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Atherosclerosis is a chronic disease of the vascular wall driven by lipid accumulation and inflammation in the intimal layer of arteries, and its main complications, myocardial infarction and stroke, are the leading cause of mortality worldwide [1], [2]. Recent studies have identified Triggering receptor expressed on myeloid cells 2 (TREM2), a lipid-sensing receptor regulating myeloid cell functions [3], to be highly expressed in macrophage foam cells in experimental and human atherosclerosis [4]. However, the role of TREM2 in atherosclerosis is not fully known. Here, we show that hematopoietic or global TREM2 deficiency increased, whereas TREM2 agonism decreased necrotic core formation in early atherosclerosis. We demonstrate that TREM2 is essential for the efferocytosis capacities of macrophages, and to the survival of lipid-laden macrophages, indicating a crucial role of TREM2 in maintaining the balance between foam cell death and clearance of dead cells in atherosclerotic lesions, thereby controlling plaque necrosis.
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Here, screen-printed carbon electrodes (SPCEs) were modified with ultrafine and mainly mono-disperse sea urchin-like tungsten oxide (SUWO3) nanostructures synthesized by a simple one-pot hydrothermal method for non-enzymatic detection of dopamine (DA) and uric acid (UA) in synthetic urine. Sea urchin-like nanostructures were clearly observed in scanning electron microscope images and WO3 composition was confirmed with XRD, Raman, FTIR and UV-Vis spectrophotometer. Modification of SPCEs with SUWO3 nanostructures via the drop-casting method clearly reduced the Rct value of the electrodes, lowered the ∆Ep and enhanced the DA oxidation current due to high electrocatalytic activity. As a result, SUWO3/SPCEs enabled highly sensitive non-enzymatic detection of DA (LOD: 51.4 nM and sensitivity: 127 µA mM-1 cm-2) and UA (LOD: 253 nM and sensitivity: 55.9 µA mM-1 cm-2) at low concentration. Lastly, SUWO3/SPCEs were tested with synthetic urine, in which acceptable recoveries for both molecules (94.02-105.8%) were obtained. Given the high selectivity, the sensor has the potential to be used for highly sensitive simultaneous detection of DA and UA in real biological samples.
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Dopamina , Nanoestructuras , Óxidos , Tungsteno , Dopamina/química , Ácido Úrico , Nanoestructuras/química , Electrodos , Carbono/química , Técnicas Electroquímicas/métodos , Ácido Ascórbico/químicaRESUMEN
Peroxidase mimicking Fe3O4@Chitosan (Fe3O4@Chi) nanozyme was synthesized and used for high-sensitive enzyme-free colorimetric detection of H2O2. The nanozyme was characterized in comparison with Fe3O4 nanoparticles (NPs) using X-ray diffraction, Fourier-transform infrared spectroscopy, dynamic light scattering, and thermogravimetric analysis. The catalytic performance of Fe3O4@Chi nanozyme was first evaluated by UV-Vis spectroscopy using 3,3',5,5'-tetramethylbenzidine. Unlike Fe3O4NPs, Fe3O4@Chi nanozyme exhibited an intrinsic peroxidase activity with a detection limit of 69 nM. Next, the nanozyme was applied to a microfluidic paper-based analytical device (µPAD) and colorimetric analysis was performed at varying concentrations of H2O2 using a machine learning-based smartphone app called "Hi-perox Sens++ ." The app with machine learning classifiers made the system user-friendly as well as more robust and adaptive against variation in illumination and camera optics. In order to train various machine learning classifiers, the images of the µPADs were taken at 30 s and 10 min by four smartphone brands under seven different illuminations. According to the results, linear discriminant analysis exhibited the highest classification accuracy (98.7%) with phone-independent repeatability at t = 30 s and the accuracy was preserved for 10 min. The proposed system also showed excellent selectivity in the presence of various interfering molecules and good detection performance in tap water.
