Showing papers by "Stefan Vogt published in 2011"
TL;DR: The mammalian egg uses a zinc-dependent switch mechanism to toggle between metaphase arrest and resumption of the meiotic cell cycle at the initiation of embryonic development.
Abstract: In last few hours of maturation, the mouse oocyte takes up over twenty billion zinc atoms and arrests after the first meiotic division, until fertilization or pharmacological intervention stimulates cell cycle progression toward a new embryo. Using chemical and physical probes, we show that fertilization of the mature, zinc-enriched egg triggers the ejection of zinc into the extracellular milieu in a series of coordinated events termed zinc sparks. These events immediately follow the well-established series of calcium oscillations within the activated egg and are evolutionarily conserved in several mammalian species, including rodents and nonhuman primates. Functionally, the zinc sparks mediate a decrease in intracellular zinc content that is necessary for continued cell cycle progression, as increasing zinc levels within the activated egg results in the reestablishment of cell cycle arrest at metaphase. The mammalian egg thus uses a zinc-dependent switch mechanism to toggle between metaphase arrest and r...
188 citations
TL;DR: Coppersensor-3 (CS3) is reported, a bright small-molecule fluorescent probe that offers the unique capability to image labile copper pools in living cells at endogenous, basal levels and opens opportunities for discovering and elucidating functions of copper in living systems.
Abstract: Dynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activation remains insufficiently explored, in large part because metals like copper and iron are typically studied as static cellular nutrients and there are a lack of direct, selective methods for monitoring their distributions in living cells. To help meet this need, we now report Coppersensor-3 (CS3), a bright small-molecule fluorescent probe that offers the unique capability to image labile copper pools in living cells at endogenous, basal levels. We use this chemical tool in conjunction with synchotron-based microprobe X-ray fluorescence microscopy (XRFM) to discover that neuronal cells move significant pools of copper from their cell bodies to peripheral processes upon their activation. Moreover, further CS3 and XRFM imaging experiments show that these dynamic copper redistributions are dependent on calcium release, establishing a link between mobile copper and major cell signaling pathways. By providing a small-molecule fluorophore that is selective and sensitive enough to image labile copper pools in living cells under basal conditions, CS3 opens opportunities for discovering and elucidating functions of copper in living systems.
168 citations
TL;DR: It was found that iron in the cortex was 34% higher than age-matched controls at an early stage, corresponding to the commencement of plaque formation, and the elevated iron was not associated with the amyloid plaques.
123 citations
TL;DR: This study demonstrates the subst a ntial biogeochemical insight that can be gained from studies of metal quotas in individual functional groups, and presents a generalized stoichiometry of Fe≈Zn>Mn≈Ni>Co in the plankton.
Abstract: The micronutrient metals Mn, Fe, Co, Ni and Zn are required for phytoplankton growth, and their availability influences ocean productivity and biogeochemistry. Here we report the first direct measurements of these metals in phytoplankton and protozoa from the equatorial Pacific Ocean. Cells representing 4 functional groups (diatoms, autotrophic flagellates, heterotrophic flagellates and autotrophic picoplankton) were collected from the surface mixed layer using trace-metal clean techniques during transects across the equator at 110°W and along the equator between 110°W and 140°W. Metal quotas were determined for individual cells with synchrotron x-ray fluorescence microscopy, and cellular stoichiometries were calculated relative to measured P and S, as well as to C calculated from biovolume. Bulk particulate (>3 μm) metal concentrations were also determined at 3 stations using inductively coupled plasma mass spectrometry for comparison to single-cell stoichiometries. Phosphorus-normalized Mn, Fe, Ni and Zn ratios were significantly higher in diatoms than other cell types, while Co stoichiometries were highest in autotrophic flagellates. The magnitude of these effects ranged from approximately 2-fold for Mn in diatoms and autotrophic flagellates to nearly an order of magnitude for Fe in diatoms and picoplankton. Variations in S-normalized metal stoichiometries were also significant but of lower magnitude (1.4 to 6-fold). Cobalt and Mn quotas were 1.6 and 3-fold higher in autotrophic than heterotrophic flagellates. Autotrophic picoplankton were relatively enriched in Ni but depleted in Zn, matching expectations based on known uses of these metals in prokaryotes and eukaryotes. Significant spatial variability in metal stoichiometries was also observed. At two stations deviations in Fe stoichiometries reflected features in the dissolved Fe distribution. At these same stations, high Ni stoichiometries in autotrophic flagellates were correlated with elevated ammonium and depressed nitrate concentrations. The spatial effects may have resulted from the passage of tropical instability waves along the equator. Comparison of bulk and single-cell results show similar Mn:P ratios at 2 of 3 stations, but Fe:P and Ni:P were systematically higher in bulk material and Co:P was lower. These results suggest an overrepresentation of diatoms or diatom-based detritus in the bulk fraction. Taken together, the analyses present a generalized stoichiometry of Fe≈Zn>Mn≈Ni>Co in the plankton. Diatom Fe quotas exceeded minimum subsistence levels, characteristic of cells growing actively on oxidized N sources. This study demonstrates the subst a ntial biogeochemical insight that can be gained from studies of metal quotas in individual functional groups.
