Spatio-temporal analysis of collective migrationin vivoby particle image velocimetry.

Collective cell migration drives the formation of complex organ systems as well as certain tumour invasions and wound healing processes. A characteristic feature of many migrating collectives is tissue-scale polarity, whereby 'leader' cells at the tissue edge guide 'followers' cells that become assembled into polarized epithelial tissues. In this study, we employed particle image velocimetry (PIV) as a tool to quantitate local dynamics underlying the migration of the posterior lateral line primordium (pLLP) in zebrafish at a short time scale. Epithelial cadherin-EGFP was the fluorescent tracer in time-lapse images for PIV analysis. At the tissue level, global speed and directionality of the primordium were extracted from spatially averaged velocity fields. Interestingly, fluctuating velocity patterns evolve at the mesoscale level, which distinguishes the pseudo-mesenchymal leading front from the epithelialized trailing edge, and superimpose to the global deceleration of the whole primordium during the separation of a protoneuromast. Local velocity fields obtained by PIV proved sensitive to estimate the migration speed and directionality of the pLLP in zebrafish, predicting protoneuromast separation at short time scales. Finally, the PIV approach may be suitable for analysing the dynamics of otherin vivomodels of collective migration.

Targeted cellular delivery of quantum dots loaded on and in biotinylated liposomes.

We describe the preparation, biophysical characterization, and receptor-mediated cellular internalization of biotinylated lipid particles (BLPs) loaded on the surface and internally with two distinct (colors) of quantum dot (QD) probes. BLPs were formulated with 1.4 and 2.7 mol % PEG-lipids containing either a fusogenic or pH-sensitive lipid to promote bilayer destabilization of endosomal membranes and favor QD cytoplasmic release. The amount of PEG was chosen to provide steric stabilization of the final construct. BLPs were loaded with a red-emitting QD(655) and surface coated with a green-emitting QD(525) conjugated to the epidermal growth factor (EGF) ligand in order to target the epidermal growth factor receptor (EGFR). The targeted and QD labeled BLPs showed strong and selective binding to EGFR-expressing tumor cell line and subsequent internalization. The dual-color QD labeling strategy and colocalization analysis allow prolonged live cell imaging of BLPs and loaded cargo independently, using a single excitation wavelength and simultaneous detection of both QDs. The chemistry of bioconjugation for the EGF ligand, the QDs, and the BLPs in a single lipid particle, involves only biotin-streptavidin interaction without requiring further purification from free EGF-QDs preformed complexes. Coupled with an encapsulated drug, the targeted and QD-labeled BLPs could provide imaging and drug delivery in a single multifunctional carrier.

Anionic Quantum Dots reveal actin-microridges in zebrafish epidermis.

Enhancement of hydrophilicity and functionalization of CdTe QDs (Quantum Dots) via surface modifications have made them suitable to be used as specific probes for cell imaging. Applications for targeting cell surfaces have been widely demonstrated in vitro but their use in animal models is not trivial. Here, we reported the interaction of mercaptosuccinic-coated (MSA) CdTe QDs with the epidermis of living and Carnoy-fixed zebrafish embryos. QDs concentrate along adherent junctions and reveal the characteristic pattern of actin microridges at the apical surface of the enveloping layer. In our study, labeling with anionic QDs is attained within few minutes at submicromolar concentrations in whole mounted Carnoy-fixed zebrafish embryos, providing a faster approach compared with immunodetection or standard Phalloidin staining of actin for visualization by fluorescence microscopy.

E-cadherin expression pattern during zebrafish embryonic epidermis development.

Background: E-cadherin is the major adhesion receptor in epithelial adherens junctions (AJs). On established epidermis, E-cadherin performs fine-tuned cell-cell contact remodeling to maintain tissue integrity, which is characterized by modulation of cell shape, size and packing density. In zebrafish, the organization and distribution of E-cadherin in AJs during embryonic epidermis development remain scarcely described. Methods: Combining classical immunofluorescence, deconvolution microscopy and 3D-segmentation of AJs in epithelial cells, a quantitative approach was implemented to assess the spatial and temporal distribution of E-cadherin across zebrafish epidermis between 24 and 72 hpf. Results: increasing levels of E-cadh protein parallel higher cell density and the appearance of hexagonal cells in the enveloping layer (EVL) as well as the establishments of new cell-cell contacts in the epidermal basal layer (EBL), being significantly between 31 and 48 hpf .Conclusions: Increasing levels of E-cadherin in AJs correlates with extensive changes in cell morphology towards hexagonal packing during the epidermis morphogenesis.