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Colorimetría , Peróxido de Hidrógeno , Inteligencia Artificial , Colorimetría/métodos , Peróxido de Hidrógeno/análisis , Peroxidasa/química , PeroxidasasRESUMEN
A growing number of sperm methylome analyses have identified genomic loci that are susceptible to paternal age effects in a variety of mammalian species, including human, bovine, and mouse. However, there is little overlap between different data sets. Here, we studied whether or not paternal age effects on the sperm epigenome have been conserved in mammalian evolution and compared methylation patterns of orthologous regulatory regions (mainly gene promoters) containing both conserved and non-conserved CpG sites in 94 human, 36 bovine, and 94 mouse sperm samples, using bisulfite pyrosequencing. We discovered three (NFKB2, RASGEF1C, and RPL6) age-related differentially methylated regions (ageDMRs) in humans, four (CHD7, HDAC11, PAK1, and PTK2B) in bovines, and three (Def6, Nrxn2, and Tbx19) in mice. Remarkably, the identified sperm ageDMRs were all species-specific. Most ageDMRs were in genomic regions with medium methylation levels and large methylation variation. Orthologous regions in species not showing this age effect were either hypermethylated (>80%) or hypomethylated (<20%). In humans and mice, ageDMRs lost methylation, whereas bovine ageDMRs gained methylation with age. Our results are in line with the hypothesis that sperm ageDMRs are in regions under epigenomic evolution and may be part of an epigenetic mechanism(s) for lineage-specific environmental adaptations and provide a solid basis for studies on downstream effects in the genes analyzed here.
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Metilación de ADN , Edad Paterna , Espermatozoides , Animales , Bovinos , Metilación de ADN/genética , Epigénesis Genética , Epigenoma , Masculino , Ratones , Espermatozoides/metabolismoRESUMEN
In the present study, iodide-mediated 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 reaction system was applied to a microfluidic paper-based analytical device (µPAD) for non-enzymatic colorimetric determination of H2O2. The proposed system is portable and incorporates a µPAD with a machine learning-based smartphone app. A smartphone app called "Hi-perox Sens" capable of image capture, cropping and processing was developed to make the system simple and user-friendly. Briefly, circular µPADs were designed and tested with varying concentrations of H2O2. Following the color change, the images of the µPADs were taken with four different smartphones under seven different illumination conditions. In order to make the system more robust and adaptive against illumination variation and camera optics, the images were first processed for feature extraction and then used to train machine learning classifiers. According to the results, TMB + KI showed the highest classification accuracy (97.8%) with inter-phone repeatability at t = 30 s under versatile illumination and maintained its accuracy for 10 minutes. In addition, the performance of the system was also comparable to two different commercially available H2O2 kits in real samples.
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Peróxido de Hidrógeno , Aplicaciones Móviles , Colorimetría , Aprendizaje Automático , Papel , Teléfono InteligenteRESUMEN
Herein, we report the fabrication of zinc oxide nanowire (ZnO NW) coated carbon fiber (CF) ultra-microelectrodes (UME). ZnO NWs were grown on commercial multifilament CFs through hydrothermal process in a teflon-lined autoclave at 90 °C for 4 h. X-ray diffraction (XRD), Raman and scanning electron microscopy characterizations showed that crystalline and well oriented NW structures were successfully obtained. The fabrication of the pH sensitive UME was carried out by a novel approach which allowed controlling the protruding length of the modified CF surface. The UME was then integrated with a metal-oxide-semiconductor field effect transistor (MOSFET) for the construction of an EGFET pH-microsensor. The present pH microsensor is expected to be useful for localized pH measurement in small volumes such single cell analysis.
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In this study, a microfluidic paper-based analytical device (µPAD) was integrated with a smartphone app capable of offline (without internet access) image processing and analysis for the rapid colorimetric detection of glucose. A self-inking stamp was used to form hydrophobic channels on a piece of paper-towel due to its superior water absorption efficiency. As demonstrated, the developed sensor was employed for the colorimetric detection of glucose in artificial saliva in the linear scope of 0 - 1 mM with a calculated detection limit of 29.65 µM. The experimental results show that the quantitative analysis of glucose with the proposed smartphone platform could be completed in less than one minute. The app developed for the smartphone platform is capable of extracting the color-changing area with an embedded image processing tool which could address the problem of color uniformity in the detection zones of µPAD. The integrated platform has great potential to be used for non-invasive measurements of glucose in body fluids, like tears, sweat and saliva.
