RESUMEN
As the second most common transition metal in the human body, zinc is of great interest to research but has few viable routes for its direct structural study in biological systems. Herein, Zn valence-to-core X-ray emission spectroscopy (VtC XES) and Zn K-edge X-ray absorption spectroscopy (XAS) are presented as a means to understand the local structure of zinc in biological systems through the application of these methods to a series of biologically relevant molecular model complexes. Taken together, the Zn K-edge XAS and VtC XES provide a means to establish the ligand identity, local geometry, and metal-ligand bond lengths. Experimental results are supported by correlation with density-functional-theory-based calculations. Combining these theoretical and experimental approaches will enable future applications to protein systems in a predictive manner.
Asunto(s)
Sondas Moleculares/química , Zinc/química , Ligandos , Espectrometría por Rayos X/métodos , Espectroscopía de Absorción de Rayos X/métodosRESUMEN
Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful method for detection and quantification of nanoparticles. Unfortunately, the linear dynamic range of single particle analysis is hindered by "unruly" transient signals, momentary pulse pile-ups at the electron multiplier detector. This study seeks to extend the dynamic range of ICP-MS nanoparticle quantification via addition of a collision gas in the collision cell of the ICP-MS. The collision gas temporally broadens the nanoparticle signal resulting in decreased pulse pile-up and increased integrated intensity, up to a point where scattering losses begin to dominate. We tested collisional broadening with a dual mode simultaneous secondary electron multiplier (pulse counting switching to analog) and the same detector configured for pulse counting only operation. With no collision gas and the detector operating in its standard dual mode, the data shows a linear response for gold nanoparticles from 20â¯nm (smallest measured size) to 150â¯nm. With the addition of helium as a collision gas in the cell, the linear range extends up to 250â¯nm. The data collected exclusively from the pulse counting mode shows that with no collision gas there is a linear response for gold nanoparticles from 20â¯nm to 60â¯nm. While the signal slightly improves with the addition of a collision gas, the linear range fails to extend up to 80â¯nm, the next largest nanoparticle size in this study. The addition of a collision gas used together with the dual mode detector shows a promising path forward towards mitigating unruly transient signals, improving the dynamic range of nanoparticle quantification.
RESUMEN
OBJECTIVE: Dynamic contrast-enhanced MRI (DCE-MRI) has become a standard component of multiparametric protocols for MRI examination of the prostate, and its use is incorporated into current guidelines for prostate MRI examination. Analysis of DCE-MRI data for the prostate is usually based on the distribution of gadolinium-based agents, such as gadodiamide, into two well-mixed compartments, and it assumes that gadodiamide does not enter into the glandular lumen. However, this assumption has not been directly tested. The purpose of this study was to use x-ray fluorescence microscopy (XFM) imaging in situ to measure the concentration of gadodiamide in the epithelia and lumens of the prostate of healthy mice after IV injection of the contrast agent. MATERIALS AND METHODS: Six C57Bl6 male mice (age, 28 weeks) were sacrificed 10 minutes after IV injection of gadodiamide (0.13 mmol/kg), and three mice were sacrificed after saline injection. Prostate tissue samples obtained from each mouse were harvested and frozen; 7-µm-thick slices were sectioned for XFM imaging, and adjacent 5-µm-thick slices were sectioned for H and E staining. Elemental concentrations were determined from XFM images. RESULTS: A mean (± SD) baseline concentration of gadolinium of 0.01 ± 0.01 mM was determined from XFM measurements of prostatic tissue samples when no gadodiamide was administered, and it was used to determine the measurement error. When gadodiamide was added, the mean concentrations of gadolinium in the epithelia and lumens in 32 prostatic glands from six mice were 1.00 ± 0.13 and 0.36 ± 0.09 mM, respectively. CONCLUSION: Our data suggest that IV administration of gadodiamide results in uptake of contrast agent by the glandular lumens of the mouse prostate. We were able to quantitatively determine gadodiamide distributions in mouse prostatic epithelia and lumens.
