RESUMO
Small molecules such as neurotransmitters are critical for biochemical functions in living systems. While conventional ultraviolet-visible spectroscopy and mass spectrometry lack portability and are unsuitable for time-resolved measurements in situ, techniques such as amperometry and traditional field-effect detection require a large ensemble of molecules to reach detectable signal levels. Here we demonstrate the potential of carbon-nanotube-based single-molecule field-effect transistors (smFETs), which can detect the charge on a single molecule, as a new platform for recognizing and assaying small molecules. smFETs are formed by the covalent attachment of a probe molecule, in our case a DNA aptamer, to a carbon nanotube. Conformation changes on binding are manifest as discrete changes in the nanotube electrical conductance. By monitoring the kinetics of conformational changes in a binding aptamer, we show that smFETs can detect and quantify serotonin at the single-molecule level, providing unique insights into the dynamics of the aptamer-ligand system. In particular, we show the involvement of G-quadruplex formation and the disruption of the native hairpin structure in the conformational changes of the serotonin-aptamer complex. The smFET is a label-free approach to analysing molecular interactions at the single-molecule level with high temporal resolution, providing additional insights into complex biological processes.
Assuntos
Aptâmeros de Nucleotídeos , Nanotubos de Carbono , Serotonina , Transistores Eletrônicos , Aptâmeros de Nucleotídeos/química , Nanotubos de Carbono/química , Cinética , Ligantes , Serotonina/química , Serotonina/metabolismo , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentaçãoRESUMO
Haemagglutinin and neuraminidase surface glycoproteins of the bat influenza H17N10 virus neither bind to nor cleave sialic acid receptors, indicating that this virus employs cell entry mechanisms distinct from those of classical influenza A viruses. We observed that certain human haematopoietic cancer cell lines and canine MDCK II cells are susceptible to H17-pseudotyped viruses. We identified the human HLA-DR receptor as an entry mediator for H17 pseudotypes, suggesting that H17N10 possesses zoonotic potential.
Assuntos
Quirópteros/virologia , Antígenos HLA-DR/genética , Orthomyxoviridae/fisiologia , Tropismo Viral , Internalização do Vírus , Animais , Cães , Células HEK293 , Antígenos HLA-DR/imunologia , Humanos , Células Madin Darby de Rim Canino , Análise em Microsséries , Receptores Virais/genética , Receptores Virais/imunologia , Zoonoses/virologiaRESUMO
Over the last 50 years, a number of important physiological changes in humans who have traveled on spaceflights have been catalogued. Of major concern are the short- and long-term radiation-induced injuries to the hematopoietic system that may be induced by high-energy galactic cosmic rays encountered on interplanetary space missions. To collect data on the effects of space radiation on the human hematopoietic system in vivo, we used a humanized mouse model. In this study, we irradiated humanized mice with 0.4 Gy of 350 MeV/n 28Si ions, a dose that has been shown to induce tumors in tumor-prone mice and a reference dose that has a relative biological effectiveness of 1 (1 Gy of 250-kVp X rays). Cell counts, cell subset frequency and cytogenetic data were collected from bone marrow spleen and blood of irradiated and control mice at short-term (7, 30 and 60 days) and long-term ( 6 - 7 months) time points postirradiation. The data show a significant short-term effect on the human hematopoietic stem cell counts imparted by both high- and low-LET radiation exposure. The radiation effects on bone marrow, spleen and blood human cell counts and human cell subset frequency were complex but did not alter the functions of the hematopoietic system. The long-term data acquired from high-LET irradiated mice showed complete recovery of the human hematopoietic system in all hematopoietic compartments. The combined results demonstrate that, in spite of early perturbation, the longer term effects of high-LET radiation are not detrimental to human hematopoiesis in our system of study.
Assuntos
Radiação Cósmica , Hematopoese/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos da radiação , Animais , Contagem de Células Sanguíneas , Medula Óssea/efeitos da radiação , Relação Dose-Resposta à Radiação , Humanos , Camundongos , Camundongos Endogâmicos NOD , Modelos Animais , Neoplasias Induzidas por Radiação/genética , Eficiência Biológica Relativa , Voo Espacial , Baço/efeitos da radiaçãoRESUMO
Single-point-functionalized carbon-nanotube field-effect transistors (CNTFETs) have been used to sense conformational changes and binding events in protein and nucleic acid structures from intrinsic molecular charge. The key to utilizing these devices as single-molecule sensors is the ability to attach a single probe molecule to an individual device. In contrast, with noncovalent attachment approaches such as those based on van der Waals interactions, covalent attachment approaches generally deliver higher stability but have traditionally been more difficult to control, resulting in low yield. Here, we present a single-point-functionalization method for CNTFET arrays based on electrochemical control of a diazonium reaction to create sp3 defects, combined with a scalable spin-casting method for fabricating large arrays of devices on arbitrary substrates. Attachment of probe DNA to the functionalized device enables single-molecule detection of DNA hybridization with complementary target, verifying the single-point functionalization. Overall, this method enables single-point defect generation with 80% yield.
