An outbreak associated with streptococcus equi subsp. zooepidemicus in layers : evidence of fecal transmission

Major disease outbreaks caused by Streptococcus equi subsp. zooepidemicus seldom are reported in poultry. Besides acute septicemia, infection can result in a subacute or chronic form of disease with described mortality rates of 11% to 80%. Previously, the source of infection in poultry was linked to horses in which this bacterium can be present as an opportunistic pathogen on mucus membranes. The main route of spreading and being maintained within a poultry flock, after entering the stable, however, remains unclear. This case report describes an outbreak associated with S. zooepidemicus affecting a flock of 28 500 layer hens housed in an aviary system with free range. Besides sudden deaths, clinical signs of depression were noticed. Between 44 and 61 wk of age a total mortality of 23% was observed. Egg production dropped from 92% to 83%. Bacterial titration revealed substantial numbers of S. zooepidemicus present in the ceca of a healthy chicken. This novel finding hypothesizes that transmission of the infection within the flock might occur through the fecal route

A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy

We present a new convenient method for quantitative three-dimensionally resolved diffusion measurements based on the photobleaching (FRAP) or photoactivation (FRAPa) of a disk-shaped area by the scanning laser beam of a multiphoton microscope. Contrary to previously reported spot-photobleaching protocols, this method has the advantage of full scalability of the size of the photobleached area and thus the range of diffusion coefficients, which can be measured conveniently. The method is compatible with low as well as high numerical aperture objective lenses, allowing us to perform quantitative diffusion measurements in three-dimensional extended samples as well as in very small volumes, such as cell nuclei. Furthermore, by photobleaching/ photoactivating a large area, diffusion along the optical axis can be measured separately, which is convenient when studying anisotropic diffusion. First, we show the rigorous mathematical derivation of the model, leading to a closed-form formula describing the fluorescence recovery/redistribution phase. Next, the ability of the multiphoton FRAP method to correctly measure absolute diffusioncoefficients is tested thoroughly onmanytest solutions of FITC-dextrans covering a wide range of diffusion coefficients. The same is done for the FRAPa method on a series of photoactivatable green fluorescent protein solutions with different viscosities. Finally, we apply the method to photoactivatable green fluorescent protein diffusing freely in the nucleus of living NIH-3T3 mouse embryo fibroblasts. © 2008 by the Biophysical Society

Targeted nanoparticles towards increased L cell stimulation as a strategy to improve oral peptide delivery in incretin-based diabetes treatment

The delivery of therapeutic peptides via the oral route remains one of biggest challenges in the pharmaceutical industry. Recently, we have described an alternative improved drug delivery system for peptide delivery via the oral route, consisting of a lipidic nanocapsule. Despite the striking effects observed, it is still essential to develop strategies to strengthen the nanocarriers' glucagon-like peptide-1 (GLP-1) secretory effect of the nanocarrier and/or prolong its antidiabetic effect in vivo to facilitate its translation into the clinic. For this purpose, we developed and compared different fatty acid-targeted lipid and polymeric nanoparticles and evaluated the L cell stimulation induced by the nanocarriers in murine L cells in vitro and in normal healthy mice in vivo. We further examined the antidiabetic effect in vivo in an obese/diabetic mouse model induced by high-fat diet feeding and examined the effect of the oral administration frequency. Among the tested nanocarriers, only lipid-based nanocarriers that were surface-modified with DSPE-PEG(2000) on the surface were able to significantly strengthen the biological effect of the nanocarriers. They increased endogenous GLP-1 levels up to 8-fold in vivo in normo-glycemic mice. Moreover, they effectively prolonged the in vivo antidiabetic effect by normalizing the plasma glucose levels in obese/diabetic mice following long-term treatment (one month). Ultimately, the targeted nanocarriers were as effective when the administration frequency was reduced from once daily to once every other day

A FRAP model to investigate reaction-diffusion of proteins within a bounded domain: a theoretical approach

