RESUMEN
Single-cell adhesion measured with atomic force microscopy (AFM) offers outstanding time and force resolution and allows the investigation of many important phenomena with unmatched precision. However, this technique suffers from serious practical limitations that hinder its effective application to a broader set of situations. Here we propose a different strategy based on the fabrication of large cantilevers and on the culture of the cells directly on them. Cantilevers are fabricated by standard micromachining, with an active area of 300 × 300 µm. A wedged structure is created so that the cantilever surface lies parallel to the substrate when mounted on an AFM system, so that the adhesion measurement probes the whole surface area at the same time. Thanks to the large area, cells can be seeded and grown on the cantilevers the day before the experiment, and let recover to optimal condition for the experiment. We used Human Embryonic Kidney cells, HEK 293A, to demonstrate the measurement of adhesion forces of up to 100 cells in parallel, and obtain a straightforward measurement of the average single cell adhesion energy. Our approach can improve significantly the cell-cell and cell-substrate adhesion statistics, reduce the experiment time and allow the investigation of the adhesion properties of cells that do not grow well in solution or on low adherent substrates, or that develop their characteristic features only after several hours or days of culture on a solid and adherent substrate.
Asunto(s)
Fenómenos Mecánicos , Microtecnología , Adhesión Celular , Humanos , Microscopía de Fuerza Atómica/métodosRESUMEN
It is known that, although the level of light is the primary determinant of pupil size, cognitive factors can also affect pupil diameter. It has been demonstrated that photographs of the sun produce pupil constriction independently of their luminance and other low-level features, suggesting that high-level visual processing may also modulate pupil response. Here, we measure pupil response to artistic paintings of the sun, moon, or containing a uniform lighting, that, being mediated by the artist's interpretation of reality and his technical rendering, require an even higher level of interpretation compared with photographs. We also study how chromatic content and spatial layout affect the results by presenting grey-scale and inverted versions of each painting. Finally, we assess directly with a categorization test how subjective image interpretation affects pupil response. We find that paintings with the sun elicit a smaller pupil size than paintings with the moon, or paintings containing no visible light source. The effect produced by sun paintings is reduced by disrupting contextual information, such as by removing color or manipulating the relations between paintings features that make more difficult to identify the source of light. Finally, and more importantly, pupil diameter changes according to observers' interpretation of the scene represented in the same stimulus. In conclusion, results show that the subcortical pupillary response to light is modulated by subjective interpretation of luminous objects, suggesting the involvement of cortical systems in charge of cognitive processes, such as attention, object recognition, familiarity, memory, and imagination.
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Luz , Pinturas , Pupila/fisiología , Percepción Visual/fisiología , Adulto , Atención/fisiología , Femenino , Humanos , Masculino , Reflejo Pupilar/fisiología , Visión Ocular/fisiología , Adulto JovenRESUMEN
Cardiac fibroblasts (CFs) are essential for preserving myocardial integrity and function. They can detect variations in cardiac tissue stiffness using various cellular mechanosensors, including the Ca2+ permeable mechanosensitive channel Piezo1. Nevertheless, how CFs adapt the mechanosensitive response to stiffness changes remains unclear. In this work we adopted a multimodal approach, combining the local mechanical stimulation (from 10 pN to 350 nN) with variations of culture substrate stiffness. We found that primary rat CFs cultured on stiff (GPa) substrates showed a broad Piezo1 distribution in the cell with particular accumulation at the mitochondria membrane. CFs displayed a force-dependent behavior in both calcium uptake and channel activation probability, showing a threshold at 300 nN, which involves both cytosolic and mitochondrial Ca2+ mobilization. This trend decreases as the myofibroblast phenotype within the cell population increases, following a possible Piezo1 accumulation at focal adhesion sites. In contrast, the inhibition of fibroblasts to myofibroblasts transition with soft substrates (kPa) considerably reduces both mechanically- and chemically-induced Piezo1 activation and expression. Our findings shed light on how Piezo1 function and expression are regulated by the substrate stiffness and highlight its involvement in the environment-mediated modulation of CFs mechanosensitivity.
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Fibroblastos , Canales Iónicos , Mecanotransducción Celular , Proteínas de la Membrana , Animales , Canales Iónicos/metabolismo , Ratas , Fibroblastos/metabolismo , Fibroblastos/citología , Células Cultivadas , Calcio/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/fisiología , Miocardio/metabolismo , Miocardio/citología , Microambiente CelularRESUMEN
Testing devices based on cell tracking are particularly interesting as diagnostic tools in medicine for antibiotics susceptibility testing and in vitro chemotherapeutic screening. In this framework, the application of nanomechanical sensors has attracted much attention, although some crucial aspects such as the effects of the viscous damping, when operating in physiological conditions environment, still need to be properly solved. To address this problem, we have designed and fabricated a nanomechanical force sensor that operates at the interface between liquid and air. Our sensor consists of a silicon chip including a 500 µm wide Si3N4 suspended membrane where three rectangular silicon nitride cantilevers are defined by a lithographically etched gap. The cantilevers can be operated in air, fully immersed in a liquid environment and in half wetting condition, with one side in contact with the solution and the opposite one in air. The formation of a water meniscus in the gap prevents the leakage of medium to the opposite side, which remained dry and is used to reflect a laser to measure the cantilever deflection. This configuration enables to keep the cells in physiological environment while operating the sensor in dry conditions. The performance of the sensor has been applied to monitor the motion and measures the forces developed by migrating breast cancer cell. The functionalization of one side of the cantilever and the use of a purposely designed chamber of measurements enable the confinement of the cell only on one side of the cantilever. Our data demonstrate that this approach can distinguish the adhesion and contraction forces developed by different cell lines and may represents valuable tool for a fast and quantitative in-vitro screening of new chemotherapeutic drugs targeting cancer cell adhesion and motility.
