RESUMO
Non-invasive methods of detecting radiation exposure show promise to improve upon current approaches to biological dosimetry in ease, speed, and accuracy. Here we developed a pipeline that employs Fourier transform infrared (FTIR) spectroscopy in the mid-infrared spectrum to identify a signature of low dose ionizing radiation exposure in mouse ear pinnae over time. Mice exposed to 0.1 to 2 Gy total body irradiation were repeatedly measured by FTIR at the stratum corneum of the ear pinnae. We found significant discriminative power for all doses and time-points out to 90 days after exposure. Classification accuracy was maximized when testing 14 days after exposure (specificity > 0.9 with a sensitivity threshold of 0.9) and dropped by roughly 30% sensitivity at 90 days. Infrared frequencies point towards biological changes in DNA conformation, lipid oxidation and accumulation and shifts in protein secondary structure. Since only hundreds of samples were used to learn the highly discriminative signature, developing human-relevant diagnostic capabilities is likely feasible and this non-invasive procedure points toward rapid, non-invasive, and reagent-free biodosimetry applications at population scales.
Assuntos
Exposição à Radiação , Radiometria , Humanos , Camundongos , Animais , Espectroscopia de Infravermelho com Transformada de Fourier , Análise de Fourier , Radiometria/métodos , Proteínas , Radiação Ionizante , Exposição à Radiação/análise , Doses de RadiaçãoRESUMO
A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the water thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration.
Assuntos
Células/química , Microfluídica/instrumentação , Espectroscopia de Infravermelho com Transformada de Fourier/instrumentação , Animais , Microfluídica/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Síncrotrons , Água/químicaRESUMO
Spatially resolved infrared spectroscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal information on functional groups in biomolecules of a sample by their characteristic vibrational modes. One difficulty in performing long-term FT-IR measurements on live cells is the competition between the strong IR absorption from water and the need to supply nutrients and remove waste. In this proof of principle study, we developed an open-channel membrane device that allows long-term continuous IR measurement of live, adherent mammalian cells. Composed of a gold-coated porous membrane between a feeding channel and a viewing chamber, it allows cells to be maintained on the upper membrane surface in a thin layer of fluid while media is replenished from the feeding channel below. Using this device, we monitored the spatiotemporal chemical changes in living colonies of PC12 cells under nerve growth factor (NGF) stimulation for up to 7 days using both conventional globar and high-resolution synchrotron radiation-based IR sources. We identified the primary chemical change cells undergo is an increase in glycogen that may be associated with secretion of glycoprotein to protect the cells from evaporative stress at the air-liquid interface. Analyzing the spectral maps with multivariate methods of hierarchical cluster analysis (HCA) and principal component analysis (PCA), we found that the cells at the boundary of the colony and in a localized region in the center of the colony tend to produce more glycogen and glycoprotein than cells located elsewhere in the colony and that the degree of spatial heterogeneity decreases with time. This method provides a promising approach for long-term live-cell spectromicroscopy on mammalian cell systems.
Assuntos
Técnicas Analíticas Microfluídicas , Animais , Adesão Celular , Análise por Conglomerados , Células PC12 , Análise de Componente Principal , Ratos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
A new experimental setup for spatially resolved ambient infrared laser ablation-mass spectrometry (AIRLAB-MS) that uses an infrared microscope with an infinity-corrected reflective objective and a continuous flow solvent probe coupled to a Fourier transform ion cyclotron resonance mass spectrometer is described. The efficiency of material transfer from the sample to the electrospray ionization emitter was determined using glycerol/methanol droplets containing 1 mM nicotine and is â¼50%. This transfer efficiency is significantly higher than values reported for similar techniques. Laser desorption does not induce fragmentation of biomolecules in droplets containing bradykinin, leucine enkephalin and myoglobin, but loss of the heme group from myoglobin occurs as a result of the denaturing solution used. An application of AIRLAB-MS to biological materials is demonstrated for tobacco leaves. Chemical components are identified from the spatially resolved mass spectra of the ablated plant material, including nicotine and uridine. The reproducibility of measurements made using AIRLAB-MS on plant material was demonstrated by the ablation of six closely spaced areas (within 2 × 2 mm) on a young tobacco leaf, and the results indicate a standard deviation of <10% in the uridine signal obtained for each area. The spatial distribution of nicotine was measured for selected leaf areas and variation in the relative nicotine levels (15-100%) was observed. Comparative analysis of the nicotine distribution was demonstrated for two tobacco plant varieties, a genetically modified plant and its corresponding wild-type, indicating generally higher nicotine levels in the mutant.