Assuntos
Técnicas Eletroquímicas , Nanotubos de Carbono/química , Transistores Eletrônicos , DNA/química , Sondas de DNA/química , Compostos de Diazônio/química , Estrutura Molecular , Hibridização de Ácido NucleicoRESUMO
The study of biomolecular interactions at the single-molecule level holds great potential for both basic science and biotechnology applications. Single-molecule studies often rely on fluorescence-based reporting, with signal levels limited by photon emission from single optical reporters. The point-functionalized carbon nanotube transistor, known as the single-molecule field-effect transistor, is a bioelectronics alternative based on intrinsic molecular charge that offers significantly higher signal levels for detection. Such devices are effective for characterizing DNA hybridization kinetics and thermodynamics and enabling emerging applications in genomic identification. In this work, we show that hybridization kinetics can be directly controlled by electrostatic bias applied between the device and the surrounding electrolyte. We perform the first single-molecule experiments demonstrating the use of electrostatics to control molecular binding. Using bias as a proxy for temperature, we demonstrate the feasibility of detecting various concentrations of 20-nt target sequences from the Ebolavirus nucleoprotein gene in a constant-temperature environment.
Assuntos
Técnicas Biossensoriais , Ebolavirus/isolamento & purificação , Hibridização de Ácido Nucleico/métodos , Nucleoproteínas/análise , Transistores Eletrônicos , Proteínas Virais/análise , Sondas de DNA/química , Cinética , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Nanotubos de Carbono/química , Nucleoproteínas/genética , Eletricidade Estática , Termodinâmica , Proteínas Virais/genéticaRESUMO
The increased threat of terrorism across the globe has raised fears that certain groups will acquire and use radioactive materials to inflict maximum damage. In the event that an improvised nuclear device (IND) is detonated, a potentially large population of victims will require assessment for radiation exposure. While photons will contribute to a major portion of the dose, neutrons may be responsible for the severity of the biologic effects and cellular responses. We investigated differences in response between these two radiation types by using metabolomics and lipidomics to identify biomarkers in urine and blood of wild-type C57BL/6 male mice. Identification of metabolites was based on a 1 Gy dose of radiation. Compared to X rays, a neutron spectrum similar to that encountered in Hiroshima at 1-1.5 km from the epicenter induced a severe metabolic dysregulation, with perturbations in amino acid metabolism and fatty acid ß-oxidation being the predominant ones. Urinary metabolites were able to discriminate between neutron and X rays on day 1 as well as day 7 postirradiation, while serum markers showed such discrimination only on day 1. Free fatty acids from omega-6 and omega-3 pathways were also decreased with 1 Gy of neutrons, implicating cell membrane dysfunction and impaired phospholipid metabolism, which should otherwise lead to release of those molecules in circulation. While a precise relative biological effectiveness value could not be calculated from this study, the results are consistent with other published studies showing higher levels of damage from neutrons, demonstrated here by increased metabolic dysregulation. Metabolomics can therefore aid in identifying global perturbations in blood and urine, and effectively distinguishing between neutron and photon exposures.
Assuntos
Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Metaboloma/efeitos da radiação , Nêutrons/efeitos adversos , Guerra Nuclear , Lesões por Radiação/metabolismo , Terrorismo , Animais , Bioensaio/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fótons/efeitos adversos , Doses de Radiação , Exposição à Radiação/efeitos adversos , Exposição à Radiação/análise , Lesões por Radiação/etiologia , Radiometria/métodosRESUMO
The current state of the art in high-throughput minimally invasive radiation biodosimetry involves the collection of samples in the field and analysis at a centralized facility. We have developed a simple biological immunoassay for radiation exposure that could extend this analysis out of the laboratory into the field. Such a forward placed assay would facilitate triage of a potentially exposed population. The phosphorylation and localization of the histone H2AX at double-stranded DNA breaks has already been proven to be an adequate surrogate assay for reporting DNA damage proportional to radiation dose. Here, we develop an assay for phosphorylated H2AX directed against minimally processed sample lysates. We conduct preliminary verification of H2AX phosphorylation using irradiated mouse embryo fibroblast cultures. Additional dosimetry is performed using human blood samples irradiated ex vivo. The assay reports H2AX phosphorylation in human blood samples in response to ionizing radiation over a range of 0-5 Gy in a linear fashion, without requiring filtering, enrichment, or purification of the blood sample.