115 citations
TL;DR: In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions and is less anisotropic than photoelectrons emission probability, consistent with photoelectron trajectory simulations.
Abstract: Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source of radiation damage. If the beam size is small relative to the photoelectron path length, then the photoelectron may escape the beam footprint, resulting in less damage in the illuminated volume. Thus, it may be possible to exploit this phenomenon to reduce radiation-induced damage during data measurement for techniques such as diffraction, spectroscopy, and imaging that use X-rays to probe both crystalline and noncrystalline biological samples. In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions. The damage rate normalized for dose was reduced by a factor of three from the largest (15.6 μm) to the smallest (0.84 μm) X-ray beam used. Radiation-induced damage to protein crystals was also mapped parallel and perpendicular to the polarization direction of an incident 1-μm X-ray beam. Damage was greatest at the beam center and decreased monotonically to zero at a distance of about 4 μm, establishing the range of photoelectrons. The observed damage is less anisotropic than photoelectron emission probability, consistent with photoelectron trajectory simulations. These experimental results provide the basis for data collection protocols to mitigate with micron-sized X-ray beams the effects of radiation damage.
114 citations
TL;DR: It is found that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway--receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active.
Abstract: Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway--receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu(C)Fe(N)Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.
72 citations
TL;DR: The change in speciation from the selenol, selenocysteine, to the diselenide,selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite.
Abstract: Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells but was only detected as a significant component between 24 and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than 2-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (μ-XANES) showed that the highly localized Se species was mostly elemental Se.
69 citations
TL;DR: Investigation of how ions are trafficked in C. moniliferum and how precipitation of (Ba,Sr)SO(4) crystals occurs in the terminal vacuoles proposes a "sulfate trap" model, which finds that the kinetics of Sr uptake and efflux depend on external Ca concentrations, and Sr, Ba, and Ca show similar intracellular localization.
57 citations
TL;DR: The present study highlights the quantitative capability, high statistical power, and versatility of the XFM technique for mapping trace metals in biological samples, e.g., brain tissue samples in order to help understand neurological processes, especially when implemented in conjunction with a high-performance detector such as Maia.
Abstract: X-ray fluorescence microscopy (XFM) facilitates high-sensitivity quantitative imaging of trace metals at high spatial resolution over large sample areas and can be applied to a diverse range of biological samples. Accurate determination of elemental content from recorded spectra requires proper calibration of the XFM instrument under the relevant operating conditions. Here, we describe the manufacture, characterization, and utilization of multi-element thin-film reference foils for use in calibration of XFM measurements of biological and other specimens. We have used these internal standards to assess the two-dimensional distribution of trace metals in a thin tissue section of a rat hippocampus. The data used in this study was acquired at the XFM beamline of the Australian Synchrotron using a new 384-element array detector (Maia) and at beamline 2-ID-E at the Advanced Photon Source. Post-processing of samples by different fixation techniques was investigated, with the conclusion that differences in solvent type and sample handling can significantly alter elemental content. The present study highlights the quantitative capability, high statistical power, and versatility of the XFM technique for mapping trace metals in biological samples, e.g., brain tissue samples in order to help understand neurological processes, especially when implemented in conjunction with a high-performance detector such as Maia.
