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Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, ß1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.
Assuntos
Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Membrana Celular/microbiologia , Membrana Celular/patologia , Endossomos/microbiologia , Endossomos/patologia , Escherichia coli Enteropatogênica/patogenicidade , Infecções por Escherichia coli/patologia , Células HeLa , HumanosRESUMO
Common fragile sites (CFSs) are genomic regions prone to breakage under replication stress conditions recurrently rearranged in cancer. Many CFSs are enriched with AT-dinucleotide rich sequences (AT-DRSs) which have the potential to form stable secondary structures upon unwinding the double helix during DNA replication. These stable structures can potentially perturb DNA replication progression, leading to genomic instability. Using site-specific targeting system, we show that targeted integration of a 3.4 kb AT-DRS derived from the human CFS FRA16C into a chromosomally stable region within the human genome is able to drive fragile site formation under conditions of replication stress. Analysis of >1300 X chromosomes integrated with the 3.4 kb AT-DRS revealed recurrent gaps and breaks at the integration site. DNA sequences derived from the integrated AT-DRS showed in vitro a significantly increased tendency to fold into branched secondary structures, supporting the predicted mechanism of instability. Our findings clearly indicate that intrinsic DNA features, such as complexed repeated sequence motifs, predispose the human genome to chromosomal instability.
Assuntos
Instabilidade Cromossômica/genética , Sítios Frágeis do Cromossomo/genética , DNA/genética , Repetições de Dinucleotídeos/genética , Replicação do DNA/genética , Genoma Humano , Humanos , Conformação de Ácido NucleicoRESUMO
We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited.
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Padronização Corporal , Heterópteros/embriologia , Animais , Padronização Corporal/genética , Divisão Celular/genética , Proliferação de Células/genética , Fase de Clivagem do Zigoto/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Heterópteros/genéticaRESUMO
Growth arrest-specific 2-like 3 (Gas2l3) is a newly discovered cell cycle protein and a cytoskeleton orchestrator that binds both actin filament and microtubule networks. Studies of cultured mammalian cells established Gas2l3 as a regulator of the cell division process, in particular cytokinesis and cell abscission. Thus far, the role of Gas2l3 in vivo remains entirely unknown. In order to investigate Gas2l3 in developing vertebrates, we cloned the zebrafish gene. Spatiotemporal analysis of gas2l3 expression revealed a ubiquitous maternal transcript as well as a zygotic transcript primarily restricted to brain tissues. We next conducted a series of loss-of-function experiments, and searched for developmental anomalies at the end of the segmentation period. Our analysis revealed abnormal brain morphogenesis and ventricle formation in gas2l3 knockdown embryos. This signature phenotype could be rescued by elevated levels of gas2l3 RNA. At the tissue level, gas2l3 downregulation interferes with cell proliferation, suggesting that the cell cycle activities of Gas2l3 are essential for brain tissue homeostasis. Altogether, this study provides the first insight into the function of gas2l3 in vivo, demonstrating its essential role in brain development.
Assuntos
Encéfalo/embriologia , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Microscopia de Fluorescência , Dados de Sequência Molecular , Morfogênese/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Proteínas de Peixe-Zebra/genéticaRESUMO
Phase retrieval is a well-established method for the recovery of an object wave from its magnitude-only measurements, with applications in fields such as material science, biology, and astronomy. In order to guarantee a stable and global solution for phase retrieval, measurement diversity is frequently used. However, this process requires taking several measurements, which hinders some of the advantages of phase retrieval compared with interferometric techniques. Here we propose a novel diversity framework that we refer to as digital resampling diversity. The proposed resampling diversity along with sparsity-constrained object reconstruction enables improved object reconstruction from a single squared magnitude image, without using object support constraint. We demonstrate this framework with simulation and experimental results.
