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The role of calcium in signal transduction relies on the precise spatial and temporal control of its concentration. The existing means to detect fluctuations in Ca2+ concentrations with adequate temporal and spatial resolution are limited. We introduce here a method to measure Ca2+ concentrations in defined locations in living cells that is based on linking the Ca2+-sensitive dye Indo-1 to SNAP-tag fusion proteins. Fluorescence spectroscopy of SNAP-Indo-1 conjugates in vitro showed that the conjugates retained the Ca2+-sensing ability of Indo-1. In a proof-of-principle experiment, local Ca2+ sensing was demonstrated in single cells dissociated from muscle of adult mice expressing a nucleus-localized SNAP-tag fusion. Ca2+ concentrations inside nuclei of resting cells were measured by shifted excitation and emission ratioing of confocal microscopic images of fluorescence. After permeabilizing the plasma membrane, changes in the bathing solution induced corresponding changes in nuclear [Ca2+] that were readily detected and used for a preliminary calibration of the technique. This work thus demonstrates the synthesis and application of SNAP-tag-based Ca2+ indicators that combine the spatial specificity of genetically encoded calcium indicators with the advantageous spectroscopic properties of synthetic indicators.
ACS Chem Biol 2009 Mar 20
PMID:Indo-1 derivatives for local calcium sensing. 1919 35

Early detection is a crucial element for the timely diagnosis and successful treatment of all human cancers but is limited by the sensitivity of current imaging methodologies. We have synthesized and studied bioresorbable calcium phosphate nanoparticles (CPNPs) in which molecules of the near-infrared (NIR) emitting fluorophore, indocyanine green (ICG), are embedded. The ICG-CPNPs demonstrate exceptional colloidal and optical characteristics. Suspensions consisting of 16 nm average diameter particles are colloidally stable in physiological solutions (phosphate buffered 0.15 M saline (PBS), pH 7.4) with carboxylate or polyethylene glycol (PEG) surface functionality. ICG-doped CPNPs exhibit significantly greater intensity at the maximum emission wavelength relative to the free constituent fluorophore, consistent with the multiple molecules encapsulated per particle. The quantum efficiency per molecule of the ICG-CPNPs is 200% greater at 0.049 +/- 0.003 over the free fluorophore in PBS. Photostability based on fluorescence half-life of encapsulated ICG in PBS is 500% longer under typical clinical imaging conditions relative to the free dye. PEGylated ICG-CPNPs accumulate in solid, 5 mm diameter xenograft breast adenocarcinoma tumors via enhanced retention and permeability (EPR) within 24 h after systemic tail vein injection in a nude mouse model. Ex situ tissue imaging further verifies the facility of the ICG-CPNPs for deep-tissue imaging with NIR signals detectable from depths up to 3 cm in porcine muscle tissue. Our ex vivo and in vivo experiments verify the promise of the NIR CPNPs for diagnostic imaging in the early detection of solid tumors.
ACS Nano 2008 Oct 28
PMID:Near-infrared emitting fluorophore-doped calcium phosphate nanoparticles for in vivo imaging of human breast cancer. 1920 41

Recent advances and progress in nanobiotechnology have demonstrated many nanoparticles (NPs) as potential and novel drug delivery vehicles, therapeutic agents, and contrast agents and luminescent biological labels for bioimaging. The emergence of new biomedical applications based on NPs signifies the need to understand, compare, and manage their cytotoxicity. In this study, we demonstrated the use of high-content screening assay (HCA) as a universal tool to probe the cytotoxicity of NPs and specifically cadmium telluride quantum dots (CdTe QDs) and gold NPs (Au NPs) in NG108-15 murine neuroblastoma cells and HepG2 human hepatocellular carcinoma cells. Neural cells represent special interest for NP-induced cytotoxicity because the optical and electrical functionalities of materials necessary for neural imaging and interfacing are matched well with the properties of many NPs. In addition, the cellular morphology of neurons is particularly suitable for automated high content screening. HepG2 cells represent a good model for high content screening studies since they are commonly used as a surrogate for human hepatocytes in pharmaceutical studies. We found the CdTe QDs to induce primarily apoptotic response in a time- and dosage-dependent manner and produce different toxicological profiles and responses in undifferentiated and differentiated neural cells. Au NPs were found to inhibit the proliferation and intracellular calcium release of HepG2 cells.
ACS Nano 2008 May
PMID:High-content screening as a universal tool for fingerprinting of cytotoxicity of nanoparticles. 1920 90

Fluorescent indicators for calcium are incredibly powerful because they enable researchers to watch the movement of calcium ions in real time in living cells. The popular small molecule indicator Indo-1 has now been targeted to a defined location, namely, the nucleus of muscle cells, using SNAP-tag technology. This combination of a chemical probe with genetic targeting expands the available options for measuring local calcium events in cells.
ACS Chem Biol 2009 Mar 20
PMID:Expanding the repertoire of fluorescent calcium sensors. 1929 92