Assuntos
Poluentes Atmosféricos/análise , Terapia a Laser/métodos , Sondas Moleculares/química , Nicotina/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Uridina/análise , Folhas de Planta/química , Plantas Geneticamente Modificadas/química , Solventes/química , Nicotiana/químicaRESUMO
The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.
Assuntos
Hidrocarbonetos/metabolismo , Metagenoma , Oceanospirillaceae/genética , Oceanospirillaceae/metabolismo , Poluição por Petróleo , Água do Mar/microbiologia , Análise de Célula Única , Transcriptoma , Archaea/genética , Archaea/fisiologia , Bactérias/genética , Biodiversidade , Golfo do México , RNA Ribossômico 16SRESUMO
Protein phosphorylation is a post-translational modification that is essential for the regulation of many important cellular activities, including proliferation and differentiation. Current techniques for detecting protein phosphorylation in single cells often involve the use of fluorescence markers, such as antibodies or genetically expressed proteins. In contrast, infrared spectroscopy is a label-free and noninvasive analytical technique that can monitor the intrinsic vibrational signatures of chemical bonds. Here, we provide direct evidence that protein phosphorylation in individual living mammalian cells can be measured with synchrotron radiation-based Fourier transform-infrared (SR-FT-IR) spectromicroscopy. We show that PC12 cells stimulated with nerve growth factor (NGF) exhibit statistically significant temporal variations in specific spectral features, correlating with changes in protein phosphorylation levels and the subsequent development of neuron-like phenotypes in the cells. The spectral phosphorylation markers were confirmed by bimodal (FT-IR/fluorescence) imaging of fluorescently marked PC12 cells with sustained protein phosphorylation activity. Our results open up new possibilities for the label-free real-time monitoring of protein phosphorylation inside cells. Furthermore, the multimolecule sensitivity of this technique will be useful for unraveling the associated molecular changes during cellular signaling and response processes.
Assuntos
Neurônios/citologia , Proteínas/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/instrumentação , Animais , Diferenciação Celular , Sobrevivência Celular , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Células PC12 , Fosforilação , Ratos , Sensibilidade e Especificidade , Síncrotrons/instrumentaçãoRESUMO
The biological effects and expected fate of the vast amount of oil in the Gulf of Mexico from the Deepwater Horizon blowout are unknown owing to the depth and magnitude of this event. Here, we report that the dispersed hydrocarbon plume stimulated deep-sea indigenous γ-Proteobacteria that are closely related to known petroleum degraders. Hydrocarbon-degrading genes coincided with the concentration of various oil contaminants. Changes in hydrocarbon composition with distance from the source and incubation experiments with environmental isolates demonstrated faster-than-expected hydrocarbon biodegradation rates at 5°C. Based on these results, the potential exists for intrinsic bioremediation of the oil plume in the deep-water column without substantial oxygen drawdown.
Assuntos
Biodegradação Ambiental , Poluição Ambiental , Gammaproteobacteria/metabolismo , Hidrocarbonetos/metabolismo , Oceanospirillaceae/metabolismo , Petróleo/metabolismo , Água do Mar/microbiologia , Biomassa , Contagem de Colônia Microbiana , Ácidos Graxos/análise , Gammaproteobacteria/classificação , Gammaproteobacteria/crescimento & desenvolvimento , Gammaproteobacteria/isolamento & purificação , Genes Bacterianos , Genes de RNAr , Dados de Sequência Molecular , Oceanospirillaceae/classificação , Oceanospirillaceae/genética , Oceanospirillaceae/isolamento & purificação , Fosfolipídeos/análise , FilogeniaRESUMO
In the present study, the antioxidant capacity of chromium-treated L-41 (human epithelial-like cells) was investigated by the ESR spin-trapping technique. The crude cell extracts of the cells grown in the presence of 2 microM (nontoxic) and 20 microM (toxic) chromium (VI) concentrations were tested in the model Fenton system with and without catalase-inhibitor sodium azide. The presented approach using the ESR technique along with inhibitors lets us discern cell extract defense capacity connected with the enzymatic activity in viable cells and the catabolic activity in dying cells.