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OBJECTIVES: The aims of this study were to develop new anti-biofilm drugs, examine their activity against Candida albicans biofilm and investigate their structure-activity relationship and mechanism of action. METHODS: A series of thiazolidinedione and succinimide derivatives were synthesized and their ability to inhibit C. albicans biofilm formation and destroy pre-formed biofilm was tested. The biofilms' structure, metabolic activity and viability were determined by XTT assay and propidium iodide and SYTO 9 live/dead stains combined with confocal microscopic analysis. The effect of the most active compounds on cell morphology, sterol distribution and cell wall morphology and composition was then determined by specific fluorescent stains and transmission electron microscopy. RESULTS: Most of the compounds were active at sub-MICs. Elongation of the aliphatic side chain resulted in reduced anti-biofilm activity and the sulphur atom contributed to biofilm killing, indicating a structure-activity relationship. The compounds differed in their effects on biofilm viability, yeast-to-hyphal form transition, hyphal morphology, cell wall morphology and composition, and sterol distribution. The most effective anti-biofilm compounds were the thiazolidinedione S8H and the succinimide NA8. CONCLUSIONS: We developed novel anti-biofilm agents that both inhibited and destroyed C. albicans biofilm. With some further development, these agents might be suitable for therapeutic purposes.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Animais , Antifúngicos/química , Biofilmes/crescimento & desenvolvimento , Candida albicans/fisiologia , Compostos Heterocíclicos/química , Ovinos , Relação Estrutura-AtividadeRESUMO
We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix-assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides.
Assuntos
Nanopartículas/química , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Técnicas de Síntese em Fase Sólida/instrumentaçãoRESUMO
Existing algorithms for automated segmentation of chromosomes and centromeres do not work well for condensed, C-banded and DAPI-stained chromosomes and centromeres. Overlapping and aggregation, which frequently occur in metaphase spreads, introduce additional challenges to the counting of chromosomes and centromeres in the Dicentrics Chromosome Assay (DCA). In this paper, we introduce adaptive algorithms, for segmentation of difficult metaphase spreads that include overlapping and aggregated chromosomes. In order to enhance and segment chromosomes, two optimizations are done: (1) the best algorithm among several options is automatically chosen based on predefined figures of merit, (2) the algorithm is automatically optimized with a binary search to modify its parameters to achieve predefined thresholds. These algorithms are designed to separate mildly or moderately aggregated chromosomal clusters. The clusters are segmented by skeleton junctions, reduction of the overall object thickness, and the watershed algorithm. The chromosomes are characterized by rules we establish, using minimal assumptions. Centromeres are detected by detecting bright spots on the surface of the chromosomes, and then using cluster analysis and shape and intensity profiles to identify them as centromeres. High sensitivity and specificity for chromosome and centromere detection were achieved.
RESUMO
IMPORTANCE: Enteropathogenic Escherichia coli (EPEC) infection is a significant cause of gastroenteritis, mainly in children. Therefore, studying the mechanisms of EPEC infection is an important research theme. EPEC modulates its host cell life by injecting via a type III secretion machinery cell death modulating effector proteins. For instance, while EspF and Map promote mitochondrial cell death, EspZ antagonizes cell death. We show that these effectors also control lysosomal exocytosis, i.e., the trafficking of lysosomes to the host cell plasma membrane. Interestingly, the capacity of these effectors to induce or protect against cell death correlates completely with their ability to induce LE, suggesting that the two processes are interconnected. Modulating host cell death is critical for establishing bacterial attachment to the host and subsequent dissemination. Therefore, exploring the modes of LE involvement in host cell death is crucial for elucidating the mechanisms underlying EPEC infection and disease.
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Amphotericin B (AMB) is an effective antifungal agent. However, its therapeutic use is hampered by its toxicity, mainly due to channel formation across kidney cell membranes and the disruption of postendocytic trafficking. We previously described a safe injectable AMB-arabinogalactan (AG) conjugate with neutralized toxicity. Here we studied the mechanism of the toxicity of free AMB and its neutralization by conjugation with AG. AMB treatment of a kidney cell line modulated the trafficking of three receptors (C-X-C chemokine receptor type 4 [CXCR4], M1 receptor, and human transferrin receptor [hTfnR]) due to an increase in endosomal pH. Similar data were also obtained in yeast but with an increase in vacuolar pH and the perturbation of Hxt2-green fluorescent protein (GFP) trafficking. The conjugation of AMB with AG neutralized all elements of the toxic activity of AMB in mammalian but not in fungal cells. Based on these results, we provide an explanation of how the conjugation of AMB with AG neutralizes its toxicity in mammalian cells and add to the knowledge of the mechanism of action of free AMB in both fungal and mammalian cells.
