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.

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.

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.

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.

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.

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.

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.

Topographical and mechanical characterization of living eukaryotic cells on opaque substrates: development of a general procedure and its application to the study of non-adherent lymphocytes.

The mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy (AFM). A robust experimental procedure was developed to overcome some features of lymphocytes, in particular their spherical shape and non-adherent character. The procedure included the immobilization of the lymphocytes on amine-functionalized substrates, the use of hydrodynamic effects on the deflection of the AFM cantilever to monitor the approaching, and the use of the jumping mode for obtaining the images. Indentation curves were analyzed according to Hertz's model for contact mechanics. The calculated values of the elastic modulus are consistent both when considering the results obtained from a single lymphocyte and when comparing the curves recorded from cells of different specimens.

Study of the influence of actin-binding proteins using linear analyses of cell deformability.

The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, α-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of α-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over cross-links during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actin-binding proteins, deformability and mechanosensing.

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.

Unexpected behavior of irradiated spider silk links conformational freedom to mechanical performance.

Silk fibers from Argiope trifasciata and Nephila inaurata orb-web weaving spiders were UV irradiated to modify the molecular weight of the constituent proteins. Fibers were characterized either as forcibly silked or after being subjected to maximum supercontraction. The effect of irradiation on supercontraction was also studied, both in terms of the percentage of supercontraction and the tensile properties exhibited by irradiated and subsequently supercontracted fibers. The effects of UV exposure at the molecular level were assessed by polyacrylamide gel electrophoresis and mass spectrometry. It is shown that UV-irradiated fibers show a steady decrease in their main tensile parameters, most notably, tensile strength and strain. The combination of the mechanical and biochemical data suggests that the restricted conformational freedom of the proteins after UV irradiation is critical in the reduction of these properties. Consequently, an adequate topological organization of the protein chains emerges as a critical design principle in the performance of spider silk.

The variability and interdependence of spider viscid line tensile properties.

True stress-true strain curves of naturally spun viscid line fibres retrieved directly from the spiral of orb-webs built by Argiope trifasciata spiders were measured using a novel methodology. This new procedure combines a method for removing the aqueous coating of the fibres and a technique that allows the accurate measurement of their cross-sectional area. Comparison of the tensile behaviour of different samples indicated that naturally spun viscid lines show a large variability, comparable to that of other silks, such as major ampullate gland silk and silkworm silk. Nevertheless, application of a statistical analysis allowed the identification of two independent parameters that underlie the variability and characterize the observed range of true stress-true strain curves. The combination of this result with previous mechanical and microstructural data suggested the assignment of these two independent effects to the degree of alignment of the protein chains and to the local relative humidity, which, in turn, depends on the composition of the viscous coating and on the external environmental conditions.

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.

Recovery in viscid line fibers.

The development of a reliable procedure for removing the viscous coating of viscid silk has allowed the accurate characterization of the tensile behavior of clean flagelliform silk (i.e., silk of the flagelliform gland without the viscous coating synthetised in the aggregate gland). For comparison, tensile tests on native viscid silk (with the viscous coating) fibers were also performed. It was found that viscid silk, either native or clean, has an elastomeric behavior when kept wet, either by immersion in water (clean fibers) or by the effect of the viscid coating (native fibers). When tested in dry environments (35% RH, relative humidity, for clean fibers and 10% RH for native fibers), their mechanical behavior was no longer elastomeric, with it being more similar to other silk fibers. Furthermore, it was noticed that flagelliform silk fibers show a ground state to which they can return independent of the previous loading history.

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.

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.

Mechanical behavior of silk during the evolution of orb-web spinning spiders.

The development of an accurate and reproducible approach to measuring the tensile behavior of spider silk has allowed characterizing and comparing the range of mechanical properties exhibited by different spider species with unprecedented detail. The comparison of silks spun by spiders belonging to different phylogenetic groups has revealed that evolution locked in many of the important properties of spider silks very early in the history of orb-web weaving spiders, despite the fact that the silk gland system is relatively isolated in physiological terms from the rest of the organism and should thus mutate quickly. The variations observed between species may be grouped in at least two patterns that are shown not to be related to phylogeny. Beyond the relevance of these results for the evolutionary biology of spiders and silks, the conservation of the basic traits observed in the mechanical behavior of spider silks is likely to set a limit to the range of properties that can be expected from artificial fibers bioinspired in natural silks.

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.