RESUMEN
The role of Influenza D virus (IDV) in bovine respiratory disease remains unclear. An in vivo experiment resulted in increased clinical signs, lesions, and pathogen replication in calves co-infected with IDV and Mycoplasma bovis (M. bovis), compared to single-infected calves. The present study aimed to elucidate the host-pathogen interactions and profile the kinetics of lipid mediators in the airways of these calves. Bronchoalveolar lavage (BAL) samples collected at 2 days post-infection (dpi) were used for proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, lipidomic analyses were performed by LC-MS/MS on BAL samples collected at 2, 7 and 14 dpi. Whereas M. bovis induced the expression of proteins involved in fibrin formation, IDV co-infection counteracted this coagulation mechanism and downregulated other acute-phase response proteins, such as complement component 4 (C4) and plasminogen (PLG). The reduced inflammatory response against M. bovis likely resulted in increased M. bovis replication and delayed M. bovis clearance, which led to a significantly increased abundance of oxylipids in co-infected calves. The identified induced oxylipids mainly derived from arachidonic acid; were likely oxidized by COX-1, COX-2, and LOX-5; and peaked at 7 dpi. This paper presents the first characterization of BAL proteome and lipid mediator kinetics in response to IDV and M. bovis infection in cattle and raises hypotheses regarding how IDV acts as a co-pathogen in bovine respiratory disease.
Asunto(s)
Enfermedades de los Bovinos , Mycoplasma bovis , Infecciones del Sistema Respiratorio , Animales , Bovinos , Deltainfluenzavirus , Cromatografía Liquida , Lipidómica , Proteómica , Espectrometría de Masas en Tándem , Interacciones Huésped-Patógeno , LípidosRESUMEN
Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the µLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/µl for 50 kb fragments and an analytical time of 50 min. Then, µLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with µLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with µLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.
Asunto(s)
Sistemas CRISPR-Cas , ADN de Plantas/genética , Genoma de Planta/genética , Medicago truncatula/genética , Análisis de Secuencia de ADN/métodos , Cromosomas Artificiales Bacterianos , Biología Computacional/métodos , ADN de Plantas/aislamiento & purificación , Electroforesis en Gel de Campo Pulsado/métodos , Reproducibilidad de los ResultadosRESUMEN
We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/µL in an operating time of 20 min. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves online concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces. Using a set of DNA ladders in the range of 100-1500 bp, we report a sizing accuracy and precision better than 3% and a concentration quantification precision of â¼20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range of 75-240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical research involving cfDNA.
Asunto(s)
Ácidos Nucleicos Libres de Células/sangre , Electroforesis Capilar/instrumentación , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/sangre , Ácidos Nucleicos Libres de Células/análisis , Diseño de Equipo , Humanos , Hidrodinámica , Límite de Detección , Neoplasias/sangre , Neoplasias/diagnóstico , Reacción en Cadena de la PolimerasaRESUMEN
Biological tissues must generate forces to shape organs and achieve proper development. Such forces often result from the contraction of an apical acto-myosin meshwork. Here we describe an alternative mechanism for tissue contraction, based on individual cell volume change. We show that during Drosophila dorsal closure (DC), a wound healing-related process, the contraction of the amnioserosa (AS) is associated with a major reduction of the volume of its cells, triggered by caspase activation at the onset of the apoptotic program of AS cells. Cell volume decrease results in a contractile force that promotes tissue shrinkage. Estimating mechanical tensions with laser dissection and using 3D biophysical modeling, we show that the cell volume decrease acts together with the contraction of the actin cable surrounding the tissue to govern DC kinetics. Our study identifies a mechanism by which tissues generate forces and movements by modulating individual cell volume during development.
Asunto(s)
Citoesqueleto de Actina/fisiología , Tamaño de la Célula , Drosophila/embriología , Embrión no Mamífero/citología , Células Epiteliales/citología , Mecanotransducción Celular , Morfogénesis/fisiología , Animales , Fenómenos Biomecánicos , Caspasas/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/metabolismo , Embrión no Mamífero/ultraestructura , Células Epiteliales/metabolismo , Miosinas/metabolismo , Fosforilación , Membrana Serosa/citología , Membrana Serosa/metabolismo , Membrana Serosa/ultraestructuraRESUMEN
Cell aggregation is frequently impaired during the growth of primary tumors and the formation of metastatic lesions. Cell aggregation depends on cell-cell adhesion; however, no rigorous approach exists to monitor and quantify it accurately in the absence of the confounding factors of cell-substrate adhesion and the resulting cell motility on the substrate. We report here a highly reproducible, automated, microscopy-based quantification of tumor-cell spheroid formation in the absence of cell-substrate adhesion and use it to characterize cell aggregation dynamics in the early steps of this process. This method is based on fluorescence and bright-field microscopy and on a custom MATLAB program to quantify automatically the cells' aggregation kinetics. We demonstrate that the cell-cell adhesion protein E-cadherin and the desmosome proteins DSG2 and DSC2 are important for aggregation. Furthermore, we show that inhibition or silencing of myosin IIa enhances aggregation, suggesting that cytoskeleton tension inhibits tumor cell aggregation. This work opens new avenues to study the principles that govern multicellular aggregation, to characterize the aggregation properties of various tumor cell types, as well as to screen for drugs that inhibit or promote aggregation.