Assuntos
Anfotericina B/análogos & derivados , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Galactanos/farmacologia , Rim/efeitos dos fármacos , Anfotericina B/química , Animais , Antifúngicos/química , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Quimiocinas CXC/metabolismo , Chlorocebus aethiops , Cães , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Galactanos/química , Humanos , Concentração de Íons de Hidrogênio , Rim/citologia , Rim/microbiologia , Células Madin Darby de Rim Canino , Testes de Sensibilidade Microbiana , Transporte Proteico/efeitos dos fármacos , Receptor Muscarínico M1/metabolismo , Receptores CXCR4/metabolismo , Receptores da Transferrina/metabolismo , Especificidade da Espécie , Transferrina/metabolismo , Células VeroRESUMO
The dicentric chromosome assay (DCA), is considered the 'gold standard' for radiation biodosimetry. Yet, DCA, as currently implemented, may be impractical for emergency response applications, especially when time is of the essence, owing to its labor-intensive and time-consuming nature. The growth of a primary lymphocyte culture for 48 h in vitro is required for DCA, and manual scoring of dicentric chromosomes (DCs) requires an additional 24-48 h, resulting in an overall processing time of 72-96 h for dose estimation. In order to improve this timing. we introduce a protocol that will detect the metaphase cells in a population of cells, and then will harvest only those metaphase cells. Our metaphase enrichment approach is based on fixed human lymphocytes incubated with monoclonal, anti-phosphorylated H3 histone (ser 10). Antibodies against this histone have been shown to be specific for mitotic cells. Colcemid is used to arrest the mitotic cells in metaphase. Following that, a flow-cytometric sorting apparatus isolates the mitotic fraction from a large population of cells, in a few minutes. These mitotic cells are then spread onto a slide and treated with our C-Banding procedure [Gonen et al. 2022], to visualize the centromeres with DAPI. This reduces the chemical processing time to ~2 h. This reduces the time required for the DCA and makes it practical for a much wider set of applications, such as emergency response following exposure of a large population to ionizing radiation.
Assuntos
Cromossomos Humanos , Radiometria , Aberrações Cromossômicas , Demecolcina , Relação Dose-Resposta à Radiação , Histonas , Humanos , Linfócitos , Metáfase , Radiometria/métodosRESUMO
Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin-rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the morphology and architecture of infected polarized monolayers. These changes are manifested by altered localization of the tight junctions and 'bulging out' morphology of the cells. Surprisingly, despite the dramatic changes in their architecture, the cells remain alive and the epithelial monolayer maintains a normal barrier function. Taken together, our results show that the EspM effectors inhibit pedestal formation and induce tight junction mislocalization as well as dramatic changes in the architecture of the polarized monolayer.