We assessed the mechanisms by which specialized hypothalamic ventromedial nucleus (VMN) neurons utilize both glucose and long-chain fatty acids as signaling molecules to alter their activity as a potential means of regulating energy homeostasis. Fura-2 calcium (Ca(2+)) and membrane potential dye imaging, together with pharmacological agents, were used to assess the mechanisms by which oleic acid (OA) alters the activity of dissociated VMN neurons from 3- to 4-wk-old rats. OA excited up to 43% and inhibited up to 29% of all VMN neurons independently of glucose concentrations. In those neurons excited by both 2.5 mM glucose and OA, OA had a concentration-dependent effective excitatory concentration (EC(50)) of 13.1 nM. Neurons inhibited by both 2.5 mM glucose and OA had an effective inhibitory concentration (IC(50)) of 93 nM. At 0.5 mM glucose, OA had markedly different effects on these same neurons. Inhibition of carnitine palmitoyltransferase, reactive oxygen species formation, long-chain acetyl-CoA synthetase and ATP-sensitive K(+) channel activity or activation of uncoupling protein 2 (UCP2) accounted for only approximately 20% of OA's excitatory effects and approximately 40% of its inhibitory effects. Inhibition of CD36, a fatty acid transporter that can alter cell function independently of intracellular fatty acid metabolism, reduced the effects of OA by up to 45%. Thus OA affects VMN neuronal activity through multiple pathways. In glucosensing neurons, its effects are glucose dependent. This glucose-OA interaction provides a potential mechanism whereby such "metabolic sensing" neurons can respond to differences in the metabolic states associated with fasting and feeding.
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PMID:Characteristics and mechanisms of hypothalamic neuronal fatty acid sensing. 1953 76

Besides the classical atom/ion/molecule based mechanism, nonclassical crystallization provides a nanoparticle-based crystallization pathway toward single crystals. However, there is a lack of experimentally established strategies for engineering a range of crystalline microstructures from common nanoparticles by nonclassical crystallization. We demonstrate that a commercial random copolymer polyelectrolyte poly(4-styrene sulfonate)-co-(maleic acid) (PSS-co-MA) considerably guides crystallization of calcium carbonate (CC) with a high versatility. The bioinspired nonclassical crystallization protocol yielded a series of calcite microstructures. Calcite single crystals obtained at low supersaturation show a pseudo-dodecahedral shape with curved faces, whereas increasing supersaturation generated calcite mesocrystals with pseudo-octahedral shapes and scalloped surfaces. Further increase of supersaturation induced the formation of polycrystalline multilayered and hollow spheres. In the initial growth stage of all these microstructures, amorphous CC nanoparticles formed as the early product. Remarkably, microparticles with minimal primitive (P)-surface were captured as the prominent intermediate indicative of liquidlike behavior. Moreover, nanogranular structures exist broadly in the as-synthesized crystals. These results demonstrate that the polyelectrolyte can effectively stabilize the amorphous CC nanoparticle precursors, impart control over the evolution from amorphous precursors via a liquid aggregate through P-surface intermediates to the final crystals, and thus allow the morphogenesis. Simple variation of calcium and polyeletrolyte concentrations enables a systematic control over the size and morphology of particles among pseudo-dodecahedra, pseudo-octahedra, multilayered spheres, and hollow spheres, which are expressed in a morphology diagram. A unifying nanoparticle aggregation formation mechanism was suggested to explain the morphogenesis by the combination of nonclassical crystallization and surface area minimization principles.
ACS Nano 2009 Jul 28
PMID:Polyelectrolyte-directed nanoparticle aggregation: systematic morphogenesis of calcium carbonate by nonclassical crystallization. 1957 17

In this article, we report a novel nanoparticle-enhanced biophysical technique that differentiates multipotent marrow stromal cells (MSCs) toward osteoblasts. We show that a brief (10 min) daily nanoparticle-facilitated exposure of MSCs to nanosecond pulse laser-induced photoacoustic (PA) stimulation enhances their differentiation toward osteoblasts. To observe osteodifferentiation under PA stimulation, tissue culture plates were seeded with MSCs without the osteogenic culture supplements (OS, 0.01 M beta-glycerophosphate, 50 mg/L ascorbic acid, 10(-8) M dexamethasone) in the presence and absence of single-walled carbon nanotubes (SWNTs) and gold nanoparticles (GNPs). The alkaline phosphatase activity, calcium content, and osteopontin secretion were monitored as indicators of MSCs' differentiation toward osteoblasts. The PA stimulated groups show up to 612% increase in calcium content compared to the controls cultured with osteogenic supplements (without PA stimulation) after 16 days. Among the PA stimulated groups, at day 16, MSCs incubated with SWNTs at 10 microg/mL concentrations showed up to 97% greater calcium content than those that did not contain SWNTs. The results demonstrated that PA stimulation not only promotes osteogenesis but also is synergistically enhanced by the presence of nanoparticles and, thus, has major implications for bone regeneration applications.
ACS Nano 2009 Aug 25
PMID:The effect of nanoparticle-enhanced photoacoustic stimulation on multipotent marrow stromal cells. 1960 49