Asunto(s)
Adhesión Celular/fisiología , Agregación Celular/fisiología , Comunicación Celular/fisiología , Neoplasias/patología , Cadherinas/metabolismo , Movimiento Celular/fisiología , Citoesqueleto/patología , Desmocolinas/metabolismo , Desmogleína 2/metabolismo , Células HCT116 , Humanos , Neoplasias/metabolismo , TransfecciónRESUMEN
A new generation of the Ephesia cell capture technology optimized for CTC capture and genetic analysis is presented, characterized in depth and compared with the CellSearch system as a reference. This technology uses magnetic particles bearing tumour-cell specific EpCAM antibodies, self-assembled in a regular array in a microfluidic flow cell. 48,000 high aspect-ratio columns are generated using a magnetic field in a high throughput (>3 ml h(-1)) device and act as sieves to specifically capture the cells of interest through antibody-antigen interactions. Using this device optimized for CTC capture and analysis, we demonstrated the capture of epithelial cells with capture efficiency above 90% for concentrations as low as a few cells per ml. We showed the high specificity of capture with only 0.26% of non-epithelial cells captured for concentrations above 10 million cells per ml. We investigated the capture behavior of cells in the device, and correlated the cell attachment rate with the EpCAM expression on the cell membranes for six different cell lines. We developed and characterized a two-step blood processing method to allow for rapid processing of 10 ml blood tubes in less than 4 hours, and showed a capture rate of 70% for as low as 25 cells spiked in 10 ml blood tubes, with less than 100 contaminating hematopoietic cells. Using this device and procedure, we validated our system on patient samples using an automated cell immunostaining procedure and a semi-automated cell counting method. Our device captured CTCs in 75% of metastatic prostate cancer patients and 80% of metastatic breast cancer patients, and showed similar or better results than the CellSearch device in 10 out of 13 samples. Finally, we demonstrated the possibility of detecting cancer-related PIK3CA gene mutation in 20 cells captured in the chip with a good correlation between the cell count and the quantitation value Cq of the post-capture qPCR.
Asunto(s)
Análisis Mutacional de ADN/métodos , Separación Inmunomagnética/métodos , Técnicas Analíticas Microfluídicas/métodos , Mutación , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Humanos , Separación Inmunomagnética/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Células Neoplásicas Circulantes/inmunología , Reproducibilidad de los ResultadosRESUMEN
In chronic degenerative syndromes, neuronal death occurs over long periods, during which cells progressively lose their axons and, ultimately, their cell bodies. Although apoptosis is recognized as a key event in neuronal death, the molecular mechanisms involved in CNS axons degeneration are poorly understood. Due to the highly polarized phenotypes of CNS neurons, the different neuronal subcompartments are likely to be targeted by light repetitive and localized aggression. Such locally initiated deleterious signal transduction pathways could theoretically spread through the cytoplasm. However, where axon-degenerative signals initiate, what these early signals are, and how they lead to axon degeneration are unanswered questions that limit our understanding of neurodegenerative diseases and our ability to identify novel therapeutic targets. Using a microfluidic culture device adapted to CNS primary neurons, allowing specific access to the axonal and somatodendritic compartments, we analyzed the molecular pathways involved in axonal degeneration of differentiated neurons. We show here that local application of proapoptotic stimuli on the somatodentritic compartment triggers a dying-back pattern involving caspase-dependent axonal degeneration. Using complementary pharmacological and genetic approaches, we further demonstrate that NAD(+) and grape wine polyphenols prevent axonal apoptosis and act via mitochondrial SirT3 activation in axons.
Asunto(s)
Apoptosis/efectos de los fármacos , Axones/metabolismo , Caspasas/metabolismo , NAD/farmacología , Sirtuina 3/metabolismo , Animales , Axones/efectos de los fármacos , Ratones , Microfluídica , Resveratrol , Estilbenos/farmacologíaRESUMEN
Various experimental models are used to study brain development and degeneration. They range from whole animal models, which preserve anatomical structures but strongly limit investigations at the cellular level, to dissociated cell culture systems that allow detailed observation of cell phenotypes but lack the highly ordered physiological neuron connection architecture. We describe here a platform comprising independent cell culture chambers separated by an array of "axonal diodes". This array involves asymmetric micro-channels, imposing unidirectional axon connectivity with 97% selectivity. It allows the construction of complex, oriented neuronal networks not feasible with earlier platforms. Different neuronal subtypes could be co-cultivated for weeks, and sequential seeding of different cell populations reproduced physiological network development. To illustrate possible applications, we created and characterized a cortico-striatal oriented network. Functional synaptic connections were established. The activation of striatal differentiation by cortical axons, and the synchronization of neural activity were demonstrated. Each neuronal population and subcompartment could be chemically addressed individually. The directionality of neural pathways being a key feature of the nervous system organization, the axon diode concept brings in a paradigmatic change in neuronal culture platforms, with potential applications for studying neuronal development, synaptic transmission and neurodegenerative disorder such as Alzheimer and Parkinson diseases at the sub-cellular, cellular and network levels.
