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In JoVE (1)
Other Publications (13)
- Analytical Chemistry
- Nature
- Cell Communication & Adhesion
- Journal of Neural Engineering
- Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
- Journal of Applied Animal Welfare Science : JAAWS
- Journal of Nanoscience and Nanotechnology
- Annals of Biomedical Engineering
- Journal of Biomedical Materials Research. Part A
- Journal of Biomechanical Engineering
- Experimental Cell Research
- Nanomedicine : Nanotechnology, Biology, and Medicine
- Journal of Nanobiotechnology
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Articles by Maribel Vazquez in JoVE
JoVE General
Флуоресцентные Анализ Скрининг на выявление каналов модуляторы GIRK
В режиме реального времени процедура скрининга для выявления наркотиков, которые взаимодействуют с G белком закрытый внутренний выпрямитель K
Other articles by Maribel Vazquez on PubMed
Electrophoretic Injection Within Microdevices
The flexibility of the microfabricated format creates unique opportunities for study of the electrophoretic process. The present work utilizes digital images to capture the motion of DNA samples during pre-electrophoretic processes. A systematic study of DNA loading and strong sample stacking (sample concentration effects) was performed in order to analyze realistic DNA analysis conditions within microdevices. Using digital imaging and microscopy, DNA sample profiles within the injector were analyzed by deconvolving the geometrical intensity profile into different velocity groups. This analysis illustrates the evolution of molecular separation into distinct migrating populations within the injector itself. The present study performed DNA injections within microfabricated devices imposing run voltages between 85 and 850 V/cm. Data from 3 different offset lengths of a double-T cross-injector, 10 different applied voltages, and 2 different sample preparation protocols are presented.
Independent Evolution of Bitter-taste Sensitivity in Humans and Chimpanzees
It was reported over 65 years ago that chimpanzees, like humans, vary in taste sensitivity to the bitter compound phenylthiocarbamide (PTC). This was suggested to be the result of a shared balanced polymorphism, defining the first, and now classic, example of the effects of balancing selection in great apes. In humans, variable PTC sensitivity is largely controlled by the segregation of two common alleles at the TAS2R38 locus, which encode receptor variants with different ligand affinities. Here we show that PTC taste sensitivity in chimpanzees is also controlled by two common alleles of TAS2R38; however, neither of these alleles is shared with humans. Instead, a mutation of the initiation codon results in the use of an alternative downstream start codon and production of a truncated receptor variant that fails to respond to PTC in vitro. Association testing of PTC sensitivity in a cohort of captive chimpanzees confirmed that chimpanzee TAS2R38 genotype accurately predicts taster status in vivo. Therefore, although Fisher et al.'s observations were accurate, their explanation was wrong. Humans and chimpanzees share variable taste sensitivity to bitter compounds mediated by PTC receptor variants, but the molecular basis of this variation has arisen twice, independently, in the two species.
Internal Fluid Flow Increases Cellular Interconnects Between Medial Collateral Ligament Fibroblasts and Cellular Extensions Within Three-dimensional Collagen Matrixes
The interconnectivity of fibroblasts within the ligamentous extracellular matrix has been largely overlooked. Studies on the cell-to-cell contacts with their neighbors via gap junctions in ligament fibroblasts, and works on the ability of fibroblasts to generate interconnected networks in vivo, suggest interfibroblastic interactions play an important role in fundamental biological processes, including homeostasis and wound healing. The current study examines how fluidic shear stresses imposed by internal flow can be used to mediate the formation of three-dimensional, interconnected fibroblast networks within collagen solutions. Several fibroblast-collagen solutions were exposed to shear stresses via Poiselle Flow. The consequent changes in cell networking, interconnections, and cell morphology within collagen matrixes exhibited by cells derived from Bovine Medial Collateral Ligaments were analyzed. Results illustrate that higher imposed stresses generate cells with more dendritic and/or branched morphologies, which form more visible three-dimensional networks within collagen matrixes than fibroblast-collagen solutions that were unexposed to shear stress.
Bio-heat Transfer Model of Deep Brain Stimulation-induced Temperature Changes
There is a growing interest in the use of chronic deep brain stimulation (DBS) for the treatment of medically refractory movement disorders and other neurological and psychiatric conditions. Fundamental questions remain about the physiologic effects of DBS. Previous basic research studies have focused on the direct polarization of neuronal membranes by electrical stimulation. The goal of this paper is to provide information on the thermal effects of DBS using finite element models to investigate the magnitude and spatial distribution of DBS-induced temperature changes. The parameters investigated include stimulation waveform, lead selection, brain tissue electrical and thermal conductivities, blood perfusion, metabolic heat generation during the stimulation and lead thermal conductivity/heat dissipation through the electrode. Our results show that clinical DBS protocols will increase the temperature of surrounding tissue by up to 0.8 degrees C depending on stimulation/tissue parameters.