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Colorimetría , Teléfono Inteligente , Glucosa , Papel , Saliva ArtificialRESUMEN
The PC12 cell line has been widely used as an in vitro model for studying neuronal differentiation and identifying the factors affecting the process. It has the ability to differentiate in the presence of nerve growth factor (NGF), resulting in neural extensions called dendrites and axons. In this study, first the impact of randomly distributed multi-walled carbon nanotubes (MWCNTs) in poly(ethylene glycol) dimethacrylate (PEGDMA) on PC12 cell differentiation was investigated in terms of neurite length, number of neurite per cell and differentiation marker gene expression profile. Then, dielectrophoretically aligned MWCNTs in PEGDMA was used to guide and support the neuronal differentiation of PC12 cells in the presence of NGF. The method is expected to be useful in revealing the nanotopographical role in fundamental studies and understanding of nanotopographical effects for biomedical applications on nerve regeneration.
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Even though large number of individually addressable electrodes can be effectively assembled in a small area, electrochemical detection methods have a relatively limited ability to detect multiple analytes compared to microdialysis probes and other analytic techniques. Here, we report a facile method for the electro-addressable functionalization of a probe comprising of closely spaced three individually addressable carbon fiber electrodes (CFEs) for the detection of nucleic acids. First, a multi electrode array probe comprising three adjacent CFEs was fabricated through pulling a three-barrel glass capillary with a single carbon fiber in each barrel. Second, electropolymerization based doping was used for the electro-addressable functionalization of the multi-electrode array probe. To demonstrate that the current strategy works, anti-miR-34a was electrografted on only one of three electrodes by the electropolymerization of pyrrole on a specific electrode. A second electrode was coated only with polypyrrole (PPy) and the third was left unmodified. The results demonstrate that the present strategy has great potential for constructing multiplex nucleic acid micro/nano biosensors for local and in situ detection of multiple nucleic acid molecules, such as miRNAs at a time.
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Técnicas Electroquímicas , MicroARNs/análisis , Electrodos , Humanos , Polímeros/química , Pirroles/químicaRESUMEN
Optimization of nanofiber (NF) surface properties is critical to achieve an adequate cellular response. Here, the impact of conjugation of biomimetic aspartic acid (ASP) and glutamic acid (GLU) templated peptides with poly(lactic-co-glycolic acid) (PLGA) electrospun NF on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Cold atmospheric plasma (CAP) was used to functionalize the NF surface and thus to mediate the conjugation. The influence of the CAP treatment following with peptide conjugation to the NF surface was assessed using water contact angle measurements, Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS). The effect of CAP treatment on morphology of NF was also checked using Scanning Electron Microscopy (SEM). Both the hydrophilicity of NF and the number of the carboxyl (-COOH) groups on the surface increased with respect to CAP treatment. Results demonstrated that CAP treatment significantly enhanced peptide conjugation on the surface of NF. Osteogenic differentiation results indicated that conjugating of biomimetic ASP templated peptides sharply increased alkaline phosphatase (ALP) activity, calcium content, and expression of key osteogenic markers of collagen type I (Col-I), osteocalcin (OC), and osteopontin (OP) compared to GLU conjugated (GLU-pNF) and CAP treated NF (pNF). It was further depicted that ASP sequences are the major fragments that influence the mineralization and osteogenic differentiation in non-collagenous proteins of bone extracellular matrix.
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Ácido Aspártico/metabolismo , Diferenciación Celular/efectos de los fármacos , Ácido Glutámico/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Nanofibras/química , Péptidos/metabolismo , Células Cultivadas , Humanos , Microscopía Electrónica de Rastreo , Osteogénesis/efectos de los fármacos , Gases em Plasma , Espectroscopía Infrarroja por Transformada de Fourier , Tensión SuperficialRESUMEN
Herein, we present an overview of recent research progress in the development of micro/nanoelectrochemical probe and chip devices for the evaluation of three-dimensional (3D) cultured cells. First, we discuss probe devices: a general outline, evaluation of O2 consumption, enzyme-modified electrodes, evaluation of endogenous enzyme activity, and the collection of cell components from cell aggregates are discussed. The next section is focused on integrated chip devices: a general outline, electrode array devices, smart electrode array devices, droplet detection of 3D cultured cells, cell manipulation using dielectrophoresis (DEP), and electrodeposited hydrogels used for fabrication of 3D cultured cells on chip devices are discussed. Finally, we provide a summary and discussion of future directions of research in this field.