Assuntos
Antioxidantes/metabolismo , Cromo/farmacologia , Catalase/antagonistas & inibidores , Catalase/metabolismo , Extratos Celulares , Linhagem Celular , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Células Epiteliais/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Glutationa/metabolismo , Humanos , Azida Sódica/farmacologiaRESUMO
In the present study, the antioxidant capacity against hydrogen peroxide (H2O2), one of the stress-inducing agents, was investigated in two distinct cell lines: L-41 (human epithelial-like cells) and HLF (human diploid lung fibroblasts), which differ in tissue origin, life span in culture, proliferate activity, and special enzyme system activity. The cell antioxidant capacity against H2O2 was estimated by the electron spin resonance (ESR) spin-trapping technique in the Fenton reaction system via Fe+2 ion action with H2O2 resulting in hydroxyl radical generation. The effects of catalase inhibitors, such as sodium azide and 3-amino-1,2,4-triazole, on the antioxidant capacity of cells were tested. Based on our observation, it can be concluded that the defensive capacity of cells against H2O2 depends on the ratio between catalase/GPx/SOD and H2O2, especially at high-stress situations, and the intracellular balance of these enzymes are more important than the influence of the single component.
Assuntos
Células Epiteliais/química , Detecção de Spin/métodos , Antioxidantes/química , Antioxidantes/metabolismo , Catalase/antagonistas & inibidores , Catalase/metabolismo , Extratos Celulares/química , Linhagem Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Diploide , Inibidores Enzimáticos/farmacologia , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Fibroblastos/química , Fibroblastos/enzimologia , Genoma Humano , Instabilidade Genômica/genética , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Leucemia Mieloide/enzimologia , Leucemia Mieloide/patologia , Pulmão/química , Pulmão/citologia , Pulmão/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Marcadores de Spin , Superóxido Dismutase/metabolismo , Superóxido Dismutase/fisiologiaRESUMO
Bacterial reduction of Cr(VI) to Cr(III) compounds may produce reactive intermediates Cr(V) and Cr(IV), which can affect the mobility and toxicity of chromium in environments. To address this important subject, we conducted an electron spin resonance (ESR) study to understand the kinetics of the formation and decomposition of Cr(V) during Cr(VI) reduction by different gram-positive Cr(VI)-tolerant bacteria, which were isolated from polluted basalts from the United States of America and the Republic of Georgia. Results from our batch experiments show that during Cr(VI) reduction, the macromolecules at the cell wall of these bacteria could act as an electron donor to Cr(VI) to form a stable square-pyramidal Cr(V) complexes, which were reduced further probably via a one-electron transfer pathway to form Cr(IV) and Cr(III) compounds. The Cr(V) peak at the ESR spectrum possessed superhyperfine splitting characteristic of the Cr(V) complexes with diol-containing molecules. It appears that the kinetics of Cr(V) formation and decomposition depended on the bacterial growth phase and on the species. Both formation and decomposition of Cr(V) occurred more quickly when Cr(VI) was added at the exponential phase. In comparison with other gram-positive bacteria from the republic of Georgia, the formation and decomposition of Cr(V) in Arthrobacter species from the Unites States was significantly slower.
Assuntos
Arthrobacter/fisiologia , Carcinógenos Ambientais/química , Cromo/química , Bactérias Gram-Positivas/fisiologia , Carcinógenos Ambientais/análise , Carcinógenos Ambientais/metabolismo , Cromo/análise , Cromo/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Minerais , SilicatosRESUMO
Synchrotron radiation-based Fourier transform infrared spectromicroscopy is a newly emerging analytical tool capable of monitoring the biochemistry within an individual living mammalian cell in real time. This unique technique provides infrared (IR) spectra, hence chemical information, with high signal to noise at spatial resolutions as fine as 3-10 microm. Mid-IR photons are too low in energy (0.05-0.5 eV) to either break bonds or to cause ionization, and the synchrotron IR beam has been shown to produce minimal sample heating. However, an important question remains, "Does the intense synchrotron beam induce any cytotoxic effects in living cells?" In this work, we present the results from a series of standard biological assays to evaluate any short- and/or long-term effects on cells exposed to the synchrotron radiation-based infrared (SR-IR) beam. Cell viability was tested using alcian blue dye exclusion and colony formation assays. Cell-cycle progression was tested with bromodeoxyuridine (BrdU) uptake during DNA synthesis. Cell metabolism was tested using a 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. All control, 5, 10, and 20 min SR-IR exposure tests (267 total and over 1000 controls) show no evidence of cytotoxic effects. Concurrent infrared spectra obtained with each experiment confirm no detectable biochemical changes between control and exposed cells.