Bio-heat Transfer Model of Deep Brain Stimulation Induced Temperature Changes
There is a growing interest in the use of chronic deep brain stimulation (DBS) for the treatment of medically refractory movement disorders and other neurological and psychiatric conditions. Fundamental questions remain about the physiologic effects and safety of DBS. Previous basic research studies have focused on the direct polarization of neuronal membranes by electrical stimulation. The goal of this paper is to provide information on the thermal effects of DBS using finite element models to investigate the magnitude and spatial distribution of DBS induced temperature changes. The parameters investigated include: stimulation waveform, lead selection, brain tissue electrical and thermal conductivity, blood perfusion, metabolic heat generation during the stimulation. Our results show that clinical deep brain stimulation protocols will increase the temperature of surrounding tissue by up to 0.8 deg C depending on stimulation/tissue parameters.
Combination Therapy Reduces Self-injurious Behavior in a Chimpanzee (Pan Troglodytes Troglodytes): a Case Report
Self-injurious behavior (SIB) remains a severe and intractable abnormal behavior for nonhuman primates in diverse settings and is a significant concern for veterinarians and behavioral scientists. To date, no single pharmacological, behavioral, social, or environmental intervention method has emerged as a reliable permanent cure for treating SIB in all, or even most, individuals. Implementation and evaluation of a combination therapeutic approach to treating SIB for nonhuman primates is rare. In May 2004, a 25-year-old male chimpanzee with severe SIB (M = 2.09 episodes/day, range = 1-4 episodes/day) underwent intensive behavioral intervention that utilized a combination of techniques. The combination therapy approach entailed the following: (a) pharmacological intervention with a gamma-aminobutyric acid (GABA) analogue to treat suspected HIV-related sensory neuropathic pain, (b) positive reinforcement training, and (c) environmental enrichment, as well as social and environmental modification. The severity of SIB warranted immediate implementation of intensive combination therapy rather than a systematic evaluation of the individual treatment options. The individually tailored, multifaceted combination therapy resulted in the virtual elimination of SIB in this chimpanzee over a 2-year period.
Liposome Delivery of Quantum Dots to the Cytosol of Live Cells
An increasing number of studies have demonstrated the multiple advantages of using nanocrystals, such as Quantum dots, for biological imaging. Quantum dots functionalized with biomolecules on their surfaces were shown to be able to bind to specific extracellular targets via specific recognition and to be internalized inside the cells, thereby allowing the imaging of intracellular pathways. However, the use of Quantum dots for live tracking of intracellular molecules is relatively limited because of the difficulties encountered during the induction of Quantum dots across living cell membranes. In this study we show that cationic liposomes can deliver low concentrations of non-targeted Quantum dots into the cytosol of living cells via a lipid-mediated fusion with the cell membrane. The intracellular Quantum dots exhibit aggregation that appears dependent upon their concentration, but does not visibly affect cell viability. Our results point towards the use of cationic liposomes as an effective delivery system for targeted Quantum dots within the cell cytosol, which would facilitate live cell imaging of the labeled molecules.
Live Cell Labeling of Glial Progenitor Cells Using Targeted Quantum Dots
This study describes the development of targeted quantum dots (T-QDs) as biomarkers for the labeling of glial progenitor cells (GPCs) that over express platelet derived growth factor (PDGF) and its receptor PDGFR (GPC(PDGF)). PDGFR plays a critical role in glioma development and growth, and is also known to affect multiple biological processes such as cell migration and embryonic development. T-QDs were developed using streptavidin-conjugated quantum dots (S-QDs) with biotinylated antibodies and utilized to label the intracellular and extracellular domains of live, cultured GPC(PDGF) cells via lipofection with cationic liposomes. Confocal studies illustrate successful intracellular and extracellular targeted labeling within live cells that does not appear to impact upstream PDGFR dynamics during real-time signaling events. Further, T-QDs were nontoxic to GPC(PDGF) cells, and did not alter cell viability or proliferation over the course of 6 days. These results raise new applications for T-QDs as ultra sensitive agents for imaging and tracking of protein populations within live cells, which that will enable future mechanistic study of oncogenic signaling events in real-time.
Flow-induced Shear Stresses Increase the Number of Cell-cell Contacts Within Extracellular Matrix
The formation of cell-cell contacts within extracellular matrix (ECM) is essential to maintain tissue homeostasis and metabolism, as well as critical toward the cell-ECM mechanotransduction that can affect intracellular organization and intercellular communication to enable cell response to external stimuli. This work illustrates the effects of shear stresses on cell-cell contacts within pre-stressed collagen ECM that were loaded in two separate conditions of constant flow (CF) and constant elution time (CET). The numbers of cell-cell contacts and cytoplasmic processes in both media and 3D ECM gels were analyzed in order to examine the shear effects of different magnitudes and time periods on 3D cell-ECM formation. The sheared collagen ECM microstructures were imaged and studied via scanning electron microscopy (SEM) to illustrate greater distances between constituent cells when larger shear stresses were applied. And the gap junction Connexin 43 expressed between networked cells that were sheared in short time period using CF loading exhibited more than those using CET loading. Notably, the number of cell-cell contacts increased when larger shear stresses were applied, suggesting these stresses may be used to increase intercellular communication within 3D matrixes.