Asunto(s)
Medios de Contraste/farmacocinética , Gadolinio DTPA/farmacocinética , Próstata/diagnóstico por imagen , Próstata/metabolismo , Animales , Medios de Contraste/administración & dosificación , Epitelio/diagnóstico por imagen , Epitelio/metabolismo , Gadolinio DTPA/administración & dosificación , Inyecciones Intravenosas , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Modelos Animales , RadiografíaRESUMEN
Contrast agents that specifically enhance cancers on magnetic resonance imaging (MRI) will allow earlier detection. Vanadium-based chelates (VCs) selectively enhance rodent cancers on MRI, suggesting selective uptake of VCs by cancers. Here we report x-ray fluorescence microscopy (XFM) of VC uptake by murine colon cancer. Colonic tumors in mice treated with azoxymethane/dextran sulfate sodium were identified by MRI. Then a gadolinium-based contrast agent and a VC were injected intravenously; mice were sacrificed and colons sectioned. VC distribution was sampled at 120 minutes after injection to evaluate the long-term accumulation. Gadolinium distribution was sampled at 10 minutes after injection due to its rapid washout. XFM was performed on 72 regions of normal and cancerous colon from five normal mice and four cancer-bearing mice. XFM showed that all gadolinium was extracellular, with similar concentrations in colon cancers and normal colon. In contrast, the average VC concentration was twofold higher in cancers versus normal tissue (p < .002). Cancers also contained numerous "hot spots" with intracellular VC concentrations sixfold higher than the concentration in normal colon (p < .0001). No hot spots were detected in normal colon. This is the first direct demonstration that VCs selectively accumulate in cancer cells and thus may improve cancer detection.
Asunto(s)
Medios de Contraste/química , Microanálisis por Sonda Electrónica , Imagen por Resonancia Magnética , Microscopía Fluorescente , Vanadio/química , Animales , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Femenino , Gadolinio/química , Glucólisis , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/patologíaRESUMEN
Rapidly-frozen hydrated (cryopreserved) specimens combined with cryo-scanning x-ray fluorescence microscopy provide an ideal approach for investigating elemental distributions in biological cells and tissues. However, because cryopreservation does not deactivate potentially infectious agents associated with Risk Group 2 biological materials, one must be concerned with contamination of expensive and complicated cryogenic x-ray microscopes when working with such materials. We employed ultraviolet germicidal irradiation to decontaminate previously cryopreserved cells under liquid nitrogen, and then investigated its effects on elemental distributions under both frozen hydrated and freeze dried states with x-ray fluorescence microscopy. We show that the contents and distributions of most biologically important elements remain nearly unchanged when compared with non-ultraviolet-irradiated counterparts, even after multiple cycles of ultraviolet germicidal irradiation and cryogenic x-ray imaging. This provides a potential pathway for rendering Risk Group 2 biological materials safe for handling in multiuser cryogenic x-ray microscopes without affecting the fidelity of the results.
Asunto(s)
Criopreservación , Células Madre Embrionarias/efectos de la radiación , Fibroblastos/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Animales , Microanálisis por Sonda Electrónica/métodos , Células Madre Embrionarias/química , Fibroblastos/química , Ratones , Microscopía FluorescenteRESUMEN
A novel approach to locate, identify and refine positions and whole areas of cell structures based on elemental contents measured by X-ray fluorescence microscopy is introduced. It is shown that, by initializing with only a handful of prototypical cell regions, this approach can obtain consistent identification of whole cells, even when cells are overlapping, without training by explicit annotation. It is robust both to different measurements on the same sample and to different initializations. This effort provides a versatile framework to identify targeted cellular structures from datasets too complex for manual analysis, like most X-ray fluorescence microscopy data. Possible future extensions are also discussed.
RESUMEN
Metalloproteins are enormously important in biology. While a variety of techniques exist for studying metals in biology, X-ray absorption spectroscopy is particularly useful in that it can determine the local electronic and physical structure around the metal center, and is one of the few avenues for studying "spectroscopically silent" metal ions like Zn(II) and Cu(I) that have completely filled valence bands. While X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) are useful for studying metalloprotein structure, they suffer the limitation that the detected signal is an average of all the various metal centers in the sample, which limits its usefulness for studying metal centers in situ or in cell lysates. It would be desirable to be able to separate the various proteins in a mixture prior to performing X-ray absorption studies, so that the derived signal is from one species only. Here we describe a method for performing X-ray absorption spectroscopy on protein bands following electrophoretic separation and western blotting.
