Indentation induces instantaneous nuclear stiffening and unfolding of nuclear envelope wrinkles.

The nuclear envelope (NE) separates genomic DNA from the cytoplasm and regulates transport between the cytosol and the nucleus in eukaryotes. Nuclear stiffening enables the cell nucleus to protect itself from extensive deformation, loss of NE integrity, and genome instability. It is known that the reorganization of actin, lamin, and chromatin can contribute to nuclear stiffening. In this work, we show that structural alteration of NE also contributes to instantaneous nuclear stiffening under indentation. In situ mechanical characterization of cell nuclei in intact cells shows that nuclear stiffening and unfolding of NE wrinkles occur simultaneously at the indentation site. A positive correlation between the initial state of NE wrinkles, the unfolding of NE wrinkles, and the stiffening ratio (stiffness fold-change) is found. Additionally, NE wrinkles unfold throughout the nucleus outside the indentation site. Finite element simulation, which involves the purely passive process of structural unfolding, shows that unfolding of NE wrinkles alone can lead to an increase in nuclear stiffness and a reduction in stress and strain levels. Together, these results provide a perspective on how cell nucleus adapts to mechanical stimuli through structural alteration of the NE.

3D Morphology Measurement for Blastocyst Evaluation From "All Angles".

Measuring the 3D morphology of spherical cell aggregates is required in both biology and medicine. Traditional methods either use fluorescent labeling, which cause cell toxicity and are unsuitable for clinical treatment, or use 2D images to roughly estimate 3D morphology. To overcome these limitations, this paper presents a quantitative label-free 3D morphology measurement technique using multi-view images. This technique, for the first time, enables the morphological evaluation of a blastocyst (Day 5 embryo) from "all angles" for IVF treatment. In this technique, a spherical rotation scale invariant feature transform (SR-SIFT) is proposed to address feature distortions for the rotation matrix calculation of the multi-view images. U-Net with generalized Dice loss is used to segment individual trophectoderm (TE) cells and the inner cell mass (ICM) of the blastocyst. Based on the rotation matrices and the segmentation results, the 3D morphological parameters of the entire blastocyst were quantified. Experimental results showed that the error of rotation angle was less than 1 °, the Dice was 95.6% for TE segmentation and 92.3% for ICM segmentation, and the overall measurement error of clinically defined blastocyst parameters was less than 6.7%.

Ultrathin and Flexible Bioelectronic Arrays for Functional Measurement of iPSC-Cardiomyocytes under Cardiotropic Drug Administration and Controlled Microenvironments.

Emerging heart-on-a-chip technology is a promising tool to establish in vitro cardiac models for therapeutic testing and disease modeling. However, due to the technical complexity of integrating cell culture chambers, biosensors, and bioreactors into a single entity, a microphysiological system capable of reproducing controlled microenvironmental cues to regulate cell phenotypes, promote iPS-cardiomyocyte maturity, and simultaneously measure the dynamic changes of cardiomyocyte function in situ is not available. This paper reports an ultrathin and flexible bioelectronic array platform in 24-well format for higher-throughput contractility measurement under candidate drug administration or defined microenvironmental conditions. In the array, carbon black (CB)-PDMS flexible strain sensors were embedded for detecting iPSC-CM contractility signals. Carbon fiber electrodes and pneumatic air channels were integrated to provide electrical and mechanical stimulation to improve iPSC-CM maturation. Performed experiments validate that the bioelectronic array accurately reveals the effects of cardiotropic drugs and identifies mechanical/electrical stimulation strategies for promoting iPSC-CM maturation.

Conductive and elastic bottlebrush elastomers for ultrasoft electronics.

Understanding biological systems and mimicking their functions require electronic tools that can interact with biological tissues with matched softness. These tools involve biointerfacing materials that should concurrently match the softness of biological tissue and exhibit suitable electrical conductivities for recording and reading bioelectronic signals. However, commonly employed intrinsically soft and stretchable materials usually contain solvents that limit stability for long-term use or possess low electronic conductivity. To date, an ultrasoft (i.e., Young's modulus <30 kPa), conductive, and solvent-free elastomer does not exist. Additionally, integrating such ultrasoft and conductive materials into electronic devices is poorly explored. This article reports a solvent-free, ultrasoft and conductive PDMS bottlebrush elastomer (BBE) composite with single-wall carbon nanotubes (SWCNTs) as conductive fillers. The conductive SWCNT/BBE with a filler concentration of 0.4 - 0.6 wt% reveals an ultralow Young's modulus (<11 kPa) and satisfactory conductivity (>2 S/m) as well as adhesion property. Furthermore, we fabricate ultrasoft electronics based on laser cutting and 3D printing of conductive and non-conductive BBEs and demonstrate their potential applications in wearable sensing, soft robotics, and electrophysiological recording.

Mechanosensitive brain tumor cells construct blood-tumor barrier to mask chemosensitivity.