54 citations
TL;DR: The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies.
Abstract: Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies. X-ray absorption spectroscopy of bulk cell pellets treated with the selenoamino acids for 24 h showed that while selenium was found exclusively in carbon-bound forms in SeMet-treated cells, a diselenide component was identified in MeSeCys-treated cells in addition to the carbon-bound selenium species. X-ray fluorescence microscopy of single cells showed that selenium accumulated with sulfur in the perinuclear region of SeMet-treated cells after 24 h, but microprobe selenium X-ray absorption near-edge spectroscopy in this region indicated that selenium was carbon-bound rather than sulfur-bound. X-ray absorption and X-ray fluorescence studies both showed that the selenium content of MeSeCys-treated cells was much lower than that of SeMet-treated cells. Selenium was distributed homogeneously throughout the MeSeCys-treated cells.
48 citations
TL;DR: Accumulation of Sr and Ba in the presence of up to five orders of magnitude excess Ca emphasizes that to address the selectivity problem, there is much to be learned and possibly gained from the strategies these organisms have evolved.
Abstract: The generation of radioactive waste and/or environmental radioactive contamination is a common side effect of activities such as nuclear power generation, medical use of radioisotopes, nuclear weapons testing, and occasionally disasters such as Chernobyl. The subsequent decontamination of waste or the environment requires the nontrivial ability to selectively separate and remove harmful radioisotopes such as Sr, a product of nuclear fission with a half-life of approximately 30 years. In the case of Sr, the chemical similarity of Ca, Sr, and Ba presents a challenge for even the most advanced ion-exchange materials. While phytoremediation approaches utilizing the accumulation of environmental contaminants by green plants are becoming increasingly popular, the effectiveness of such approaches for Sr sequestration are drastically reduced in the presence of Ca, due to the indiscriminate transport of Ca, Sr, and Ba exhibited by most organisms. Surprisingly, there are a small number of organisms that selectively sequester Sr and/or Ba in biominerals. For example, the marine radiolarian acantharea builds an endoskeleton from celestite (SrSO4), and the desmid [5] and stonewort green algae deposit barite (BaSO4) in vacuoles. Accumulation of Sr and Ba in the presence of up to five orders of magnitude excess Ca emphasizes that to address the selectivity problem, there is much to be learned and possibly gained from the strategies these organisms have evolved. Here, we quantitatively demonstrate the incorporation of up to 45 mol % Sr in barite crystals deposited by desmid green algae. The unicellular desmid green algae are ubiquitous in fresh water habitats and robust in culture, and as such are particularly suitable as a model system for Sr/Ba biomineralization and as a potential candidate for phytoremediation. In the desmid Closterium moniliferum, BaSO4 crystals are found in small terminal vacuoles at the tips of the crescent-shaped cells (Figure 1). Scanning electron microscopy and energy dispersive spectroscopy (SEM/EDS) analysis of BaSO4 crystals in ashed cells reveals clusters of mixed rhombic and hexagonal crystals of 0.5–1 mm diameter with 0.1–0.5 mm thickness (Figure 1), which exhibit strong Ba and S signals and little Ca (Supporting Information), consistent with previous descriptions.
TL;DR: MR is characterized by the reduced utilization of selenium due to a specific defect in GPx1 expression, and its selenoproteome was sequenced to characterize its liver transcriptome.
TL;DR: In this paper, the authors used synchrotron based x-ray fluorescence microscopy (SXRF) to study how the cellular Fe, Si, P and S contents of natural diatoms respond to additions of Fe and Si in the eastern equatorial Pacific (EEP), a major natural source of CO2 to the atmosphere.