Assuntos
Histonas/sangue , Histonas/efeitos da radiação , Radiometria/métodos , Animais , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Relação Dose-Resposta à Radiação , Ensaio de Imunoadsorção Enzimática/métodos , Raios gama/efeitos adversos , Histonas/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Fosforilação/efeitos da radiaçãoRESUMO
Current advances in single cell sequencing, gene expression and proteomics require the isolation of single cells, frequently from a very small source population. In this work we describe the design and characterization of a manually operated microfluidic cell sorter that 1) can accurately sort single or small groups of cells from very small cell populations with minimal losses, 2) that is easy to operate and that can be used in any laboratory that has a basic fluorescent microscope and syringe pump, 3) that can be assembled within minutes, 4) that can sort cells in very short time (minutes) with minimum cell stress, 5) that is cheap and reusable. This microfluidic sorter is made from hard plastic material (PMMA) into which microchannels are directly milled with hydraulic diameter of 70 µm. Inlet and outlet reservoirs are drilled through the chip. Sorting occurs through hydrodynamic switching ensuring low hydrodynamic shear stresses, which were modeled or experimentally confirmed to be below the cell damage threshold. Manually operated, the maximum sorting frequencies were approximately 10 cells per minute. Experiments verified that cell sorting operations could be achieved in as little as 15 minutes, including the assembly and testing of the sorter. In only one out of 10 sorting experiments the sorted cells were contaminated with another cell type. This microfluidic cell sorter represents an important capability for protocols requiring fast isolation of single cells from small number of rare cell populations.
RESUMO
PURPOSE: Previously, we showed that microRNA (miRNA) signatures derived from the peripheral blood of mice are highly specific for both radiation energy (γ-rays or high linear energy transfer [LET] (56)Fe ions) and radiation dose. Here, we investigate to what extent miRNA expression signatures derived from mouse blood can be used as biomarkers for exposure to 600 MeV proton radiation. MATERIALS AND METHODS: We exposed mice to 600 MeV protons, using doses of 0.5 or 1.0 Gy, isolated total RNA at 6 h or 24 h after irradiation, and used quantitative real-time polymerase chain reaction (PCR) to determine the changes in miRNA expression. RESULTS: A total of 26 miRNA were differentially expressed after proton irradiation, in either one (77%) or multiple conditions (23%). Statistical classifiers based on proton, γ, and (56)Fe-ion miRNA expression signatures predicted radiation type and proton dose with accuracies of 81% and 88%, respectively. Importantly, gene ontology analysis for proton-irradiated cells shows that genes targeted by radiation-induced miRNA are involved in biological processes and molecular functions similar to those controlled by miRNA in γ ray- and (56)Fe-irradiated cells. CONCLUSIONS: Mouse blood miRNA signatures induced by proton, γ, or (56)Fe irradiation are radiation type- and dose-specific. These findings underline the complexity of the miRNA-mediated radiation response.
Assuntos
Bioensaio/métodos , Biomarcadores/sangue , Raios gama , Íons Pesados , MicroRNAs/sangue , MicroRNAs/efeitos da radiação , Prótons , Animais , Relação Dose-Resposta à Radiação , Ferro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doses de Radiação , Irradiação Corporal TotalRESUMO
When cells are exposed to a dose of radiation large enough to cause chromosome aberrations, they become arrested at the G(2)/M checkpoint, facilitating DNA repair. Defects in checkpoint control genes can impart radiosensitivity. Arrest kinetics were monitored in mouse embryo fibroblasts at doses ranging from 10 mGy to 5.0 Gy of γ radiation over a time course of 0 to 12 h. We observe no significant checkpoint engagement at doses below 100 mGy. The checkpoint is only fully activated at doses where most of the cells are either bound for mitotic catastrophe or are reproductively dead. Atm null cells with ablated checkpoint function exhibited no robust arrest. Surprisingly, haploinsufficiency for ATM alone or in combination with other radioresistance genes did not alter checkpoint activation. We have shown previously that haploinsufficiency for several radioresistance genes imparts intermediate phenotypes for several end points including apoptosis, transformation and survival. These findings suggest that checkpoint control does not contribute toward these intermediate phenotypes and that different biological processes can be activated at high doses compared to low doses.
