Methodologies for the isolation of alternative binders with improved clinical potentiality over conventional antibodies

The availability of binders to different functional domains of the same protein or to physiologically co-operating proteins allows for the simultaneous inhibition of independent downstream signaling pathways. This multi-target approach represents a promising therapeutic strategy, as demonstrated in the case of the synergistic effect of anti-Her2 treatment based on the combined use of the trastuzumab and pertuzumab monoclonal antibodies that induce cellular cytotoxicity and impair the receptor dimerization, respectively. Therefore, a reliable selection method for the recovery of epitope-specific antibodies is highly needed. Animal immunization with short peptides resembling the epitope sequence for raising conventional antibodies represents an alternative. Panning phage displayed libraries of recombinant antibodies such as scFvs and nanobodies or of other peptide collections is another option. Although recombinant antibodies can provide the same specificity as conventional antibodies, they offer at least two further advantages: i) the protocols for the selection of epitope-specific antibodies can be rationally designed, and ii) their expression as multivalent, bispecific and biparatopic molecules is feasible. This review will analyze the recent literature concerning technical aspects related to the isolation, the expression as multivalent molecules, and the therapeutic applications of binders able to interfere with antigen functional domains. The term binder will be preferred when possible to include those molecules, such as peptides or affibodies, with at least some proven practical uses.     

A Rapid and Inexpensive Enzymatic Assay for Total Cyanide in Cassava (Manihot esculenta Crantz) and Cassava Products

The well-known alkaline picrate test for cyanide has been improved by incorporating an enzymatic step to make the assay much more specific and quantitative. The sensitivity or detection limit of this method was found to be 0.16 μg/cm³ while the precision as indicated by the coefficient of variation was 3%. The method was, in addition, found to be rapid, simple, inexpensive and ideally suited for the analysis of large number of cassava tissues and products, such as may be encountered in cassava agronomy and breeding work or in industrial quality control laboratories. A trained operator working alone consistently analyzed at least 700 samples/day using this assay method.     

Tangential cell migration during layer formation of chick optic tectum

The laminated structure of the optic tectum is formed by radial and tangential cell migration during development. Studies of developing chick optic tectum have revealed two streams of tangential cell migration in the middle and superficial layers, which have distinctive origins, migratory paths, modes of migration, and destinations. We will review the process of the two types of tangential migrations, in order to elucidate their roles in the formation of the optic tectum layers.     

VEGF-C attenuates renal damage in salt-sensitive hypertension

Salt-sensitive hypertension is a major risk factor for renal impairment leading to chronic kidney disease. High-salt diet leads to hypertonic skin interstitial volume retention enhancing the activation of the tonicity-responsive enhancer-binding protein (TonEBP) within macrophages leading to vascular endothelial growth factor C (VEGF-C) secretion and NOS3 modulation. This promotes skin lymphangiogenesis and blood pressure regulation. Whether VEGF-C administration enhances renal and skin lymphangiogenesis and attenuates renal damage in salt-sensitive hypertension remains to be elucidated. Hypertension was induced in BALB/c mice by a high-salt diet. VEGF-C was administered subcutaneously to high-salt-treated mice as well as control animals. Analyses of kidney injury, inflammation, fibrosis, and biochemical markers were performed in vivo. VEGF-C reduced plasma inflammatory markers in salt-treated mice. In addition, VEGF-C exhibited a renal anti-inflammatory effect with the induction of macrophage M2 phenotype, followed by reductions in interstitial fibrosis. Antioxidant enzymes within the kidney as well as urinary RNA/DNA damage markers were all revelatory of abolished oxidative stress under VEGF-C. Furthermore, VEGF-C decreased the urinary albumin/creatinine ratio and blood pressure as well as glomerular and tubular damages. These improvements were associated with enhanced TonEBP, NOS3, and lymphangiogenesis within the kidney and skin. Our data show that VEGF-C administration plays a major role in preserving renal histology and reducing blood pressure. VEGF-C might constitute an interesting potential therapeutic target for improving renal remodeling in salt-sensitive hypertension.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

Septins regulate border cell surface geometry,shape, and motility downstream of Rho in Drosophila

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