Abstract: The elemental content of diatoms determines in part their productivity and their influence on biogeochemical cycles. We used a cell-specific technique, synchrotron based x-ray fluorescence microscopy (SXRF), to study for the first time how the cellular Fe, Si, P and S contents of natural diatoms respond to additions of Fe and Si in the eastern equatorial Pacific (EEP), a major natural source of CO2 to the atmosphere. We then compare these measurements to ratios of silicic acid, nitrate and phosphate drawdown in the experiments and to measurements of dissolved silicic acid and iron in the EEP. Addition of silicic acid (20 μM) resulted in increases of 25-50% in cell volume and cellular silica content in two experiments, but there was no significant change in either variable during a third experiment. No other effects of Si addition on cellular stoichiometry were observed. Cellular Fe content and molar ratios of Fe:P and Fe:S increased by 24- 14- and 17- fold 48 hours after addition of 2 nM Fe, but then declined to 7-, 4- and 6-fold higher than those in Control after 96 hours. Cellular P and S (a proxy for cell protein) both increased by up to 2-fold in response to Fe addition, and cell volume increased by 50-80%. Cellular Si content was not affected by Fe addition, but Si:P and Si:S ratios declined due to the effects of Fe on P and S. Our results suggest that Fe supply affects the ratios at which silicic acid and nitrate are utilized by blooming diatoms in the EEP. However, the production of biogenic silica per cell will not be affected by Fe supply. Consequently, in the EEP Fe availability is unlikely to influence the density and vertical transport of cellular material by affecting the amount of silica mineral ballast in cells. The relationship between silica content of diatoms and supply of silicic acid may help dampen variability in dissolved silicic acid concentrations in the EEP.
TL;DR: In this paper, the formation of Au-anomalous calcrete can be biomediated through the activity of resident microorganisms, and may not simply be the result of passive nucleation on inactive cells or evapotransporative processes.
TL;DR: The original and ongoing work with this tissue archive is presented here as a case study of a more general trend in the radiobiology megastudies.
Abstract: Between 1952 and 1992, more than 200 large radiobiology studies were conducted in research institutes throughout Europe, North America, and Japan to determine the effects of external irradiation and internal emitters on the lifespan and tissue toxicity development in animals. At Argonne National Laboratory, 22 external beam studies were conducted on nearly 700 beagle dogs and 50,000 mice between 1969 and 1992. These studies helped to characterize the effects of neutron and gamma irradiation on lifespan, tumorigenesis, and mutagenesis across a range of doses and dosing patterns. The records and tissues collected at Argonne during that time period have been carefully preserved and redisseminated. Using these archived data, ongoing statistical work has been done and continues to characterize quality of radiation, dose, dose rate, tissue, and gender-specific differences in the radiation responses of exposed animals. The ongoing application of newly-developed molecular biology techniques to the archived tissues has revealed gene-specific mutation rates following exposure to ionizing irradiation. The original and ongoing work with this tissue archive is presented here as a case study of a more general trend in the radiobiology megastudies. These experiments helped form the modern understanding of radiation responses in animals and continue to inform development of new radiation models. Recent archival efforts have facilitated open access to the data and materials produced by these studies, and so a unique opportunity exists to expand this continued research.
TL;DR: In this paper, a novel experimental approach was proposed to derive quantitative concentration map of light elements in whole cells by combining two complementary nano-probe methods: X-ray fluorescence microscopy (XRFM) and atomic force microscopy(AFM) and the concentration was derived by normalizing point-by-point the elemental spatial distribution obtained by XRFM, by the thickness measured using AFM.
TL;DR: PC12 cells can differentiate molecular and polymeric forms of plutonium, and molecular plutonium is taken up by PC12 cells and mostly co-localizes with iron but aged polymeric plutonium is not internalized by the cells.
Abstract: Purpose: To study the cellular responses to molecular and polymeric forms of plutonium using PC12 cells derived from a rat pheochromocytoma.Materials and methods: Serum starved PC12 cells were exposed to polymeric and molecular forms of plutonium for 3 h. Cells were washed with 10 mM ethylene glycol tetraacetic acid (EGTA), 100 mM NaCl at pH 7.4 to remove surface sorbed plutonium. Localization of plutonium in individual cell was quantitatively analyzed by synchrotron X-ray fluorescence (XRF) microscopy.Results: Molecular plutonium complexes introduced to cell growth media in the form of nitrilotriacetic acid (NTA), citrate, or transferrin complexes were taken up by PC12 cells, and mostly colocalized with iron within the cells. Aged polymeric plutonium prepared separately was not internalized by PC12 cells but it was always found on the cell surface as big agglomerates; however, polymeric plutonium formed in situ was mostly found within the cells as agglomerates.Conclusions: PC12 cells can differen...
