Enhancing micropipette aspiration with artificial-intelligence analysis.

The micropipette-aspiration technique is commonly used in the field of mechanobiology, offering a variety of measurement types. To extract biophysical parameters from the experiments, numerical analysis is required. Although previous works have developed techniques for the partial automation of these analyses, these approaches are relatively time consuming for the researchers. In this article, we describe the development and application of an artificial-intelligence tool for the completely automatic analysis of micropipette-aspiration experiments. The use of this tool is compared with previous methods and the impressive reduction in the time required for these analyses is discussed. The new tool opens new possibilities for the micropipette-aspiration technique by enabling dealing with large numbers of experiments and real-time measurements.

Aging is accompanied by T-cell stiffening and reduced interstitial migration through dysfunctional nuclear organization.

Age-associated changes in T-cell function play a central role in immunosenescence. The role of aging in the decreased T-cell repertoire, primarily because of thymic involution, has been extensively studied. However, increasing evidence indicates that aging also modulates the mechanical properties of cells and the internal ordering of diverse cell components. Cellular functions are generally dictated by the biophysical phenotype of cells, which itself is also tightly regulated at the molecular level. Based on previous evidence suggesting that the relative nuclear size contributes to variations of T-cell stiffness, here we examined whether age-associated changes in T-cell migration are dictated by biophysical parameters, in part through nuclear cytoskeleton organization and cell deformability. In this study, we first performed longitudinal analyses of a repertoire of 111 functional, biophysical and biomolecular features of the nucleus and cytoskeleton of mice CD4+ and CD8+ T cells, in both naive and memory state. Focusing on the pairwise correlations, we found that age-related changes in nuclear architecture and internal ordering were correlated with T-cell stiffening and declined interstitial migration. A similarity analysis confirmed that cell-to-cell variation was a direct result of the aging process and we applied regression models to identify biomarkers that can accurately estimate individuals' age. Finally, we propose a biophysical model for a comprehensive understanding of the results: aging involves an evolution of the relative nuclear size, in part through DNA-hypomethylation and nuclear lamin B1, which implies an increased cell stiffness, thus inducing a decline in cell migration.

Possibilities of using T-cell biophysical biomarkers of ageing.

Ageing is interrelated with the development of immunosenescence. This article focuses on one of the cell sets of the adaptive immune system, T cells, and provides a review of the known changes in T cells associated with ageing. Such fundamental changes affect both cell molecular content and internal ordering. However, acquiring a complete description of the changes at these levels would require extensive measurements of parameters and, furthermore, important fine details of the internal ordering that may be difficult to detect. Therefore, an alternative approach for the characterisation of cells consists of the performance of physical measurements of the whole cell, such as deformability measurements or migration measurements: the physical parameters, complementing the commonly used chemical biomarkers, may contribute to a better understanding of the evolution of T-cell states during ageing. Mechanical measurements, among other biophysical measurements, have the advantage of their relative simplicity: one single parameter agglutinates the complex effects of the variety of changes that gradually appear in cells during ageing.

Mitochondrial DNA insertions into nuclear DNA affecting chromosome segregation: Insights for a novel mechanism of immunosenescence in mice.

Mitochondrial DNA sequences were found inserted in the nuclear genome of mouse peritoneal T lymphocytes that increased progressively with aging. These insertions were preferentially located at the pericentromeric heterochromatin. In the same individuals, binucleated T-cells with micronuclei showed a significantly increased frequency associated with age. Most of them were positive for centromere sequences, reflecting the loss of chromatids or whole chromosomes. The proliferative capacity of T lymphocytes decreased with age as well as the glutathione reductase activity, whereas the oxidized glutathione and malondialdehyde concentrations exhibited a significant increase. These results may point to a common process that provides insights for a new approach to understanding immunosenescence. We propose a novel mechanism in which mitochondrial fragments, originated by the increased oxidative stress status during aging, accumulate inside the nuclear genome of T lymphocytes in a time-dependent way. The primary entrance of mitochondrial fragments at the pericentromeric regions may compromise chromosome segregation, causing genetic loss that leads to micronuclei formation, rendering aneuploid cells with reduced proliferation capacity, one of the hallmark of immunosenescence. Future experiments deciphering the mechanistic basis of this phenomenon are needed.

Basic Principles in the Design of Spider Silk Fibers.