Asunto(s)
Metaloproteínas/análisis , Espectroscopía de Absorción de Rayos X/métodos , Animales , Bovinos , Caballos , Electroforesis en Gel de Poliacrilamida Nativa , Conejos , Espectrometría por Rayos X , Estadística como AsuntoRESUMEN
X-ray fluorescence (XRF) microscopy is an important tool for studying trace metals in biology, enabling simultaneous detection of multiple elements of interest and allowing quantification of metals in organelles without the need for subcellular fractionation. Currently, analysis of XRF images is often done using manually defined regions of interest (ROIs). However, since advances in synchrotron instrumentation have enabled the collection of very large data sets encompassing hundreds of cells, manual approaches are becoming increasingly impractical. We describe here the use of soft clustering to identify cell ROIs based on elemental contents, using data collected over a sample of the malaria parasite Plasmodium falciparum as a test case. Soft clustering was able to successfully classify regions in infected erythrocytes as "parasite," "food vacuole," "host," or "background." In contrast, hard clustering using the k-means algorithm was found to have difficulty in distinguishing cells from background.While initial tests showed convergence on two or three distinct solutions in 60% of the cells studied, subsequent modifications to the clustering routine improved results to yield 100% consistency in image segmentation. Data extracted using soft cluster ROIs were found to be as accurate as data extracted using manually defined ROIs, and analysis time was considerably improved.
Asunto(s)
Microanálisis por Sonda Electrónica/métodos , Metales/análisis , Orgánulos/química , Plasmodium falciparum/químicaRESUMEN
Dynamic fluxes in the concentration of ions and small molecules are fundamental features of cell signaling, differentiation, and development. Similar roles for fluxes in transition metal concentrations are less well established. Here, we show that massive zinc fluxes are essential in the infection cycle of an intracellular eukaryotic parasite. Using single-cell quantitative imaging, we show that growth of the blood-stage Plasmodium falciparum parasite requires acquisition of 30 million zinc atoms per erythrocyte before host cell rupture, corresponding to a 400% increase in total zinc concentration. Zinc accumulates in a freely available form in parasitophorous compartments outside the food vacuole, including mitochondria. Restriction of zinc availability via small molecule treatment causes a drop in mitochondrial membrane potential and severely inhibits parasite growth. Thus, extraordinary zinc acquisition and trafficking are essential for parasite development.
Asunto(s)
Eritrocitos/efectos de los fármacos , Simulación de Dinámica Molecular , Plasmodium falciparum/efectos de los fármacos , Zinc/farmacología , Relación Dosis-Respuesta a Droga , Eritrocitos/química , Humanos , Plasmodium falciparum/crecimiento & desarrollo , Relación Estructura-Actividad , Zinc/químicaRESUMEN
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 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 increased incubation times, EGFR targeted nanoconjugates could also be found colocalized with EGFR within the cell nucleus. This suggests that EGFR-targeted nanoconjugates can bind the receptor at the cell membrane, which leads to the internalization of NC-receptor complexes and the subsequent transport of nanoconjugates into the nucleus.
RESUMEN
The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.
Asunto(s)
Diferenciación Celular , Núcleo Celular/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Zinc/metabolismo , Activinas/farmacología , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Cromosomas Humanos/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Endodermo/citología , Endodermo/efectos de los fármacos , Humanos , Mitosis , Células Madre Pluripotentes/efectos de los fármacos , Compuestos Policíclicos/metabolismo , Tretinoina/farmacologíaRESUMEN
We employed solid state (2)H NMR, complemented by computer simulations, to compare molecular organization in model membranes composed of 1-elaidoyl-2-stearoylphosphatidylcholine (t18:1-18:0PC), 1-oleoyl-2-stearoylphosphatidylcholine (c18:1-18:0PC), and 1,2-distearoylphosphatidylcholine (18:0-18:0PC). These phospholipids have elaidic acid (EA) containing a trans double bond, oleic acid (OA) containing a cis double bond, and saturated stearic acid (SA), respectively, at the sn-1 position and were synthesized with perdeuterated SA at the sn-2 position. The temperature of the chain melting transition is depressed less for t18:1-18:0PC (31.5 degrees C) than c18:1-18:0PC (7 degrees C) relative to 18:0-18:0PC (53 degrees C), reflecting the smaller deviation from the linear conformation produced by a trans as opposed to cis double bond. Acyl chain order in t18:1-18:0PC (S(CD) = 0.135) in the liquid crystalline state is much closer to that of c18:1-18:0PC (S(CD) = 0.128) than that of the substantially more ordered 18:0-18:0PC (S(CD) > 0.156), which is attributed to the reduced energy barrier to rotation about the C-C single bonds next to either a trans or cis carbon double bond. A conformation that somewhat resembles a saturated chain and an intrinsic disorder approaching that of a cis unsaturated chain characterize EA and, we speculate, may play a role in the adverse impact dietary trans fatty acids (TFA) have on biological function.
