Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity.Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing.Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS.Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures.Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

Background Phosphorylation plays an essential role in regulating the voltage-gated sodium (Nav) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Nav channels. Herein, we posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Nav channels. Methods We used patch-clamp electrophysiology to record sodium currents from Nav1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues. Results We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Nav1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Nav1.2-encoded currents. Neither mRNA nor total protein expression were changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Nav1.2 channel indicates that cell surface expression of CD4-Nav1.2-Ctail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3β phosphorylates T1966 at the C-terminal tail of Nav1.2. Conclusion These findings provide evidence for a new mechanism by which GSK3 modulate Nav channel function via its C-terminal tail. General Significance These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.

Despite considerable progress in the field of automated neurite arbor reconstruction from two-dimensional (2-D) or three-dimensional (3-D) images acquired using optical microscopes, 1 even the best available automated systems of today have a non-zero error rate, implying the continued need for visual proofreading and corrective editing systems. Currently available systems require the user to visually detect potential errors, and perform corrective edits serially, one at a time. Consequently, it is not uncommon for the task of proofreading an automated reconstruction to take longer than de novo manual reconstruction in the hands of a skilled operator, 2 appearing to defeat the utility of automated reconstruction systems. There is a compelling need in the neuroscience research community for smarter and more scalable proofreading tools that can significantly accelerate the process, and reduce the tedium and manpower cost.This need is especially compelling in large-scale and highthroughput studies that require large numbers of neurite arbor measurements. Meeting the above-mentioned need calls for a well-integrated combination of methods for (i) detailed visualization of reconstructions overlaid on the source images (usually large 2-D/3-D, and multi-channel image data); (ii) rapid identification of tracing errors; and (iii) rapid and minimal-effort correction of errors using interactive graphical tools.There is a parallel need among computer science researchers who are developing new and improved algorithms for automated reconstruction. Each newly developed algorithm must be validated, and its performance quantified in order to determine the extent to which it improves upon previously developed systems. Detailed performance evaluation data can enable algorithm developers to focus efforts on the most important algorithm deficiencies. Traditionally, this goal has been met by comparing automatically generated reconstructions against pre-established "gold standard" reconstructions. Currently available gold standards are generated by manual reconstruction, and sometimes proofread by multiple human observers to generate a consensus of reconstructions. This process is expensive, slow, and not scalable. Manual reconstructions are simply unavailable in an operational setting such as a highthroughput study. These considerations suggest the need for alternative performance evaluation and validation methodologies.In this paper, we advocate edit-based methodologies for validation and performance assessment. These methods become practically appropriate as and when the performance of automated algorithms is close to being optimal, i.e., the differences between automated reconstructions and human reconstructions are less than 10%. Under these 0 Peng H., Long F., Zhao T., Myers E.W., (2010) Proof-editing is the bottleneck of 3D neuron reconstruction: The problem and solutions, Neuroinformatics, published online: 17 th Dec. favorable conditions, a more efficient alternative methodology can, and should be, considered. In this alternativ...

Articles you may be interested inPreparation of graphene oxide/epoxy nanocomposites with significantly improved mechanical properties Enhanced dielectric properties of BaTiO3/poly(vinylidene fluoride) nanocomposites for energy storage applications J. Appl. Phys. 113, 034105 (2013); 10.1063/1.4776740 Influence of Cr2O3 nanoparticles on the physical properties of polyvinyl alcohol J. Appl. Phys. 112, 093525 (2012); 10.1063/1.4764864Modeling of hyaluronic acid containing anti-cancer drugs-loaded polylactic-co-glycolic acid bioconjugates for targeted delivery to cancer cells AIP Conf.Polymer-matrix nanocomposites based on Poly(lactic-co-glycolic) acid (PLGA) and Graphene platelets (GNPs) were studied. GNPs, nanomaterials with a 2D flat surface, were chosen with or without chemical modification in PLGA/GNP nanocomposites and their microstructure, thermal property, and their compatibility as scaffolds for cell growth were investigated. PLGA/GNP nanocomposites (0, 1, and 5 wt. % of GNPs) were prepared using a solution based technique. Transmission electron microscopy, X-ray diffraction, Differential scanning calorimeter, and Thermogravimetric analyzer were used to analyze morphology and thermal properties. This work demonstrated the role of GNPs flat surface to provide a favorable platform resulting in an enhanced PLGA crystallization. Functionalized GNPs suppress both the thermal stability and the crystallization of PLGA. Finally, to determine the potential usefulness of these scaffolds for biomedical applications, mammalian cells were cultured on various PLGA/GNP nanocomposites (0, 1, and 5 wt. % GNPs). 1 wt. % PLGA/GNP nanocomposites showed better biocompatibility for cell growth with/without graphenes functionalization compared to pure PLGA and 5 wt. % PLGA/GNP. The function of GNPs in PLGA/GNPs (1 wt. %) composites is to provide a stage for PLGA crystallization where cell growth is favored. These results provide strong evidence for a new class of materials that could be important for biomedical applications. V C 2014 AIP Publishing LLC.