The prominence of spider silk as a hallmark in biomimetics relies not only on its unrivalled mechanical properties, but also on how these properties are the result of a set of original design principles. In this sense, the study of spider silk summarizes most of the main topics relevant to the field and, consequently, offers a nice example on how these topics could be considered in other biomimetic systems. This review is intended to present a selection of some of the essential design principles that underlie the singular microstructure of major ampullate gland silk, as well as to show how the interplay between them leads to the outstanding tensile behavior of spider silk. Following this rationale, the mechanical behavior of the material is analyzed in detail and connected with its main microstructural features, specifically with those derived from the semicrystalline organization of the fibers. Establishing the relationship between mechanical properties and microstructure in spider silk not only offers a vivid image of the paths explored by nature in the search for high performance materials, but is also a valuable guide for the development of new artificial fibers inspired in their natural counterparts.

Single-cell biophysical study reveals deformability and internal ordering relationship in T cells.

Deformability and internal ordering are key features related to cell function, particularly critical for cells that routinely undergo large deformations, like T cells during extravasation and migration. In the measurement of cell deformability, a considerable variability is typically obtained, masking the identification of possible interrelationships between deformability, internal ordering and cell function. We report the development of a single-cell methodology that combines measurements of living-cell deformability, using micropipette aspiration, and three-dimensional confocal analysis of the nucleus and cytoskeleton. We show that this single-cell approach can serve as a powerful tool to identify appropriate parameters that characterize deformability within a population of cells, not readably discernable in population-averaged data. By applying this single-cell methodology to mouse CD4+ T cells, our results demonstrate that the relative size of the nucleus, better than other geometrical or cytoskeletal features, effectively determines the overall deformability of the cells within the population.

Programmable ROS-Mediated Cancer Therapy via Magneto-Inductions.

Reactive oxygen species (ROS), a group of oxygen derived radicals and derivatives, can induce cancer cell death via elevated oxidative stress. A spatiotemporal approach with safe and deep-tissue penetration capabilities to elevate the intracellular ROS level is highly desirable for precise cancer treatment. Here, a mechanical-thermal induction therapy (MTIT) strategy is developed for a programmable increase of ROS levels in cancer cells via assembly of magnetic nanocubes integrated with alternating magnetic fields. The magneto-based mechanical and thermal stimuli can disrupt the lysosomes, which sequentially induce the dysfunction of mitochondria. Importantly, intracellular ROS concentrations are responsive to the magneto-triggers and play a key role for synergistic cancer treatment. In vivo experiments reveal the effectiveness of MTIT for efficient eradication of glioma and breast cancer. By remote control of the force and heat using magnetic nanocubes, MTIT is a promising physical approach to trigger the biochemical responses for precise cancer treatment.

Soft-Lithography of Polyacrylamide Hydrogels Using Microstructured Templates: Towards Controlled Cell Populations on Biointerfaces.

Polyacrylamide hydrogels are interesting materials for studying cells and cell-material interactions, thanks to the possibility of precisely adjusting their stiffness, shear modulus and porosity during synthesis, and to the feasibility of processing and manufacturing them towards structures and devices with controlled morphology and topography. In this study a novel approach, related to the processing of polyacrylamide hydrogels using soft-lithography and employing microstructured templates, is presented. The main novelty relies on the design and manufacturing processes used for achieving the microstructured templates, which are transferred by soft-lithography, with remarkable level of detail, to the polyacrylamide hydrogels. The conceived process is demonstrated by patterning polyacrylamide substrates with a set of vascular-like and parenchymal-like textures, for controlling cell populations. Final culture of amoeboid cells, whose dynamics is affected by the polyacrylamide patterns, provides a preliminary validation of the described strategy and helps to discuss its potentials.

Remote Control of Mechanical Forces via Mitochondrial-Targeted Magnetic Nanospinners for Efficient Cancer Treatment.

In cells, mechanical forces play a key role in impacting cell behaviors, including adhesion, differentiation, migration, and death. Herein, a 20 nm mitochondria-targeted zinc-doped iron oxide nanocube is designed as a nanospinner to exert mechanical forces under a rotating magnetic field (RMF) at 15 Hz and 40 mT to fight against cancer. The nanospinners can efficiently target the mitochondria of cancer cells. By means of the RMF, the nanocubes assemble in alignment with the external field and produce a localized mechanical force to impair the cancer cells. Both in vitro and in vivo studies show that the nanospinners can damage the cancer cells and reduce the brain tumor growth rate after the application of the RMF. This nanoplatform provides an effective magnetomechanical approach to treat deep-seated tumors in a spatiotemporal fashion.