Assuntos
Escherichia coli Êntero-Hemorrágica/patogenicidade , Escherichia coli Enteropatogênica/patogenicidade , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Proteínas de Escherichia coli/fisiologia , Fatores de Virulência/fisiologia , Linhagem Celular , Sobrevivência Celular , Humanos , Fibras de Estresse/metabolismo , Junções ÍntimasRESUMO
In this paper, we characterize the dynamic nature of the full amyloid beta (1-40) (Aß (1-40)) aggregates. We labeled the peptide with either 5-carboxytetramethylrhodamine (TAMRA) or with fluorescein-isothiocyanate (FITC). The labeled peptides were mixed after separate fibrillization, and the dynamic changes in the structure of the fibrils were imaged using confocal microscopy. Fluorescence resonance energy transfer (FRET) measurements showed that the Aß (1-40) peptides detach from and reattach to the fibrils in a biologically relevant timescale (days). With time, the two peptides mix at the molecular level. This process is concentration dependent and occurs primarily in the external parts of the aggregates with a half time between 4 and 7 days. This study shows that the combination of confocal microscopy and FRET analysis is a facile method for studying dynamic processes in supra-molecular aggregates.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Isotiocianatos/química , Microscopia Confocal , Rodaminas/químicaRESUMO
A synthetic "phage-like" system was designed for screening mixtures of small molecules in live cells. The core of the system consists of 2 mum diameter cross-linked monodispersed microspheres bearing a panel of fluorescent tags and peptides or small molecules either directly synthesized or covalently conjugated to the microspheres. The microsphere mixtures were screened for affinity to cell line PC-3 (prostate cancer model) by incubation with live cells, and as was with phage-display peptide methods, unbound microspheres were removed by repeated washings followed by total lysis of cells and analysis of the bound microspheres by flow-cytometry. Similar to phage-display peptide screening, this method can be applied even in the absence of prior information about the cellular targets of the candidate ligands, which makes the system especially interesting for selection of molecules with high affinity for desired cells, tissues, or tumors. The advantage of the proposed system is the possibility of screening synthetic non-natural peptides or small molecules that cannot be expressed and screened using phage display libraries. A library composed of small molecules synthesized by the Ugi reaction was screened, and a small molecule, Rak-2, which strongly binds to PC-3 cells was found. Rak-2 was then individually synthesized and validated in a complementary whole cell-based binding assay, as well as by live cell microscopy. This new system demonstrates that a mixture of molecules bound to subcellular sized microspheres can be screened on plated cells. Together with other methods using subcellular sized particles for cellular multiplexing, this method represents an important milestone toward high throughput screening of mixtures of small molecules in live cells and in vivo with potential applications in the fields of drug delivery and diagnostic imaging.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Neoplasias da Próstata/patologia , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Química Combinatória , Reagentes de Ligações Cruzadas/química , Diagnóstico por Imagem/métodos , Sistemas de Liberação de Medicamentos/métodos , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/química , Humanos , Masculino , MicroesferasRESUMO
Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-beta-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia.
Assuntos
Colesterol/metabolismo , Endocitose/fisiologia , Microdomínios da Membrana/metabolismo , Animais , Caveolina 1/metabolismo , Linhagem Celular , Polaridade Celular , Cães , Endossomos/metabolismo , Humanos , Imunoglobulina A/química , Imunoglobulina A/metabolismo , Ligantes , Receptores Fc/metabolismo , Receptores da Transferrina/metabolismo , Transdução de Sinais/fisiologia , beta-Ciclodextrinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The deterioration of field potential (FP) recording quality and yield by in vivo multielectrode arrays (MEA) within days to weeks of implantation severely limits progress in basic and applied brain research. The prevailing hypothesis is that implantation of MEA platforms initiate and perpetuate inflammatory processes which culminate in the formation of scar tissue (the foreign body response, FBR) around the implant. The FBR leads to progressive degradation of the recording qualities by displacing neurons away from the electrode surfaces, increasing the resistance between neurons (current source) and the sensing pads and by reducing the neurons' excitable membrane properties and functional synaptic connectivity through the release of pro-inflammatory cytokines. Meticulous attempts to causally relate the cellular composition, cell density, and electrical properties of the FBR have failed to unequivocally correlate the deterioration of recording quality with the histological severity of the FBR. Based on confocal and electron microscope analysis of thin sections of polyimide based MEA implants along with the surrounding brain tissue at different points in time after implantation, we propose that abrupt FP amplitude attenuation occurs at the implant/brain-parenchyma junction as a result of high seal resistance insulation formed by adhering microglia to the implant surfaces. In contrast to the prevailing hypothesis, that FP decrease occurs across the encapsulating scar of the implanted MEA, this mechanism potentially explains why no correlations have been found between the dimensions and density of the FBR and the recording quality. Recognizing that the seal resistance formed by adhering-microglia to the implant constitutes a downstream element undermining extracellular FP recordings, suggests that approaches to mitigate the formation of the insulating glial could lead to improved recording quality and yield.