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Fenómenos Mecánicos , Línea Celular , Adhesión Celular , Movimiento (Física)RESUMEN
(1) Background: Several types of hearing aids are available for the rehabilitation of vestibular-schwannoma (VS)-related hearing loss. There is a lack of recently published papers regarding this theme. The aim of the present work is to organize current knowledge. (2) Methods: A review of the literature regarding the topics "vestibular schwannoma", "hearing loss", and "hearing aid" was performed. Nineteen studies were thus considered. (3) Results: Conventional hearing aids, contralateral routing of signal (CROS) aids, bone anchored hearing aids (BAHA), and others are available options for hearing rehabilitation in VS patients. The speech discrimination score (SDS) is considered the best measure to assess candidacy for rehabilitation with hearing aids. The best hearing rehabilitative conditions in VS patients when using conventional hearing aid devices are a mild-moderate hearing loss degree with good word recognition (more than 50% SDS). CROS-Aid and BAHA are reported to be beneficial. CROS-Aid expands on the area of receiving hearing. BAHA aids use direct bone-conduction stimulation. Unfortunately, there are no available studies focused specifically on VS patients that compare CROS and BAHA technologies. (4) Conclusions: Hearing aids, CROS, and BAHA are viable options for rehabilitating hearing impairment in VS, but require an accurate case-by-case audiological evaluation for rehabilitating hearing impairment in VS. Further studies are needed to prove if what is currently known about similar hearing illnesses can be confirmed, particularly in the case of VS.
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The deposition of biomolecules on biosensing surface platforms plays a key role in achieving the required sensitivity and selectivity for biomolecular interactions analysis. Controlling the interaction between the surface and biomolecules is increasingly becoming a crucial design tool to modulate the surface properties needed to improve the performance of the assay and the detection outcome. Carboxymethyl-dextran (CMD) coating can be exploited to promote chemical grafting of proteins, providing a hydrophilic, bioinert, nonfouling surface and a high surface density of immobilized proteins. In the present work, we developed and optimized a technique to produce a cost-effective CMD-based patterned surface for the immobilization of biomolecules to be used on standard protocols optimization. They consist of silicon or glass substrates with patterned bioactive areas able to efficiently confine the sampling solution by simply exploiting hydrophilic/hydrophobic patterning of the surface. The fabrication process involves the use of low-cost instruments and techniques, compatible with large scale production. The devices were validated through a chemiluminescence assay we recently developed for the analysis of binding of DNA nanoassemblies modified with an affinity binder to target proteins immobilized on the bioactive areas. Through this assay we were able to characterize the chemical reactivity of two target proteins toward a dextran matrix on patterned surfaces and to compare it with model CMD-based surface plasmon resonance (SPR) surfaces. We found a high reproducibility and selectivity in molecular recognition, consistent with results obtained on SPR sensor surfaces. The suggested approach is straightforward, cheap, and provides the means to assess patterned functionalized surfaces for bioanalytical platforms.
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Dextranos , Resonancia por Plasmón de Superficie , Dextranos/química , Proteínas , Reproducibilidad de los Resultados , Silicio , Resonancia por Plasmón de Superficie/métodos , Propiedades de SuperficieRESUMEN
The long-known role of cell migration in physiological and pathological contexts still requires extensive research to be fully understood, mainly because of the intricate interaction between moving cells and their surroundings. While conventional assays fail to capture this complexity, recently developed 3D platforms better reproduce the cellular micro-environment, although often requiring expensive and time-consuming imaging approaches. To overcome these limitations, we developed a novel approach based on 2D micro-patterned substrates, compatible with conventional microscopy analysis and engineered to create micro-gaps with a length of 150 µm and a lateral size increasing from 2 to 8 µm, where a curved water-air interface is created on which cells can adhere, grow, and migrate. The resulting hydrophilic/hydrophobic interfaces, variable surface curvatures, spatial confinements, and size values mimic the complex micro-environment typical of the extracellular matrix in which aggressive cancer cells proliferate and migrate. The new approach was tested with two breast cancer cell lines with different invasive properties. We observed that invasive cells (MDA-MB-231) can align along the pattern and modify both their morphology and their migration rate according to the size of the water meniscus, while non-invasive cells (MCF-7) are only slightly respondent to the surrounding micro-environment. Moreover, the selected pattern highlighted a significative matrix deposition process connected to cell migration. Although requiring further optimizations, this approach represents a promising tool to investigate cell migration in complex environments.