A Microfluidic Device to Establish Concentration Gradients Using Reagent Density Differences
Microfabrication has become widely utilized to generate controlled microenvironments that establish chemical concentration gradients for a variety of engineering and life science applications. To establish microfluidic flow, the majority of existing devices rely upon additional facilities, equipment, and excessive reagent supplies, which together limit device portability as well as constrain device usage to individuals trained in technological disciplines. The current work presents our laboratory-developed bridged μLane system, which is a stand-alone device that runs via conventional pipette loading and can operate for several days without need of external machinery or additional reagent volumes. The bridged μLane is a two-layer polydimethylsiloxane microfluidic device that is able to establish controlled chemical concentration gradients over time by relying solely upon differences in reagent densities. Fluorescently labeled Dextran was used to validate the design and operation of the bridged μLane by evaluating experimentally measured transport properties within the microsystem in conjunction with numerical simulations and established mathematical transport models. Results demonstrate how the bridged μLane system was used to generate spatial concentration gradients that resulted in an experimentally measured Dextran diffusivity of (0.82 ± 0.01) × 10(-6) cm(2)/s.
Migration of Connective Tissue-derived Cells is Mediated by Ultra-low Concentration Gradient Fields of EGF
The directed migration of cells towards chemical stimuli incorporates simultaneous changes in both the concentration of a chemotactic agent and its concentration gradient, each of which may influence cell migratory response. In this study, we utilized a microfluidic system to examine the interactions between epidermal growth factor (EGF) concentration and EGF gradient in stimulating the chemotaxis of connective tissue-derived fibroblast cells. Cells seeded within microfluidic devices were exposed to concentration gradients established by EGF concentrations that matched or exceeded those required for maximum chemotactic responses seen in transfilter migration assays. The migration of individual cells within the device was measured optically after steady-state gradients had been experimentally established. Results illustrate that motility was maximal at EGF concentration gradients between .01- and 0.1-ng/(mL.mm) for all concentrations used. In contrast, the number of motile cells continually increased with increasing gradient steepness for all concentrations examined. Microfluidics-based experiments exposed cells to minute changes in EGF concentration and gradient that were in line with the acute EGFR phosphorylation measured. Correlation of experimental data with established mathematical models illustrated that the fibroblasts studied exhibit an unreported chemosensitivity to minute changes in EGF concentration, similar to that reported for highly motile cells, such as macrophages. Our results demonstrate that shallow chemotactic gradients, while previously unexplored, are necessary to induce the rate of directed cellular migration and the number of motile cells in the connective tissue-derived cells examined.
Targeted Extracellular Nanoparticles Enable Intracellular Detection of Activated Epidermal Growth Factor Receptor in Living Brain Cancer Cells
Mechanistic study of biological processes via Quantum Dots (QDs) remain constrained by inefficient QD delivery methods and consequent altered cell function. Here the authors present a rapid method to label activated receptor populations in live cancer cells derived from medulloblastoma and glioma tumors. The authors used QDs to bind the extracellular domain of Epidermal Growth Factor Receptor (EGF-R) proteins and then induced receptor activation to facilitate specific detection of intracellular, activated EGF-R subpopulations. Such labeling enables rapid identification of biological markers characteristic of tumor type, grade and chemotherapy resistance. FROM THE CLINICAL EDITOR: In this paper, a rapid, quantum dot-based method is presented with the goal of labeling activated receptor populations in live cancer cells. More accurate characterization of medulloblastoma and glioma cancer cells using this biomarker detection technique may lead to a more specific targeted therapy.
Sendai Virus-based Liposomes Enable Targeted Cytosolic Delivery of Nanoparticles in Brain Tumor-Derived Cells
ABSTRACT: BACKGROUND: Nanotechnology-based bioassays that detect the presence and/or absence of a combination of cell markers are increasingly used to identify stem or progenitor cells, assess cell heterogeneity, and evaluate tumor malignancy and/or chemoresistance. Delivery methods that enable nanoparticles to rapidly detect emerging, intracellular markers within cell clusters of biopsies will greatly aid in tumor characterization, analysis of functional state and development of treatment regimens. RESULTS: Experiments utilized the Sendai virus to achieve in vitro, cytosolic delivery of Quantum Dots (Qdots) in cells cultured from Human brain tumors. Using fluorescence microscopy and Transmission Electron Microscopy (TEM), in vitro experiments illustrate that our virus-based liposomes (VBL) decreased the amount of non-specifically endocytosed nanoparticles by 50% in the Human glioblastoma (GBM) and medulloblastoma (MB) samples studied. Significantly, VBL delivery also facilitated targeted binding of Qdots to cytosolic Epidermal Growth Factor Receptor (EGFR) within cultured cells, focal to the early detection and characterization of malignant brain tumors. CONCLUSIONS: These findings are the first to utilize the Sendai virus to achieve cytosolic, targeted intracellular binding of Qdots within Human brain tumor cells. The results are significant to the continued applicability of nanoparticles used for the molecular labeling of cancer cells to determine tumor heterogeneity, grade, and chemotherapeutic resistivity.