Assuntos
Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Haploinsuficiência/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Pontos de Checagem da Fase M do Ciclo Celular/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Embrião de Mamíferos , Feminino , Fibroblastos/metabolismo , Camundongos , GravidezRESUMO
The relative radiation sensitivities of the various compartments of the heart are poorly characterized. Cardiac fibrosis is a common side effect of radiotherapy, suggesting that endothelial barrier function is an important factor in radiation-induced pathology. We employed Electric Cell Substrate Impedance Sensing (ECIS) to assess cytoskeletal rearrangement, permeability changes and endothelial barrier function changes in response to radiation in studies of human coronary arterial endothelial cells (HCAECs). A 5-Gy dose of γ radiation resulted in a significant sixfold transient decrease in transmonolayer resistance 3 h postirradiation (P = 0.001). This decrease in resistance coincided with changes in fluorescent tracer flux (P = 0.05) and display of an actin bundling phenotype. After irradiation, decreases in wound healing (P = 0.03) and micromotion within the monolayer (P = 0.02) were also observed. Time-lapse video studies confirmed that the monolayer is dynamic and showed that cells are extruded from the monolayer at a higher frequency after irradiation. These findings suggest that perturbed endothelial barrier function in the heart can occur at lower doses of γ radiation than previously reported.
Assuntos
Vasos Coronários/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/efeitos da radiação , Movimento Celular/efeitos da radiação , Citoesqueleto/efeitos da radiação , Impedância Elétrica , Endotélio Vascular/citologia , Humanos , Modelos Biológicos , Imagem Molecular , Permeabilidade/efeitos da radiação , Fatores de TempoRESUMO
PURPOSE: MicroRNAs (miRNAs), a class of noncoding small RNAs that regulate gene expression, are involved in numerous physiologic processes in normal and malignant cells. Our in vivo study measured miRNA and gene expression changes in human blood cells in response to ionizing radiation, to develop miRNA signatures that can be used as biomarkers for radiation exposure. METHODS AND MATERIALS: Blood from 8 radiotherapy patients in complete remission 1 or 2 was collected immediately before and 4 hours after total body irradiation with 1.25 Gy x-rays. Both miRNA and gene expression changes were measured by means of quantitative polymerase chain reaction and microarray hybridization, respectively. Hierarchic clustering, multidimensional scaling, class prediction, and gene ontology analysis were performed to investigate the potential of miRNAs to serve as radiation biomarkers and to elucidate their likely physiologic roles in the radiation response. RESULTS: The expression levels of 45 miRNAs were statistically significantly upregulated 4 hours after irradiation with 1.25 Gy x-rays, 27 of them in every patient. Nonirradiated and irradiated samples form separate clusters in hierarchic clustering and multidimensional scaling. Out of 223 differentially expressed genes, 37 were both downregulated and predicted targets of the upregulated miRNAs. Paired and unpaired miRNA-based classifiers that we developed can predict the class membership of a sample with unknown irradiation status, with accuracies of 100% when all 45 upregulated miRNAs are included. Both miRNA control of and gene involvement in biologic processes such as hemopoiesis and the immune response are increased after irradiation, whereas metabolic processes are underrepresented among all differentially expressed genes and the genes controlled by miRNAs. CONCLUSIONS: Exposure to ionizing radiation leads to the upregulation of the expression of a considerable proportion of the human miRNAome of peripheral blood cells. These miRNA expression signatures can be used as biomarkers of radiation exposure.
Assuntos
Células Sanguíneas/efeitos da radiação , Perfilação da Expressão Gênica/métodos , MicroRNAs/efeitos da radiação , Irradiação Corporal Total , Adulto , Biomarcadores , Células Sanguíneas/metabolismo , Regulação para Baixo , Feminino , Humanos , Leucemia/sangue , Linfoma/sangue , Masculino , MicroRNAs/metabolismo , Análise em Microsséries/métodos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Adulto JovemRESUMO
The alpha1beta1 and alpha2beta1 integrins belong to a family of cell-surface molecules involved in structural contacts and signal-transduction events across the cell membrane. Employing two-dimensional substrates coated with fluorescently labeled type I collagen, we have discovered a novel subcellular matrix remodeling event that is particular to cells that express the fibrillar collagen receptor alpha2beta1. Cells expressing alpha1beta1 also perform this collagen organization process, but less proficiently. This work will provide a basis for subsequent studies of cell-mediated collagen fibril assembly.