Whole cantaloupes either not inoculated or inoculated with Salmonella Poona were submerged in water, 180 ppm of chlorine, acidified calcium sulfate (ACS: 1.2% Safe(2)O-ACS50), 1,000 ppm of acidified sodium chlorite (ASC), 80 ppm of peroxyacetic acid (PAA), and a combination of ACS and PAA for 10 min. Although only ASC and the combination of ACS and PAA significantly reduced the aerobic plate count of samples taken from the surface of whole cantaloupe (compared with samples taken from cantaloupe submerged in water only), all treatments reduced yeast and mold counts on the whole cantaloupe. However, none of the treatments of whole cantaloupes consistently reduced yeast and mold counts for the samples of fresh-cut cantaloupes. The aerobic plate counts for fresh-cut cantaloupe were reduced by 1 to 2 log CFU/g by sanitization of whole fruit with ASC, ACS, and the combination of ACS and PAA. The low bacterial population on the fresh-cut fruit was maintained during 14 days of storage at 4 degrees C. All treatments had a limited effect on the population of Salmonella, achieving no more than a 1.5-log reduction of the pathogen inoculated on the surface of the whole cantaloupes. Salmonella was nondetectable via direct plating (with a detection limit of 0.4 log CFU/g) in fresh-cut cantaloupes prepared from whole cantaloupes treated with any of the sanitizers. However, after enrichment, Salmonella often was detectable. Color, texture, soluble solids, pH, ascorbic acid, and drip loss of cut cantaloupes were not consistently affected by any of the whole-fruit treatments. Overall, treatments of whole cantaloupe with ASC, ACS, and the combination of ACS and PAA at the concentrations tested permitted a significant reduction in Salmonella and native microflora of whole and cut fruit; however, Salmonella still could be found in cut cantaloupes from all treatments.
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PMID:Use of chemical sanitizers to reduce microbial populations and maintain quality of whole and fresh-cut cantaloupe. 2000 25

The establishment of verifiably safe nanotechnology requires the development of assessment tools to identify hazardous nanomaterial properties that could be modified to improve nanomaterial safety. While there is a lot of debate of what constitutes appropriate safety screening methods, one approach is to use the assessment of cellular injury pathways to collect knowledge about hazardous material properties that could lead to harm to humans and the environment. We demonstrate the use of a multiparameter cytotoxicity assay that evaluates toxic oxidative stress to compare the effects of titanium dioxide (TiO(2)), cerium oxide (CeO(2)), and zinc oxide (ZnO) nanoparticles in bronchial epithelial and macrophage cell lines. The nanoparticles were chosen on the basis of their volume of production and likelihood of spread to the environment. Among the materials, dissolution of ZnO nanoparticles and Zn(2+) release were capable of ROS generation and activation of an integrated cytotoxic pathway that includes intracellular calcium flux, mitochondrial depolarization, and plasma membrane leakage. These responses were chosen on the basis of the compatibility of the fluorescent dyes that contemporaneously assess their response characteristics by a semiautomated epifluorescence procedure. Purposeful reduction of ZnO cytotoxicity was achieved by iron doping, which changed the material matrix to slow Zn(2+) release. In summary, we demonstrate the utility of a rapid throughput, integrated biological oxidative stress response pathway to perform hazard ranking of a small batch of metal oxide nanoparticles, in addition to showing how this assay can be used to improve nanosafety by decreasing ZnO dissolution through Fe doping.
ACS Nano 2010 Jan 26
PMID:Use of a rapid cytotoxicity screening approach to engineer a safer zinc oxide nanoparticle through iron doping. 2004 40

The use of delivery templates makes it possible to fabricate shaped, millimeter-thick heterogeneously patterned films of ionotropic hydrogels. These structures include two-dimensional (2-D) patterns of a polymer cross-linked by different ions (e.g., alginic acid cross-linked with Ca2+ and Fe3+) and patterns of step gradients in the concentration of a single cross-linking ion. The delivery templates consist of stacked sheets of chromatography paper patterned with hydrophobic barriers (waterproof tape, transparency film, or toner deposited by a color laser printer). Each layer of paper serves as a reservoir for a different solution of cross-linking ions, while the hydrophobic barriers prevent solutions on adjacent sheets from mixing. Holes cut through the sheets expose different solutions of cross-linking ions to the surface of the templates. Films with shaped regions of hydrogels cross-linked by paramagnetic ions can be oriented with a bar magnet. Variations in the concentrations of cations used to cross-link the gel can control the mechanical properties of the film: for single alginate films composed of areas cross-linked with different concentrations of Fe3+, the regions cross-linked with high concentrations of Fe3+ are more rigid than regions cross-linked with low concentrations of Fe3+. The heterogeneous hydrogel films can be used to culture bacteria in various 2-D designs. The pattern of toxic and nontoxic ions used to cross-link the polymer determines the pattern of viable colonies of Escherichia coli within the film.
ACS Appl Mater Interfaces 2009 Aug
PMID:Heterogeneous films of ionotropic hydrogels fabricated from delivery templates of patterned paper. 2004 55


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