Comparison of cell mechanical measurements provided by Atomic Force Microscopy (AFM) and Micropipette Aspiration (MPA).

A comparative analysis of T-lymphocyte mechanical data obtained from Micropipette Aspiration (MPA) and Atomic Force Microscopy (AFM) is presented. Results obtained by fitting the experimental data to simple Hertz and Theret models led to non-Gaussian distributions and significantly different values of the elastic moduli obtained by both techniques. The use of more refined models, taking into account the finite size of cells (simplified double contact and Zhou models) reduces the differences in the values calculated for the elastic moduli. Several possible sources for the discrepancy between the techniques are considered. The analysis suggests that the local nature of AFM measurements compared with the more general character of MPA measurements probably contributed to the differences observed.

Advances in Micropipette Aspiration: Applications in Cell Biomechanics, Models, and Extended Studies.

With five decades of sustained application, micropipette aspiration has enabled a wide range of biomechanical studies in the field of cell mechanics. Here, we provide an update on the use of the technique, with a focus on recent developments in the analysis of the experiments, innovative microaspiration-based approaches, and applications in a broad variety of cell types. We first recapitulate experimental variations of the technique. We then discuss analysis models focusing on important limitations of widely used biomechanical models, which underpin the urge to adopt the appropriate ones to avoid misleading conclusions. The possibilities of performing different studies on the same cell are also considered.

Straining Flow Spinning of Artificial Silk Fibers: A Review.

This work summarizes the main principles and some of the most significant results of straining flow spinning (SFS), a technology developed originally by the authors of this work. The principles on which the technology is based, inspired by the natural spinning system of silkworms and spiders, are presented, as well as some of the main achievements of the technique. Among these achievements, spinning under environmentally friendly conditions, obtaining high-performance fibers, and imparting the fibers with emerging properties such as supercontraction are discussed. Consequently, SFS appears as an efficient process that may represent one of the first realizations of a biomimetic technology with a significant impact at the production level.

Probing the effect of tip pressure on fungal growth: Application to Aspergillus nidulans.

The study of fungal cells is of great interest due to their importance as pathogens and as fermenting fungi and for their appropriateness as model organisms. The differential pressure between the hyphal cytoplasm and the bordering medium is essential for the growth process, because the pressure is correlated with the growth rate. Notably, during the invasion of tissues, the external pressure at the tip of the hypha may be different from the pressure in the surrounding medium. We report the use of a method, based on the micropipette-aspiration technique, to study the influence of this external pressure at the hyphal tip. Moreover, this technique makes it possible to study hyphal growth mechanics in the case of very thin hyphae, not accessible to turgor pressure probes. We found a correlation between the local pressure at the tip and the growth rate for the species Arpergillus nidulans. Importantly, the proposed method allows one to measure the pressure at the tip required to arrest the hyphal growth. Determining that pressure could be useful to develop new medical treatments for fungal infections. Finally, we provide a mechanical model for these experiments, taking into account the cytoplasm flow and the wall deformation.

Cell Mechanosensors and the Possibilities of Using Magnetic Nanoparticles to Study Them and to Modify Cell Fate.

The use of magnetic nanoparticles (MNPs) is a promising technique for future advances in biomedical applications. This idea is supported by the availability of MNPs that can target specific cell components, the variety of shapes of MNPs and the possibility of finely controlling the applied magnetic forces. To examine this opportunity, here we review the current developments in the use of MNPs to mechanically stimulate cells and, specifically, the cell mechanotransduction systems. We analyze the cell components that may act as mechanosensors and their effect on cell fate and we focus on the promising possibilities of controlling stem-cell differentiation, inducing cancer-cell death and treating nervous-system diseases.

Elongated Nanoparticle Aggregates in Cancer Cells for Mechanical Destruction with Low Frequency Rotating Magnetic Field.