Temporally and spatially resolved measurements of protein transport inside cells provide important clues to the functional architecture and dynamics of biological systems. Fluorescence Recovery After Photobleaching (FRAP) technique has been used over the past three decades to measure the mobility of macromolecules and protein transport and interaction with immobile structures inside the cell nucleus. A theoretical model is presented that aims to describe protein transport inside the nucleus, a process which is influenced by the presence of a boundary (i.e. membrane). A set of reaction-diffusion equations is employed to model both the diffusion of proteins and their interaction with immobile binding sites. The proposed model has been designed to be applied to biological samples with a Confocal Laser Scanning Microscope (CLSM) equipped with the feature to bleach regions characterised by a scanning beam that has a radially Gaussian distributed profile. The proposed model leads to FRAP curves that depend on the on- and off-rates. Semi-analytical expressions are used to define the boundaries of on- (off-) rate parameter space in simplified cases when molecules move within a bounded domain. The theoretical model can be used in conjunction to experimental data acquired by CLSM to investigate the biophysical properties of proteins in living cells.Comment: 25 pages. Abstracts Proceedings, The American Society for Cell Biology, 46th Annual Meeting, December 9-13, 2006, San Dieg

Protein Diffusion in Mammalian Cell Cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS

Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient

In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the “added value” we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms

Sustained Oscillations of NF-κB Produce Distinct Genome Scanning and Gene Expression Profiles

NF-κB is a prototypic stress-responsive transcription factor that acts within a complex regulatory network. The signaling dynamics of endogenous NF-κB in single cells remain poorly understood. To examine real time dynamics in living cells, we monitored NF-κB activities at multiple timescales using GFP-p65 knock-in mouse embryonic fibroblasts. Oscillations in NF-κB were sustained in most cells, with several cycles of transient nuclear translocation after TNF-α stimulation. Mathematical modeling suggests that NF-κB oscillations are selected over other non-oscillatory dynamics by fine-tuning the relative strengths of feedback loops like IκBα. The ability of NF-κB to scan and interact with the genome in vivo remained remarkably constant from early to late cycles, as observed by fluorescence recovery after photobleaching (FRAP). Perturbation of long-term NF-κB oscillations interfered with its short-term interaction with chromatin and balanced transcriptional output, as predicted by the mathematical model. We propose that negative feedback loops do not simply terminate signaling, but rather promote oscillations of NF-κB in the nucleus, and these oscillations are functionally advantageous

Dioctadecyldimethylammonium:monoolein nanocarriers for efficient in vitro gene silencing

This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.This work was supported by FEDER through POFC - COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBM.A), PEst-C/FIS/UI0607/2011 (CFUM), and PTDC/QUI/69795/2006, while Ana Oliveira holds scholarship SFRH/BD/68588/2010. Eloi Feitosa thanks FAPESP (2011/03566-0) and CNPq (303030/2012-7), and Renata D. Adati thanks FAPESP for scholarship (2011/07414-0). K. Raemdonck is a postdoctoral fellow of the Research Foundation - Flanders (FWO-Vlaanderen). We acknowledge NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment

In Vivo Monitoring of mRNA Movement in Drosophila Body Wall Muscle Cells Reveals the Presence of Myofiber Domains

Background: In skeletal muscle each muscle cell, commonly called myofiber, is actually a large syncytium containing numerous nuclei. Experiments in fixed myofibers show that mRNAs remain localized around the nuclei in which they are produced. Methodology/Principal Findings: In this study we generated transgenic flies that allowed us to investigate the movement of mRNAs in body wall myofibers of living Drosophila embryos. We determined the dynamic properties of GFP-tagged mRNAs using in vivo confocal imaging and photobleaching techniques and found that the GFP-tagged mRNAs are not free to move throughout myofibers. The restricted movement indicated that body wall myofibers consist of three domains. The exchange of mRNAs between the domains is relatively slow, but the GFP-tagged mRNAs move rapidly within these domains. One domain is located at the centre of the cell and is surrounded by nuclei while the other two domains are located at either end of the fiber. To move between these domains mRNAs have to travel past centrally located nuclei. Conclusions/Significance: These data suggest that the domains made visible in our experiments result from prolonged interactions with as yet undefined structures close to the nuclei that prevent GFP-tagged mRNAs from rapidly moving between the domains. This could be of significant importance for the treatment of myopathies using regenerative cellbase