TL;DR: XFM data are consistent with previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β-cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast.
Abstract: Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β-cells and showed that, in the presence of MnCl(2), glucose-activated pancreatic islets yield significant signal enhancement in T(1)-weigheted MR images. In this study, we exploited for the first time the unique capabilities of X-ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β-cells at cellular and subcellular levels. MIN-6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 × 10(-11)µg/µm(2), homogenously distributed across the cell. Exposure to 2 mM glucose and 50 µM MnCl(2) for 20 min resulted in nonglucose-dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 × 10(-11) µg/µm(2). When cells were activated by incubation in 16 mM glucose in the presence of 50 µM MnCl(2), a significant increase in cytoplasmic Mn was measured, reaching 2.57 ± 1.34 × 10(-10) µg/µm(2). A further rise in intracellular concentration was measured following KCl-induced depolarization, with concentrations totaling 1.25 ± 0.33 × 10(-9) and 4.02 ± 0.71 × 10(-10) µg/µm(2) in the cytoplasm and nuclei, respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β-cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast.
TL;DR: Microprobe X-ray absorption near edge structure measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1-μm(2) areas within single rat pheochromocytoma cells (PC12).
Abstract: Microprobe X-ray absorption near edge structure (μ-XANES) measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1-μm2 areas within single rat pheochromocytoma cells (PC12). The living cells were incubated in vitro for 3 h in the presence of Pu added to the media in different oxidation states (Pu(III), Pu(IV), and Pu(VI)) and in different chemical forms. Regardless of the initial oxidation state or chemical form of Pu presented to the cells, the XANES spectra of the intracellular Pu deposits were always consistent with tetravalent Pu even though the intracellular milieu is generally reducing.
TL;DR: In this paper, the authors showed that the uptake of the PtII-terpymbolidine (terpy) unit in a functionalized closo-carborane cage results in an increase in Cl, K, and Cu and a decrease in Fe within the treated cells.
Abstract: Treatment of A549 human lung carcinoma cells with a DNA metallointercalator complex containing a PtII-terpy (terpy = 2,2′:6′,2′′-terpyridine) unit linked to a functionalized closo-carborane cage results in the uptake of the complex within the cells, as determined by synchrotron X-ray fluorescence (XRF) imaging. Although a significant cellular uptake of Pt existed, there was no significant accumulation of the element within the cell nuclei. Other statistically significant changes from the XRF data included an increase in Cl, K, and Cu and a decrease in Fe within the treated cells.
TL;DR: The elemental composition of single cells of Nitrosomonas europaea was studied via synchrotron X-ray fluorescence microscopy (XFM) as a function of inhibition by divalent copper (Cu(II)) and batch growth phase to find out the impact of Cu inferred from specific oxygen uptake rate (sOUR) measurements at the two physiological states.
Abstract: The elemental composition of single cells of Nitrosomonas europaea 19718 was studied via synchrotron X-ray fluorescence microscopy (XFM) as a function of inhibition by divalent copper (Cu(II)) and batch growth phase. Based on XFM, the intracellular Cu concentrations in exponential phase cultures of N. europaea exposed to Cu(II) were statistically higher than in stationary phase cultures at the 95% confidence interval (α = 0.05). However, the impact of Cu inferred from specific oxygen uptake rate (sOUR) measurements at the two physiological states was statistically not dissimilar at the Cu(II) doses tested, except at 1000 µM Cu(II), at which exponential phase cultures were significantly more inhibited. Furthermore, the elemental composition in uninhibited exponential and stationary phase N. europaea cultures was similar. Notably, the molar fractions of Cu and Fe, relative to other elements in N. europaea cultures were statistically higher than those recently reported in Pseudomonas fluorescens possibly owing to the preponderance of metal cofactor rich catalytic enzymes (such as ammonia monooxygenase) and electron transport mechanisms in N. europaea.
09 Sep 2011
TL;DR: Inside cells imaged by XFM, the complex elemental signature of nanoconjugates (Ti-Co-Fe-Au) always co-registered in the 2D elemental map of the cell and retained its integrity as shown by X-ray fluorescence microscopy.