Self-assembling supramolecular complexes by single-stranded extension from plasmid DNA.

Self-assembling supramolecular complexes are of great interest for bottom-up research like nanotechnology. DNA is an inexpensive building block with sequence-specific self-assembling capabilities through Watson-Crick and/or Hoogsteen base pairing and could be used for applications in surface chemistry, material science, nanomechanics, nanoelectronics, nanorobotics, and of course in biology. The starting point is usually single-stranded DNA, which is rather easily accessible for base pairing and duplex formation. When long stretches of double-stranded DNA are desirable, serving either as genetic codes or electrical wires, bacterial expansion of plasmids is an inexpensive approach with scale-up properties. Here, we present a method for using double-stranded DNA of any sequence for generating simple structures, such as junctions and DNA lattices. It is known that supercoiled plasmids are strand-invaded by certain DNA analogs. Here we add to the complexity by using "Self-assembling UNiversal (SUN) anchors" formed by DNA analog oligonucleotides, synthesized with an extension, a "sticky-end" that can be used for further base pairing with single-stranded DNA. We show here how the same set of SUN anchors can be utilized for gene therapy, plasmid purification, junction for lattices, and plasmid dimerization through Watson-Crick base pairing. Using atomic force microscopy, it has been possible to characterize and quantify individual components of such supra-molecular complexes.

A simple and effective method to improve intrasplenic rat hepatocyte transplantation.

Transplanted hepatocytes integrate, survive, and express their specific functions in the liver parenchyma. The aim of this study was to determine whether a large number of hepatocytes could move from the spleen to the liver when the cells are injected together with sodium nitroprusside, and if the improved hepatocyte migration may be related with portal vein dilatation. Wistar rats were transplanted in the spleen with fluorescent-labeled hepatocytes alone or together with sodium nitroprusside. At 1, 3, 6, and 24 h after the transplant, the liver from recipient animals was removed and morphometric analyses were performed. Portal and arterial pressures were also measured immediately after intrasplenic injection of a solution of sodium nitroprusside, hepatocytes alone, or hepatocytes plus sodium nitroprusside. Intrasplenically injected sodium nitroprusside produced a transient drop in arterial pressure and a sustained reduction in portal pressure. During hepatocyte transplantation it increased the number of transplanted cells migrating to the liver after 3 h. Sodium nitroprusside simultaneously injected with hepatocytes in the spleen allowed more cells to migrate into the liver of the host animal without risk in animal survival.

Assembly, characterization, and delivery of quantum dot labeled biotinylated lipid particles.

Lipid nanoparticles composed of mixtures of PEGylated-lipids; cationic and neutral lipids prepared by detergent dialysis can encapsulate biological active molecules and show considerable potential as systemic therapeutic agents. Addition of biotinylated lipids to this formulation allows surface modification of these particles with a suitable ligand or probe conjugated to streptavidin for specific cell targeting. Monitoring long circulating particles and cellular uptake requires stable and bright fluorescent probes. Quantum dots (QDs) constitute a relatively new class of fluorescent probes that overcome the limitations of organic fluorophores in biological imaging applications. Here, a protocol for the encapsulation of QD655 (red) in biotinylated lipid particles (BLPs) prepared by a detergent dialysis technique is presented followed by characterization of the loaded liposomal vehicles. Then, a protocol for BLPs surface modification via biotin-streptavidin linkage with preformed complexes of ligand-QD525 (green) for specific cell targeting of the nanoparticle is detailed. Conditions for cell binding and uptake of two colors QD labeled BLPs as well as basic microscopic settings for confocal live cell imaging are described.

A modular perfused chamber for low- and normal-temperature microscopy of living cells.

