An incremental approach to automated protein localisation

Tscherepanow M, Jensen N, Kummert F. An incremental approach to automated protein localisation. BMC Bioinformatics. 2008;9(1): 445.Background: The subcellular localisation of proteins in intact living cells is an important means for gaining information about protein functions. Even dynamic processes can be captured, which can barely be predicted based on amino acid sequences. Besides increasing our knowledge about intracellular processes, this information facilitates the development of innovative therapies and new diagnostic methods. In order to perform such a localisation, the proteins under analysis are usually fused with a fluorescent protein. So, they can be observed by means of a fluorescence microscope and analysed. In recent years, several automated methods have been proposed for performing such analyses. Here, two different types of approaches can be distinguished: techniques which enable the recognition of a fixed set of protein locations and methods that identify new ones. To our knowledge, a combination of both approaches – i.e. a technique, which enables supervised learning using a known set of protein locations and is able to identify and incorporate new protein locations afterwards – has not been presented yet. Furthermore, associated problems, e.g. the recognition of cells to be analysed, have usually been neglected. Results: We introduce a novel approach to automated protein localisation in living cells. In contrast to well-known techniques, the protein localisation technique presented in this article aims at combining the two types of approaches described above: After an automatic identification of unknown protein locations, a potential user is enabled to incorporate them into the pre-trained system. An incremental neural network allows the classification of a fixed set of protein location as well as the detection, clustering and incorporation of additional patterns that occur during an experiment. Here, the proposed technique achieves promising results with respect to both tasks. In addition, the protein localisation procedure has been adapted to an existing cell recognition approach. Therefore, it is especially well-suited for high-throughput investigations where user interactions have to be avoided. Conclusion: We have shown that several aspects required for developing an automatic protein localisation technique – namely the recognition of cells, the classification of protein distribution patterns into a set of learnt protein locations, and the detection and learning of new locations – can be combined successfully. So, the proposed method constitutes a crucial step to render image-based protein localisation techniques amenable to large-scale experiments

Formulation and in-vitro release studies on chitosan-alginate microcapsules modified for fish vaccine delivery

La présente étude examine la libération in vitro de Vibrio bacterin, un vaccin pour les poissons produit à partir de microcapsules chitosan-alginate modifiées de HPMCAS pour l\'administration par voie orale chez les poissons. Les microcapsules ont été préparées avec la méthode de coacervation counterion en utilisant un générateur électrostatique de gouttelettes. Le noyau alginate de microcapsules a été modifié avec HPMCAS à divers ratios d\'alginate (A) : HPMCAS(H) de 1 :1 (AH) ; 1 :2 (AH2) et 2:1 (A2H). La libération du vaccin a été étudiée in vitro à différents pH du milieu d\'élution étalant le pH de l\'intestin du poisson, et les données obtenues ont été insérées dans différents types de libération. Les microcapsules d\'AH ont produit la plus faible quantité de libération de vaccin de 18,2% après 9h et la quantité était très différente (p < 0,05) du témoin. On a trouvé que la libération du vaccin était dans l\'ordre suivant : AH < A2H < AH2 0,05) en termes de libération du vaccin entre les microcapsules modifiées et le témoin au pH 3, 6 et 8. Cela était attribué à l\'effet entérique de HPMCAS. On a aussi noté que la libération de protéine des microcapsules était contrôlée par la diffusion. La modification de microcapsules de chitosan-alginate est une méthode potentielle pour la préparation de formules de vaccin par voie orale pour les poissons.This study examines the in vitro release of Vibrio bacterin, a fish vaccine, from HPMCAS modified chitosan-alginate microcapsules designed for oral delivery in fish. The microcapsules were prepared by the counterion coacervation method using an electrostatic droplet generator. The alginate core of the microcapsules was modified with HPMCAS at varying alginate (A): HPMCAS (H) ratios of 1:1 (AH), 1:2 (AH2) and 2:1 (A2H). Vaccine release was studied in vitro at different pH of the elution medium spanning the pH of the fish gut, and the data obtained were fitted into different release models. AH microcapsules effected the lowest amount of vaccine release of 18.2% after 9 h and was significantly different (p 0.05) in vaccine release between the modified microcapsules and the control at pH 3, 6 and 8. This was attributed to the enteric effect of HPMCAS. Protein release from the microcapsules was found to be diffusion-controlled. Modification of chitosan-alginate microcapsules is a potential approach for preparing suitable oral fish vaccine formulations. Keywords: Microcapsules, Vibrio bacterin, chitosan-alginate, vibriosis, fish vaccine delivery.Bulletin of Animal Health and Production in Africa Vol. 56 (2) 2008: pp. 98-10

Immobilization/encapsulation of cells using electrostatic droplet generation - Experiments and theory

The mechanism of alginate droplet formation as well as experimental parameters for producing very small polymer microbeads (i,e, less then 100 microns diameter) using an electrostatic droplet generator were investigated, It was found that microbead size was a function of needle diameter, charge arrangement (i,e, electrode geometry and spacing) and strength of electric field. The process of alginate droplet formation under the influence of electrostatic forces was assessed with an image analysis/video system and revealed distinct stages; After a voltage was applied the liquid meniscus at the needle tip was distorted from a spherical shape into an inverted cone-like shape. Alginate solution flowed into this cone at an increasing rate causing formation of a neck-like filament, When this filament broke away, producing small droplets, the meniscus relaxed back to a spherical shape until now of the polymer caused the process to start again, A mathematical model of droplet formation at the electrified needle was developed from an analysis of the forces acting on a charged droplet, and agreed well with experimental results. Finally, to assess the effect of an electric field on animal cell viability, an insect cell suspension was subjected to a high voltage, There was no detectable loss in cell viability after the voltage was applied

Cell immobilisation by electrostatic droplet generation

This paper reviews the feasibility of electrostatic droplet generation for the production of uniform hydrogel microbeads and applications of this technique for cell immobilization. This is a novel extrusion technique that uses electrostatic forces to disrupt a liquid surface at the capillary/needle tip and form a charged stream of small droplets. Experimental parameters which are critical for production of polymer microbeads (in the range of 0.1 to 1 mm in diameter), as well as mechanisms of alginate droplet formation are presented here. It was shown that microbead size was a function of applied potential, polymer surface tension, needle size and electrode geometry. In addition, this technique was applied for immobilization of several cell types (yeast, mammalian and plant cells). There was no detectable loss in viability of these cell cultures after exposure to high electrostatic potentials. Cultivation studies of cells immobilized by electrostatic droplet generation showed good maintenance of cell viability and activity, indicating broad potential of this technique for the immobilization of a variety of cell types for applications in different fields of biotechnology, pharamceuticals and medicine