Magnetic nanoparticles (MNPs) functionalized with targeting moieties can recognize specific cell components and induce mechanical actuation under magnetic field. Their size is adequate for reaching tumors and targeting cancer cells. However, due to the nanometric size, the force generated by MNPs is smaller than the force required for largely disrupting key components of cells. Here, we show the magnetic assembly process of the nanoparticles inside the cells, to form elongated aggregates with the size required to produce elevated mechanical forces. We synthesized iron oxide nanoparticles doped with zinc, to obtain high magnetization, and functionalized with the epidermal growth factor (EGF) peptide for targeting cancer cells. Under a low frequency rotating magnetic field at 15 Hz and 40 mT, the internalized EGF-MNPs formed elongated aggregates and generated hundreds of pN to dramatically damage the plasma and lysosomal membranes. The physical disruption, including leakage of lysosomal hydrolases into the cytosol, led to programmed cell death and necrosis. Our work provides a novel strategy of designing magnetic nanomedicines for mechanical destruction of cancer cells.

Production of High Performance Bioinspired Silk Fibers by Straining Flow Spinning.

In the last years, there has been an increasing interest in bioinspired approaches for different applications, including the spinning of high performance silk fibers. Bioinspired spinning is based on the natural spinning system of spiders and worms and requires combining changes in the chemical environment of the proteins with the application of mechanical stresses. Here we present the novel straining flow spinning (SFS) process and prove its ability to produce high performance fibers under mild, environmentally friendly conditions, from aqueous protein dopes. SFS is shown to be an extremely versatile technique which allows controlling a large number of processing parameters. This ample set of parameters allows fine-tuning the microstructure and mechanical behavior of the fibers, which opens the possibility of adapting the fibers to their intended uses.

Improved Measurement of Elastic Properties of Cells by Micropipette Aspiration and Its Application to Lymphocytes.

Mechanical deformability of cells is an important property for their function and development, as well as a useful marker of cell state. The classical technique of micropipette aspiration allows single-cell studies and we provide here a method to measure the two basic mechanical parameters, elastic modulus and Poisson's ratio. The proposed method, developed from finite-element analysis of micropipette aspiration experiments, may be implemented in future technologies for the automated measurement of mechanical properties of cells, based on the micropipette aspiration technique or on the cell transit through flow constrictions. We applied this method to measure the elastic parameters of lymphocytes, in which the mechanical properties depend on their activation state. Additionally, we discuss in this work the accuracy of previous models to estimate the elastic modulus of cells, in particular the analytical model by Theret et al., widely used in the field. We show the necessity of using an improved model, taking into account the finite size of the cells, to obtain new insights that may remain hidden otherwise.

Biomimetic spinning of artificial spider silk from a chimeric minispidroin.

Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.

Material properties of evolutionary diverse spider silks described by variation in a single structural parameter.

Spider major ampullate gland silks (MAS) vary greatly in material properties among species but, this variation is shown here to be confined to evolutionary shifts along a single universal performance trajectory. This reveals an underlying design principle that is maintained across large changes in both spider ecology and silk chemistry. Persistence of this design principle becomes apparent after the material properties are defined relative to the true alignment parameter, which describes the orientation and stretching of the protein chains in the silk fiber. Our results show that the mechanical behavior of all Entelegynae major ampullate silk fibers, under any conditions, are described by this single parameter that connects the sequential action of three deformation micromechanisms during stretching: stressing of protein-protein hydrogen bonds, rotation of the ß-nanocrystals and growth of the ordered fraction. Conservation of these traits for over 230 million years is an indication of the optimal design of the material and gives valuable clues for the production of biomimetic counterparts based on major ampullate spider silk.

Mechanical behaviour and formation process of silkworm silk gut.

High performance silk fibers were produced directly from the silk glands of silkworms (Bombyx mori) following an alternative route to natural spinning. This route is based on a traditional procedure that consists of soaking the silk glands in a vinegar solution and stretching them by hand leading to the so called silkworm guts. Here we present, to the authors' best knowledge, the first comprehensive study on the formation, properties and microstructure of silkworm gut fibers. Comparison of the tensile properties and microstructural organization of the silkworm guts with those of naturally spun fibers allows gain of a deeper insight into the mechanisms that lead to the formation of the fiber, as well as the relationship between the microstructure and properties of these materials. In this regard, it is proved that an acidic environment and subsequent application of tensile stress in the range of 1000 kPa are sufficient conditions for the formation of a silk fiber.