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We report on the application of surface plasmon resonance (SPR), based on Fourier transform infrared spectroscopy in the mid-infrared wavelength range, for real-time and label-free sensing of transferrin-induced endocytic processes in human melanoma cells. The evanescent field of the mid-infrared surface plasmon penetrates deep into the cell, allowing highly sensitive SPR measurements of dynamic processes occurring at significant cellular depths. We monitored in real-time, infrared reflectivity spectra in the SPR regime from living cells exposed to human transferrin (Tfn). We show that although fluorescence microscopy measures primarily Tfn accumulation in recycling endosomes located deep in the cell's cytoplasm, the SPR technique measures mainly Tfn-mediated formation of early endocytic organelles located in close proximity to the plasma membrane. Our SPR and fluorescence data are very well described by a kinetic model of Tfn endocytosis, suggested previously in similar cell systems. Hence, our SPR data provide further support to the rather controversial ability of Tfn to stimulate its own endocytosis. Our analysis also yields what we believe is novel information on the role of membrane cholesterol in modulating the kinetics of endocytic vesicle biogenesis and consumption.
Assuntos
Endocitose/efeitos dos fármacos , Melanoma/metabolismo , Modelos Biológicos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Ressonância de Plasmônio de Superfície/métodos , Transferrina/farmacologia , Vesículas Transportadoras/metabolismo , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Vesículas Transportadoras/efeitos dos fármacosRESUMO
AIM: To elucidate the interactions, uptake mechanisms and cytotoxicity profile of glucose-functionalized gold nanoparticles (2GF-GNPs), for expanding and advancing the recently proposed technology of metabolic-based cancer detection to a variety of cancer diseases. METHODS: Several cell types with different metabolic features were used to assess the involvement of GLUT-1 and different endocytosis pathways in 2GF-GNP uptake, and the cytotoxicity profile of 2GF-GNPs. RESULTS: Cellular uptake of 2GF-GNP strongly correlated with GLUT-1 surface expression, and occurred mainly through clathrin-mediated endocytosis. 2GF-GNPs showed no toxic effect on cell cycle and proliferation. CONCLUSION: These findings promote development of metabolic-based cancer detection technologies, and suggest that 2GF-GNPs may enable specific cancer detection in a wide range of tumors characterized by high GLUT-1 expression.
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Meios de Contraste/administração & dosagem , Transportador de Glucose Tipo 1/genética , Nanopartículas Metálicas/administração & dosagem , Neoplasias/diagnóstico por imagem , Células A549 , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Contraste/química , Citocalasina B/farmacologia , Endocitose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/química , Glucose/metabolismo , Transportador de Glucose Tipo 1/antagonistas & inibidores , Ouro/química , Ouro/farmacologia , Humanos , Nanopartículas Metálicas/química , Neoplasias/genética , Neoplasias/patologia , Tomografia Computadorizada por Raios XRESUMO
In this manuscript we present a novel super resolving approach based upon projection of a random speckle pattern onto samples observed through a microscope. The projection of the speckle pattern is created by coherent illumination of the inspected pattern through a diffuser. Due to local interference of the coherent wave front with itself, a random speckle pattern is superimposed on the sample. This speckle pattern can be scanned over the object. A super-resolved image can be extracted from a temporal sequence of images by appropriate digital processing of the image stream. The resulting resolution is significantly higher than the diffraction limitation of the microscope objective. The new super-resolution method is demonstrated by application to fluorescence of biological samples.
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In this paper, we present a new approach providing super resolved images exceeding the geometrical limitation given by the detector pixel size of the imaging camera. The concept involves the projection of periodic patterns on top of the sample, which are then investigated under a microscope. Combining spatial scanning together with proper digital post-processing algorithm yields the improved geometrical resolution enhancement. This new method is especially interesting for microscopic imaging when the resolution of the detector is lower than the resolution due to diffraction.