkers important in cancer are differentially expressed molecules located on the outside of the cell membranes.

Molecular markers of glial tumors: current targeting strategies.

Diagnosis and therapy for malignant gliomas represents one of the most challenging problems in clinical oncology. Current treatment of malignant glioma is multimodal, involving surgical resection, radiotherapy and chemotherapy. Even with these combined therapies, patients usually die within 1 to 2 years after onset of symptoms. Clearly, improved strategies for selective delivery of therapeutic agents to gliomas are needed to combat these devastating and usually fatal cancers. This review summarizes current knowledge concerning targetable molecular markers on the surface of glial tumor cells and tumor vasculature. Such markers are altered or up-regulated in gliomas compared to normal tissues, or they might be glioma-restricted. These markers include growth factor receptors, cell-surface adhesion molecules, and membrane-type matrix metalloproteinases. Current approaches that utilize growth factor peptides and peptide/antibodies identified via phage display technology as carrier ligands for targeting malignant gliomas are discussed.

Flow microsphere immunoassay-based method of virus quantitation.

A sensitive assay for adenovirus quantitation in vitro was developed using the flow microsphere immunoassay (FMIA) approach. Polystyrene microspheres were covalently coated with purified anti-adenoviral antibodies and incubated with virus-containing samples. After incubation, the samples were stained with DNA-specific fluorescent dyes, and microsphere-associated fluorescence was quantitated with a flow cytometer. The adsorption of virus to microspheres was examined under different experimental conditions. The flow cytometric assay was determined to be as accurate in detecting adenovirus as titering on 293 cells. The proposed method can be used to quantify virus in viral stocks and in biological samples.

Elucidation of muscle-binding peptides by phage display screening.

Muscle makes up the largest tissue volume of the body, yet its size makes muscle-specific therapy difficult. This becomes particularly relevant when approaches to gene therapy for inherited myopathies are evaluated. Thus, a mechanism to target constructs or pharmaceuticals to muscle following intravenous injection would be advantageous. By screening a random phage display library we have identified a heptapeptide sequence, ASSLNIA, with enhanced in vivo skeletal and cardiac muscle binding. Phage carrying this peptide showed a 9- to 20-fold (depending on control tissue) increase in muscle selectivity compared with phage with no insert. When the injected individual phage clone was localized by immunohistochemistry, it was found within focal areas of the membrane of myofibers. Thus, the peptide identified represents a ligand that is capable of accessing skeletal and cardiac muscle from the lumen of blood vessels and could therefore readily be exploited for targeted delivery to muscle cells.

The flow karyotype of Arabidopsis thaliana interphase chromosomes.

The DNA contents of various aneuploid lines of Arabidopsis thaliana were measured by flow cytometry of 4',6-diamidino-2-phenylindole-stained interphase nuclei in suspensions and compared with each other as well as with the wild-type. The fluorescence intensifies for all lines were highly reproducible as were the deviations from the wild-type. The results allowed the estimation of the relative DNA contents of each Arabidopsis chromosome and of chromosomes arms. The sum of the surplus values for all trisomics was close to the value expected for the haploid (2C) DNA content. Only the line with the smallest telotrisome (Tr 3A) did not significantly differ in DNA content from that of the wild-type. It is concluded that approximately 3% of the genome represents the limit for resolution of differences in DNA content in this system. Thus, the approach allows a fast and reliable screening for duplications and deficiencies extending to 3% of the Arabidopsis genome. Regarding chromosomes sizes a comparison of the flow karyotype with existing karyotypes revealed differences which are discussed.

Tick-borne encephalitis (TBE) epidemiology in the Brest Province of the Republic of Belarus.

Prevalence of tick-borne encephalitis (TBE) and number of TBE infected ticks was analysed in the Brest Province since 1955. They revealed increasing tendency, which reached the highest values in the last years. TBE incidence was the highest in June and it was observed predominantly in people between 30 and 39 of age. The studies of TBE incidence rate regularities have disclosed its cyclical nature.