Asunto(s)
Membrana Dobles de Lípidos/química , Fosfatidilcolinas/química , Ácidos Grasos trans/química , Algoritmos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Ácido Oléico/química , Ácidos Oléicos , Transición de Fase , Temperatura , Temperatura de TransiciónRESUMEN
This study examined the effects of an external nasal dilator (END) on sedentary and aerobically trained women using the blood lactate threshold as a measure of aerobic performance. Three groups of women (sedentary, pre-season, in-season) participated in the study: nine sedentary college students (age 19 +/- 1.0 y), eight pre-season college athletes (age 20 +/- 2.3 y), and six in-season college rowers (age 20 +/- 1.7 y). A two-way repeated-measures design was used with subjects in each group being exposed to both conditions (with END and without END). The first two groups performed two incremental exercise tests in random order on a cycle ergometer, and the third group performed the tests on a rowing ergometer. Participants in each group wore an END strip for only one of the tests. Venous blood was collected at rest, during the final 30 seconds of each stage, and 1 and 3 minutes into the recovery period for the determination of blood lactate concentration and identification of the blood lactate threshold. No significant differences (P = 0.05) were found in blood lactate concentration at the lactate threshold between conditions for either group (sedentary: with END 2.51 +/- 1.18 mmol x L(-1), without END 2.56 +/- 0.84 mmol x L(-1); pre-season: with END 2.93 +/- 0.97 mmol x L(-1), without END 2.81 +/- 1.15 mmol x L(-1); and in-season: with END 3.93 +/- 0.50 mmol x L(-1), without END 3.49 +/- 0.387 mmol x L(-1)). We conclude that (a) the END did not improve the lactate threshold in either sedentary or trained college-age women, and (b) the END did not result in lower blood lactate levels during moderate to high-intensity exercise in the three groups examined in this study.
Asunto(s)
Dilatación/instrumentación , Ergometría , Ejercicio Físico/fisiología , Lactatos/sangre , Esfuerzo Físico/fisiología , Adulto , Umbral Anaerobio , Análisis de Varianza , Estudios Cruzados , Diseño de Equipo , Femenino , Humanos , Nariz , Consumo de Oxígeno/fisiología , Probabilidad , Valores de Referencia , Mecánica Respiratoria , Sensibilidad y Especificidad , DeportesRESUMEN
Organic-coated superparamagnetic iron oxide nanoparticles (OC-SPIONs) were synthesized and characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. OC-SPIONs were transferred from organic media into water using poly(amidoamine) dendrimers modified with 6-TAMRA fluorescent dye and folic acid molecules. The saturation magnetization of the resulting dendrimer-coated SPIONs (DC-SPIONs) was determined, using a superconducting quantum interference device, to be 60 emu/g Fe versus 90 emu/g Fe for bulk magnetite. Selective targeting of the DC-SPIONs to KB cancer cells in vitro was demonstrated and quantified using two distinct and complementary imaging modalities: UV-visible and X-ray fluorescence; confocal microscopy confirmed internalization. The results were consistent between the uptake distribution quantified by flow cytometry using 6-TAMRA UV-visible fluorescence intensity and the cellular iron content determined using X-ray fluorescence microscopy.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Compuestos Férricos , Ácido Fólico/farmacocinética , Imagen por Resonancia Magnética/métodos , Nanomedicina/métodos , Nanoestructuras , Línea Celular Tumoral , Medios de Contraste/farmacocinética , Cristalización/métodos , Portadores de Fármacos/química , Compuestos Férricos/farmacocinética , Ácido Fólico/química , Humanos , Sustancias Macromoleculares/química , Magnetismo , Ensayo de Materiales , Conformación Molecular , Nanoestructuras/química , Nanoestructuras/ultraestructura , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The ever-increasing amount of trans fatty acids in the human diet has been linked to a variety of afflictions, most notably coronary heart disease and arteriosclerosis. The mechanism of why the replacement of cis fatty acids with their trans counterparts can be detrimental to the health of an individual remains a mystery. Here, we compare the differences in membrane physical properties including molecular dynamics, lateral lipid packing, thermotropic phase behavior, "fluidity", lateral mobility, and permeability between model membranes (lipid monolayers and bilayers) composed of cis- and trans-containing phosphatidylcholines (PCs). The PCs tested have a total of zero, one, two, or four cis (oleic or linoleic) or trans (elaidic or linoelaidic) double bonds. These experiments all confirm the basic hypothesis that trans fatty acids produce membrane properties more similar to those of saturated chains than to those of acyl chains containing cis double bonds; i.e., cis double bonds induce much larger membrane perturbations than trans double bonds.