Spinning Disk Confocal Microscopy for Optimized and Quantified Live Imaging of 3D Mitochondrial Network.

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

Fusion of Wild-Type Mesoangioblasts with Myotubes of mtDNA Mutation Carriers Leads to a Proportional Reduction in mtDNA Mutation Load.

In 25% of patients with mitochondrial myopathies, pathogenic mitochondrial DNA (mtDNA) mutation are the cause. For heteroplasmic mtDNA mutations, symptoms manifest when the mutation load exceeds a tissue-specific threshold. Therefore, lowering the mutation load is expected to ameliorate disease manifestations. This can be achieved by fusing wild-type mesoangioblasts with mtDNA mutant myotubes. We have tested this in vitro for female carriers of the m.3271T>C or m.3291T>C mutation (mutation load >90%) using wild-type male mesoangioblasts. Individual fused myotubes were collected by a newly-developed laser capture microdissection (LCM) protocol, visualized by immunostaining using an anti-myosin antibody. Fusion rates were determined based on male-female nuclei ratios by fluorescently labelling the Y-chromosome. Using combined 'wet' and 'air dried' LCM imaging improved fluorescence imaging quality and cell yield. Wild-type mesoangioblasts fused in different ratios with myotubes containing either the m.3271T>C or the m.3291T>C mutation. This resulted in the reduction of the mtDNA mutation load proportional to the number of fused wild-type mesoangioblasts for both mtDNA mutations. The proportional reduction in mtDNA mutation load in vitro after fusion is promising in the context of muscle stem cell therapy for mtDNA mutation carriers in vivo, in which we propose the same strategy using autologous wild-type mesoangioblasts.

Dystrobrevin is required postsynaptically for homeostatic potentiation at the Drosophila NMJ.

Evolutionarily conserved homeostatic systems have been shown to modulate synaptic efficiency at the neuromuscular junctions of organisms. While advances have been made in identifying molecules that function presynaptically during homeostasis, limited information is currently available on how postsynaptic alterations affect presynaptic function. We previously identified a role for postsynaptic Dystrophin in the maintenance of evoked neurotransmitter release. We herein demonstrated that Dystrobrevin, a member of the Dystrophin Glycoprotein Complex, was delocalized from the postsynaptic region in the absence of Dystrophin. A newly-generated Dystrobrevin mutant showed elevated evoked neurotransmitter release, increased bouton numbers, and a readily releasable pool of synaptic vesicles without changes in the function or numbers of postsynaptic glutamate receptors. In addition, we provide evidence to show that the highly conserved Cdc42 Rho GTPase plays a key role in the postsynaptic Dystrophin/Dystrobrevin pathway for synaptic homeostasis. The present results give novel insights into the synaptic deficits underlying Duchenne Muscular Dystrophy affected by a dysfunctional Dystrophin Glycoprotein complex.

Solubility of phenytoin in aqueous mixtures of ethanol and sodium dodecyl sulfate at 298 K / Solubilidad de la fenitoína en mezclas acuosas de etanol y dodecil-sulfato sódico a 298 K

The solubility of phenytoin in binary mixtures of ethanol + water at 298.2 K in the presence of three different concentrations of sodium dodecyl sulfate (SDS) was reported. The Jouyban-Acree model was used for correlating the generated data and the obtained mean relative deviation was 7.3 %. When all data points of phenytoin in binary solvents at various SDS concentrations were fitted to the model, the obtained mean relative deviation was 10.1 %.

Se presenta la solubilidad de la fenitoína a 298.2 K en mezclas binarias de etanol y agua en presencia de tres diferentes concentraciones de dodecil-sulfato sódico (DSS). Se utiliza el modelo de Jouyban-Acree para correlacionar los datos generados obteniendo una desviación media relativa de 7,3 %. Al ajustar al modelo todos los datos obtenidos de la fenitoína en los solventes binarios y en las diferentes concentraciones de DSS la desviación media relativa obtenida fue de 10,1%.

Cellular toxicity of nanogenomedicine in MCF-7 cell line: MTT assay.

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

Characterization and astrocytic modulation of system L transporters in brain microvasculature endothelial cells.

Brain trafficking of amino acids is mainly mediated by amino acids transport machineries of the blood-brain barrier (BBB), where astrocytes play a key maintenance role. However, little is known about astrocytes impacts on such transport systems, in particular system L that consists of large and small neutral amino acids (NAAs) transporters, that is, LAT1/4F2hc and LAT2/4F2hc, respectively. In the current investigation, functionality and expression of system L were studied in the immortalized mouse brain microvascular endothelial b.End3 cells cocultured with astrocytes or treated with astrocyte-conditioned media (ACM). LAT2/4F2hc mediated luminal uptake of L-phenylalanine and L-leucine resulted in significantly decreased affinity of system L in b.End3 cells treated with ACM, while LAT2/4F2hc mediated luminal uptake of L-alanine remained unchanged. Gene expression analysis revealed marked upregulation of LAT1 and 4F2hc, but downregulation of LAT2 in b.End3 cells cultured with ACM. The basal to apical transport of L-phenylalanine and L-alanine appeared to be significantly greater than that of the apical to basal direction in b.End3 cells indicating an efflux functionality of system L. No marked influence was observed for transport of L-phenylalanine in b.End3 cells cocultured with astrocytes, while a slight decrease was seen for L-alanine in the basal to apical direction. Based on our findings, we propose that system L functions as influx and/or efflux transport machinery displaying a greater propensity for the outward transport of large and small NAAs. Astrocytes appeared to modulate the transcriptic expression and uptake functionalities of system L, but not the transport activities.

Microarray analysis of the toxicogenomics and the genotoxic potential of a cationic lipid-based gene delivery nanosystem in human alveolar epithelial a549 cells.

ABSTRACT Viral and nonviral vectors have been widely used in gene therapy as delivery reagents for nucleic acids. Toxicity with viral vectors has increasingly led to the search for suitable nonviral vectors, such as cationic lipids/polymers, as potentially safer alternatives. However, little is known about the genomic toxicity of these delivery systems in target cells/tissues. In the current investigation, we report on the toxicogenomics and genotoxicity of cationic lipid Oligofectamine (OF) nanosystems in human alveolar epithelial A549 cells. To investigate the nature and the ontology of the gene expression changes in A549 cells upon treatment with OF nanoliposomes, microarray gene expression profiling methodology was utilized. For microarray analysis, cyanine (Cy3/Cy5)-labeled cDNA samples from treated and untreated cells were hybridized on target arrays housing 200 genes. Both OF and OF-DNA lipoplex induced significant gene expression changes belonging to the different genomic ontologies such as cell defense and apoptosis pathways. Flow cytometry analyses revealed induction of apoptosis in A549 cells treated with these nanosystems that is likely due to interactions and/or deterioration of the cell membranes. However, no DNA damage was detected by the Comet assay. These data suggest that cationic nanoliposomes in the absence of direct DNA damage elicit multiple gene expression changes in A549 cells that may compromise the main goals of gene medicine where only therapy-defined gene changes are required.