Abstract: A multimodal nanocomposite was designed, synthesized with super‐paramagnetic core (CoFe2O4), noble metal corona (Au), and semiconductor shell (TiO2). The sizes of core, core‐corona, and core‐corona‐shell particles were determined by TEM. This multimodal nanocrystal showed promise as a contrast agent for two of the most widely used biomedical imaging techniques: magnetic resonance imaging (MRI) and x‐ray computed tomography (CT). Finally, these nanocomposites were coated with a peptide SN‐50. This led to their ready uptake by the cultured cells and targeted the nanocomposites to the pores of nuclear membrane. Inside cells, this nanocomposite retained its integrity as shown by x‐ray fluorescence microscopy (XFM). Inside cells imaged by XFM we found the complex elemental signature of nanoconjugates (Ti‐Co‐Fe‐Au) always co‐registered in the 2D elemental map of the cell.
01 Sep 2011
TL;DR: Interestingly, with increased incubation times, EGFR targeted nanoconjugates could also be found colocalized with EGFR within the cell nucleus, which suggests that EGFR-targeted nanoconjongates can bind the receptor at the cell membrane, which leads to the internalization of NC-receptor complexes and the subsequent transport of nanconjugates into the nucleus.
Abstract: We are developing TiO2 nanoconjugates that can be used as therapeutic and diagnostic agents. Nanoscale TiO2 can be surface conjugated with various molecules and has the unique ability to induce the production of reactive oxygen species after radiation activation. One way to improve the potential clinical usefulness of TiO2 nanoparticles is to control their delivery to malignant cells by targeting them to cancer‐cell‐specific antigens. Epidermal growth factor receptor (EGFR) is one potential target that is enriched in epithelial cancers and is rapidly internalized after ligand binding. Hence, we have synthesized TiO2 nanoparticles and functionalized them with a short EGFR‐binding peptide to create EGFR‐targeted NCs. X‐ray fluorescence microscopy was used to image nanoconjugates within EGFR‐positive HeLa cells. Further labeling of fixed cells with antibodies against EGFR and Protein A nanogold showed that TiO2 nanoconjugates can colocalize with receptors at the cell’s plasma membrane. Interestingly, with in...
01 Jan 2011
TL;DR: It is reported that iron oxide–titanium dioxide core-shell nanocomposites can serve as efficient carriers for doxorubicin to overcome this common mechanism of drug resistance in cancer cells.
Abstract: Resistance to anthracyclines and other chemotherapeutics due to P-glycoprotein (pgp)-mediated export is a frequent problem in cancer treatment. Here, we report that iron oxide–titanium dioxide core-shell nanocomposites can serve as efficient carriers for doxorubicin to overcome this common mechanism of drug resistance in cancer cells. Doxorubicin nanocarriers (DNC) increased effective drug uptake in drug-resistant ovarian cells. Mechanistically, doxorubicin bound to the TiO2 surface by a labile bond that was severed upon acidification within cell endosomes. Upon its release, doxorubicin traversed the intracellular milieu and entered the cell nucleus by a route that evaded pgp-mediated drug export. Confocal and X-ray fluorescence microscopy and flow cytometry were used to show the ability of DNCs to modulate transferrin uptake and distribution in cells. Increased transferrin uptake occurred through clathrin-mediated endocytosis, indicating that nanocomposites and DNCs may both interfere with removal of transferrin from cells. Together, our findings show that DNCs not onlyprovideanalternativerouteofdeliveryofdoxorubicintopgp-overexpressingcancercellsbutalsomayboost the uptake of transferrin-tagged therapeutic agents. Cancer Res; 72(3); 769–78. � 2011 AACR.
16 Sep 2011
TL;DR: In this paper, a methodology that combines scanning x-ray fluorescencee microscopy and atomic force microscopy is presented to determine a concentration map of Mg in whole (not sectioned) cells.
Abstract: We present in this paper a novel methodology that combines scanning x‐ray fluorescencee microscopy and atomic force microscopy. The combination of these two techniques allows the determination of a concentration map of Mg in whole (not sectioned) cells.