An inexpensive modular perfused chamber (MPC) designed for low- and normal-temperature live-cell imaging is presented. The device consists of four lathed pieces of stainless steel assembled as a cylindrical open chamber that can hold either round or square glass coverslips. The chamber is connected to a thermal-bath operating with recirculation. For image acquisition at 4°C, cooled air is blown toward the coverslip surface to prevent condensation. Principal advantages of this device are thermal stability in the sample environment, rapid response to changes in temperature set point, and easy sample insertion. The device enables the study of dynamic processes in cells governed by large temperature differences such as those imposed by hypothermic preservation of cells (0-4°C) followed by rewarming to normothermia (37°C). The capabilities of the MPC were demonstrated by monitoring the internalization of fluorescent quantum dots (QDs) in rat hepatocytes after hypothermic storage and during rewarming with an inverted microscope.

Estudio de la variante aint del antígeno A / Study of a antigen aint variant

Se definen como A intermedio (Aint) los hematíes que comparten caracteres con A1 y A2. Existen diferencias cualitativas y cuantitativas en los epitopes A. Los hematíes A1 son aglutinados por la lectina de Ulex Europaeus (anti-H), en tanto los hematíes Aint son débilmente aglutinados por ambas de manera inesperada. Se realizó una búsqueda de individuos Aint en una población hospitalaria de acuerdo con la reacción con las lectinas mencionadas. Se evaluó el proceso de aglutinación producida mediante una técnica cinética. Se estudiaron 557 muestras, resultando 285 O (51,1 por ciento), 216 A (38,8 por ciento), 45 B (8,1 por ciento), 8 A1B (1,4 por ciento) y 3 A2B (0,6 por ciento). Las muestras A se subdividieron en 173 A1, (80,2 por ciento), 31 A2 (14,3 por ciento) y 12 Aint (5,5 por ciento). Se observó, en las curvas de cinética, diferencia de reacción entre los hematíes de grupo A1 Aint y A2. Nuestros resultados concuerdan con observaciones previas sobre la marcada heterogeneidad de los hematíes Aint. El reconocimiento de de variantes débiles del grupo A reviste importancia cuando se presentan reacciones transfusionales hemolíticas y en la práctica forense. Importa señalar que el valor de este estudio es relevante, en Inmunogenética Poblacional, por su contribución al conocimiento del mestizaje con poblaciones negras

Frecuencia de la variante aint del antígeno A / Frecuency of aint antigen

Se definen como A intermedio (Aint) los hematíes que comparten caracteres con A1 y A2. Existen diferencias cualitativas y cuantitativas en los epitopes A. Los hematíes A, son aglutinados por la lectina de Dolichus biflorus (anti-A1), los A2 por la lectina Ulex europaeus (anti-H), en tanto los hematíes Aint son variablemente aglutinados por ambas de manera inesperada. Se realizó una búsqueda de individuos Aint en una población hospitalaria de acuerdo con la reacción con las lectinas mencionadas evaluando el proceso de aglutinación producida mediante una técnica cinética. Se caracterizó la actividad aglutinante de la lectina de Amaranthus hypochondriacus (específica para N-acetil-D-galactosamina) antes y después de la incubación con saliva A y frente a eritrocitos A1, Aint y A2. Se estudiaron 458 muestras, resultando 242 O (52,8 por ciento), 170 A (37,1 por ciento), 38 B (8,3 por ciento), 5 A1B (1,1 por ciento) y 3 A2B (0,7 por ciento). Las muestras A se subdividieron en 138 A1 (81,2 por ciento), 23 A2 (13,5 por ciento) y 9 Aint (5,3 por ciento). La comparación de las curvas de cinética obtenidos permite diferenciar los hematíes de grupos A1, Aint y A2. Nuestros resultados concuerdan con observaciones previas sobre la marcada heterogeneidad de los hematíes Aint. El reconocimiento de variantes débiles del grupo A reviste importancia cuando se presentan reacciones transfusionales hemolíticas y en la práctica forense. Este estudio es relevante, en inmunogenética poblacional, por su contribución al conocimiento del mestizaje con poblaciones negras.