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Células Cultivadas , Electrodos , Dispositivos Laboratorio en un Chip , Nanotecnología , Animales , Agregación Celular , Línea Celular , Perros , Electroforesis , Enzimas/química , Células Hep G2 , Humanos , Hidrogeles , Células de Riñón Canino Madin Darby , Oxígeno/análisisRESUMEN
In nanotechnological devices, mass transport can be initiated by pressure driven flow, diffusion or by employing molecular motors. As the scale decreases, molecular motors can be helpful as they are not limited by increased viscous resistance. Moreover, molecular motors can move against diffusion gradients and are naturally fitted for nanoscale transportation. Among motor proteins, kinesin has particular potential for lab-on-a-chip applications. It can be used for sorting, concentrating or as a mechanical sensor. When bound to a surface, kinesin motors propel microtubules in random directions, depending on their landing orientation. In order to circumvent this complication, the microtubule motion should be confined or guided. To this end, dielectrophoretically aligned multi-walled-carbon nanotubes (MWCNT) can be employed as nanotracks. In order to control more precisely the spatial repartition of the MWCNTs, a screening method has been implemented and tested. Polygonal patterns have been fabricated with the aim of studying the guiding and the microtubule displacement between MWCNT segments. Microtubules are observed to transfer between MWCNT segments, a prerequisite for the guiding of microtubules in MWCNT circuit-based biodevices. The effect of the MWCNT organization (crenellated or hexagonal) on the MT travel distance has been investigated as well.
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Microtúbulos/química , Nanotubos de Carbono/química , Diseño de Equipo , Cinesinas/metabolismo , Microscopía Fluorescente , Peso MolecularRESUMEN
We fabricated a platinum-based double barrel probe for scanning electrochemical microscopy-scanning ion conductance microscopy (SECM-SICM) by electrodepositing platinum onto the carbon nanoelectrode of the double barrel probe. The deposition conditions were optimized to attain highly sensitive electrochemical measurements and imaging. Simultaneous SECM-SICM imaging of electrochemical features and noncontact topography by using the optimized probe afforded high-resolution images of epidermal growth factor receptors (EGFR) on the membrane surface of the A431 cells.
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In this study, various amounts of oxygen were added to Ti-10Cr (mass%) alloys. It is expected that a large changeable Young's modulus, caused by a deformation-induced ω-phase transformation, can be achieved in Ti-10Cr-O alloys by the appropriate oxygen addition. This "changeable Young's modulus" property can satisfy the otherwise conflicting requirements for use in spinal implant rods: high and low moduli are preferred by surgeons and patients, respectively. The influence of oxygen on the microstructures and mechanical properties of the alloys was examined, as well as the bending springback and cytocompatibility of the optimized alloy. Among the Ti-10Cr-O alloys, Ti-10Cr-0.2O (mass%) alloy shows the largest changeable Young's modulus following cold rolling for a constant reduction ratio. This is the result of two competing factors: increased apparent ß-lattice stability and decreased amounts of athermal ω phase, both of which are caused by oxygen addition. The most favorable balance of these factors for the deformation-induced ω-phase transformation occurred at an oxygen concentration of 0.2mass%. Ti-10Cr-0.2O alloy not only exhibits high tensile strength and acceptable elongation, but also possesses a good combination of high bending strength, acceptable bending springback and great cytocompatibility. Therefore, Ti-10Cr-0.2O alloy is a potential material for use in spinal fixture devices.
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Aleaciones , Materiales Biocompatibles , Cromo/química , Oxígeno/química , Columna Vertebral/cirugía , Titanio/química , Ensayo de MaterialesRESUMEN
We present a chip device with an array of 900 gourd-shaped microwells designed to pair single cells of different types. The device consists of interdigitated array (IDA) electrodes and uses positive dielectrophoresis to trap cells within the microwells. Each side of a microwell is on a different comb of the IDA, so that cells of different types are trapped on opposite sides of the microwells, leading to close cell pairing. Using this device, a large number of cell pairs can be formed easily and rapidly, making it a highly attractive tool for controllable cell pairing in a range of biological applications.