Asunto(s)
Axones/fisiología , Técnicas Analíticas Microfluídicas , Red Nerviosa/citología , Neuronas/citología , Compuestos de Anilina/química , Animales , Calcio/metabolismo , Diferenciación Celular , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Ratones Transgénicos , Red Nerviosa/metabolismo , Red Nerviosa/fisiología , Neuronas/metabolismo , Xantenos/químicaRESUMEN
A broad range of microfluidic applications, ranging from cell culture to protein crystallization, requires multilevel devices with different heights and feature sizes (from micrometers to millimeters). While state-of-the-art direct-writing techniques have been developed for creating complex three-dimensional shapes, replication molding from a multilevel template is still the preferred method for fast prototyping of microfluidic devices in the laboratory. Here, we report on a "dry and wet hybrid" technique to fabricate multilevel replication molds by combining SU-8 lithography with a dry film resist (Ordyl). We show that the two lithography protocols are chemically compatible with each other. Finally, we demonstrate the hybrid technique in two different microfluidic applications: (1) a neuron culture device with compartmentalization of different elements of a neuron and (2) a two-phase (gas-liquid) global micromixer for fast mixing of a small amount of a viscous liquid into a larger volume of a less viscous liquid.
RESUMEN
We propose a strategy for optimizing distribution of flow in a microfluidic chamber for microreactor, lateral flow assay and immunocapture applications. It is aimed at maximizing flow throughput, while keeping footprint, cell thickness, and shear stress in the distribution channels at a minimum, and offering a uniform flow field along the whole analysis chamber. In order to minimize footprint, the traditional tree-like or "rhombus" design, in which distribution microchannels undergo a series of splittings into two subchannels with equal lengths and widths, was replaced by a design in which subchannel lengths are unequal, and widths are analytically adapted within the Hele-Shaw approximation, in order to keep the flow resistance uniform along all flow paths. The design was validated by hydrodynamic flow simulation using COMSOL finite element software. Simulations show that, if the channel is too narrow, the Hele-Shaw approximation loses accuracy, and the flow velocity in the chamber can fluctuate by up to 20%. We thus used COMSOL simulation to fine-tune the channel parameters, and obtained a fluctuation of flow velocity across the whole chamber below 10%. The design was then implemented into a PDMS device, and flow profiles were measured experimentally using particle tracking. Finally, we show that this system can be applied to cell sorting in self-assembling magnetic arrays, increasing flow throughput by a factor 100 as compared to earlier reported designs.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Modelos Teóricos , Línea Celular Tumoral , Diseño de Equipo , Humanos , Separación Inmunomagnética , CinéticaRESUMEN
Degeneration of central axons may occur following injury or due to various diseases and it involves complex molecular mechanisms that need to be elucidated. Existing in vitro axotomy models are difficult to perform, and they provide limited information on the localization of events along the axon. We present here a novel experimental model system, based on microfluidic isolation, which consists of three distinct compartments, interconnected by parallel microchannels allowing axon outgrowth. Neurons cultured in one compartment successfully elongated their axons to cross a short central compartment and invade the outermost compartment. This design provides an interesting model system for studying axonal degeneration and death mechanisms, with a previously impossible spatial and temporal control on specific molecular pathways. We provide a proof-of-concept of the system by reporting its application to a well-characterized experimental paradigm, axotomy-induced Wallerian degeneration in primary central neurons. Using this model, we applied localized central axotomy by a brief, isolated flux of detergent. We report that mouse embryonic cortical neurons exhibit rapid Wallerian-like distal degeneration but no somatic death following central axotomy. Distal axons show progressive degeneration leading to axonal beading and cytoskeletal fragmentation within a few hours after axotomy. Degeneration is asynchronous, reminiscent of in vivo Wallerian degeneration. Axonal cytoskeletal fragmentation is significantly delayed with nicotinamide adenine dinucleotide pretreatment, but it does not change when distal calpain or caspase activity is inhibited. These findings, consistent with previous experiments in vivo, confirm the power and biological relevance of this microfluidic architecture.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Corteza Cerebral/patología , Microfluídica/métodos , Neuronas/patología , Degeneración Walleriana/patología , Animales , Axotomía/métodos , Sistema Nervioso Central/citología , Sistema Nervioso Central/patología , Corteza Cerebral/citología , Corteza Cerebral/embriología , Ratones , Neuronas/citologíaRESUMEN
We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.