The retina, as the only visually accessible tissue in the central nervous system, has attracted significant attention for evaluating it as a biomarker for neurodegenerative diseases. Yet, most of studies focus on characterizing the loss of retinal ganglion cells (RGCs) and degeneration of their axons. There is no integrated analysis addressing temporal alterations of different retinal cells in the neurovascular unit (NVU) in particular retinal vessels. Here we assessed NVU changes in two mouse models of tauopathy, P301S and P301L transgenic mice overexpressing the human tau mutated gene, and evaluated the therapeutic effects of a tau oligomer monoclonal antibody (TOMA). We found that retinal edema and breakdown of blood–retina barrier were observed at the very early stage of tauopathy. Leukocyte adhesion/infiltration, and microglial recruitment/activation were constantly increased in the retinal ganglion cell layer of tau transgenic mice at different ages, while Müller cell gliosis was only detected in relatively older tau mice. Concomitantly, the number and function of RGCs progressively decreased during aging although they were not considerably altered in the very early stage of tauopathy. Moreover, intrinsically photosensitive RGCs appeared more sensitive to tauopathy. Remarkably, TOMA treatment in young tau transgenic mice significantly attenuated vascular leakage, inflammation and RGC loss. Our data provide compelling evidence that abnormal tau accumulation can lead to pathology in the retinal NVU, and vascular alterations occur more manifest and earlier than neurodegeneration in the retina. Oligomeric tau-targeted immunotherapy has the potential to treat tau-induced retinopathies. These data suggest that retinal NVU may serve as a potential biomarker for diagnosis and staging of tauopathy as well as a platform to study the molecular mechanisms of neurodegeneration.

Chemical burns due to alkalis cause extensive damage to the ocular surface leading to blindness. Assessment of ocular burn could be challenging due to severe opacity, inflammation, and angiogenesis. Anterior segment optical coherence tomography (AS-OCT) and OCT angiography (OCTA) may provide fast, non-invasive deep tissue visualization of pathology with high sensitivity in conjunction with slit-lamp analysis.Methods: C57-BL/6J mice were anesthetized with ketamine/dexmedetomidine, and corneal alkali burn was induced (n = 6) by placing filter paper soaked in 1-M sodium hydroxide for 30 seconds on the right eye while the left eye was kept as control. Longitudinal imaging was done with AS-OCT/OCTA and fluorescein angiography at various time intervals for 14 days.Results: AS-OCT showed characteristic pathological changes in alkali-burned eyes with high sensitivity. Although OCT/OCTA showed three-dimensional and cross-sectional views of the anterior chamber and angiogenesis, fluorescein angiography showed nascent vessels with active leakage. Corneal swelling progressively increased by 125.26% on day 12 with a high prevalence of epithelial bullae, stromal cysts, stromal splitting, and Descemet's membrane detachment. Neovascularization was noted as early as day 4 in the burned eyes by both methods. Severe corneal opacity and anterior chamber inflammation were also detected by AS-OCT/OCTA. Conclusions: AS-OCT/OCTA is a promising, noninvasive, high-resolution imaging modality that can provide both qualitative and quantitative information regarding deep tissue pathology at a structural level.Translational Relevance: Noninvasive AS-OCT/OCTA and fluorescein methods show promise in clinical pathology evaluation for ocular injury management and prognostic indications, as the early presence of Descemet's membrane detachment and corneal swelling appears to be correlated with the severity and localization of corneal neovascularization.

No abstract

No abstract