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Electroforesis/instrumentación , Análisis de Matrices Tisulares/instrumentación , Células 3T3 , Animales , Electrodos , Células Madre Embrionarias/citología , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Microscopía FluorescenteRESUMEN
A large scale integration (LSI)-based amperometric sensor is used for electrochemical evaluation and real-time monitoring of the alkaline phosphatase (ALP) activity of mouse embryoid bodies (EBs). EBs were prepared by the hanging drop culture of embryonic stem (ES) cells. The ALP activity of EBs with various sizes was electrochemically detected at 400 measurement points on a Bio-LSI chip. The electrochemical measurements revealed that the relative ALP activity was low for large EBs and decreased with progress of the differentiation level of the ES cells. The ALP activity of the EBs was successfully monitored in real time for 3.5h, and their ALP activity in a glucose-free buffer decreased after 2h. To the best of our knowledge, this is the first report on the application of an LSI-based amperometric sensor for real-time cell monitoring over 3h. The chip is expected to be useful for the evaluation of cell activities.
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Fosfatasa Alcalina/metabolismo , Técnicas Biosensibles/instrumentación , Técnicas Electroquímicas/instrumentación , Cuerpos Embrioides/enzimología , Animales , Diferenciación Celular , Supervivencia Celular , Cuerpos Embrioides/citología , Pruebas de Enzimas/instrumentación , Diseño de Equipo , RatonesRESUMEN
A lab-on-a-chip device is described for the electrochemical detection of alkaline phosphatase (ALP) secreted by transformed single HeLa cells. Detection on the chip device is based on local redox cycling at 256 individually addressable sensor points. Ring-disk electrodes (generator/collector) are arranged at individual sensor points to amplify the signal due to redox-cycling with only 32 connector pads. The surface of each sensor point is modified with antibodies for secreted alkaline phosphatase (SEAP) immobilization, which facilitates separation and detection of SEAP. Separation of SEAP from HeLa cells enables elimination of endogenous ALP and prevents HeLa cells from damage due to exposure to high level pH used during electrochemical detection. The large number of sensor points enables the simultaneous analysis of a large amount of single cells using the chip. The system is useful for gene reporter assays and for the detection of several types of secreted proteins.
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Fosfatasa Alcalina/análisis , Técnicas Electroquímicas , Genes Reporteros , Técnicas Analíticas Microfluídicas , Análisis de la Célula Individual , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Técnicas Electroquímicas/instrumentación , Electrodos , Células HeLa , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Oxidación-Reducción , Análisis de la Célula Individual/instrumentación , Propiedades de SuperficieRESUMEN
A new electrochemical assay for the detection of secreted alkaline phosphatase (SEAP) from transfectant HeLa cells is proposed using a microarray device and scanning electrochemical microscopy (SECM). The assay consists of two steps: the first is the incubation of a transfected cell in a microarray culture device covered with a substrate modified with anti-SEAP under physiological conditions without any additives. The array device consists of a 4 × 4 array of microwells having a size of 100 µm × 100 µm (diameter × depth). The second step is SECM measurement of secreted SEAP at the antibody-immobilized substrate. This assay ensures accuracy and intactness because the undesired influence of endogeneous ALP is eliminated and the transfected cells are incubated in a culture device under suitable conditions. We successfully detected the expression of SEAP from intact cells at the single-cell level using this assay. The system is useful as a cell-based gene-expression assay.
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Fosfatasa Alcalina/metabolismo , Técnicas Electroquímicas/métodos , Perfilación de la Expresión Génica/métodos , Genes Reporteros , Células HeLa , Humanos , Análisis por Micromatrices , TransfecciónRESUMEN
Boron is an industrially and biologically important element. However, the mechanisms of boron tolerance and its transport in bacteria and many other living systems are still not clearly understood. In this study, the boron resistance level of a boron-tolerant bacterium, Bacillus boroniphilus DSM 17376, was improved up to 300 mmol l(-1) boron, by employing an in vivo evolutionary engineering strategy based on batch selection under continuous exposure to gradually increasing boron stress levels. The resistance was heterogeneous within the final mutant population which ranged from about 1- to 16-fold of the wild type resistance at 150 mmol l(-1) boron stress level. Boron-resistant mutants had significant cross-resistance to iron and copper stresses, and were also cross-resistant to salt (NaCl) stress, suggesting a common resistance mechanism between these stress types. Additionally, highly boron-resistant mutants had up to 2.8-fold higher boron contents than the wild-type, when exposed to high levels of (150 mmol l(-1)) continuous boron stress throughout their cultivation. It was shown that evolutionary engineering is a successful approach to significantly increase bacterial boron resistance and investigate the complex mechanism of boron tolerance and transport in microbial systems.