Major obstacles in brain cancer treatment include the blood-tumor barrier (BTB), which limits the access of most therapeutic agents, and quiescent tumor cells, which resist conventional chemotherapy. Here, we show that Sox2+ tumor cells project cellular processes to ensheathe capillaries in mouse medulloblastoma (MB), a process that depends on the mechanosensitive ion channel Piezo2. MB develops a tissue stiffness gradient as a function of distance to capillaries. Sox2+ tumor cells perceive substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion to promote cellular process growth and cell surface sequestration of ß-catenin. Piezo2 knockout reverses WNT/ß-catenin signaling states between Sox2+ tumor cells and endothelial cells, compromises the BTB, reduces the quiescence of Sox2+ tumor cells, and markedly enhances the MB response to chemotherapy. Our study reveals that mechanosensitive tumor cells construct the BTB to mask tumor chemosensitivity. Targeting Piezo2 addresses the BTB and tumor quiescence properties that underlie treatment failures in brain cancer.

Microrobotic swarms for selective embolization.

Inspired by the collective intelligence in natural swarms, microrobotic agents have been controlled to form artificial swarms for targeted drug delivery, enhanced imaging, and hyperthermia. Different from these well-investigated tasks, this work aims to develop microrobotic swarms for embolization, which is a clinical technique used to block blood vessels for treating tumors, fistulas, and arteriovenous malformations. Magnetic particle swarms were formed for selective embolization to address the low selectivity of the present embolization technique that is prone to cause complications such as stroke and blindness. We established an analytical model that describes the relationships between fluid viscosity, flow rate, branching angle, magnetic field strength, and swarm integrity, based on which an actuation strategy was developed to maintain the swarm integrity inside a targeted region under fluidic flow conditions. Experiments in microfluidic channels, ex vivo tissues, and in vivo porcine kidneys validated the efficacy of the proposed strategy for selective embolization.

A Carbon-Based Biosensing Platform for Simultaneously Measuring the Contraction and Electrophysiology of iPSC-Cardiomyocyte Monolayers.

Heart beating is triggered by the generation and propagation of action potentials through the myocardium, resulting in the synchronous contraction of cardiomyocytes. This process highlights the importance of electrical and mechanical coordination in organ function. Investigating the pathogenesis of heart diseases and potential therapeutic actions in vitro requires biosensing technologies which allow for long-term and simultaneous measurement of the contractility and electrophysiology of cardiomyocytes. However, the adoption of current biosensing approaches for functional measurement of in vitro cardiac models is hampered by low sensitivity, difficulties in achieving multifunctional detection, and costly manufacturing processes. Leveraging carbon-based nanomaterials, we developed a biosensing platform that is capable of performing on-chip and simultaneous measurement of contractility and electrophysiology of human induced pluripotent stem-cell-derived cardiomyocyte (iPSC-CM) monolayers. This platform integrates with a flexible thin-film cantilever embedded with a carbon black (CB)-PDMS strain sensor for high-sensitivity contraction measurement and four pure carbon nanotube (CNT) electrodes for the detection of extracellular field potentials with low electrode impedance. Cardiac functional properties including contractile stress, beating rate, beating rhythm, and extracellular field potential were evaluated to quantify iPSC-CM responses to common cardiotropic agents. In addition, an in vitro model of drug-induced cardiac arrhythmia was established to further validate the platform for disease modeling and drug testing.

Microengineered platforms for characterizing the contractile function of in vitro cardiac models.

Emerging heart-on-a-chip platforms are promising approaches to establish cardiac cell/tissue models in vitro for research on cardiac physiology, disease modeling and drug cardiotoxicity as well as for therapeutic discovery. Challenges still exist in obtaining the complete capability of in situ sensing to fully evaluate the complex functional properties of cardiac cell/tissue models. Changes to contractile strength (contractility) and beating regularity (rhythm) are particularly important to generate accurate, predictive models. Developing new platforms and technologies to assess the contractile functions of in vitro cardiac models is essential to provide information on cell/tissue physiologies, drug-induced inotropic responses, and the mechanisms of cardiac diseases. In this review, we discuss recent advances in biosensing platforms for the measurement of contractile functions of in vitro cardiac models, including single cardiomyocytes, 2D monolayers of cardiomyocytes, and 3D cardiac tissues. The characteristics and performance of current platforms are reviewed in terms of sensing principles, measured parameters, performance, cell sources, cell/tissue model configurations, advantages, and limitations. In addition, we highlight applications of these platforms and relevant discoveries in fundamental investigations, drug testing, and disease modeling. Furthermore, challenges and future outlooks of heart-on-a-chip platforms for in vitro measurement of cardiac functional properties are discussed.

Integrated Assembly and Photopreservation of Topographical Micropatterns.

Micromanipulation techniques that are capable of assembling nano/micromaterials into usable structures such as topographical micropatterns (TMPs) have proliferated rapidly in recent years, holding great promise in building artificial electronic and photonic microstructures. Here, a method is reported for forming TMPs based on optoelectronic tweezers in either "bottom-up" or "top-down" modes, combined with in situ photopolymerization to form permanent structures. This work demonstrates that the assembled/cured TMPs can be harvested and transferred to alternate substrates, and illustrates that how permanent conductive traces and capacitive circuits can be formed, paving the way toward applications in microelectronics. The integrated, optical assembly/preservation method described here is accessible, versatile, and applicable for a wide range of materials and structures, suggesting utility for myriad microassembly and microfabrication applications in the future.

An organ-on-a-chip model for pre-clinical drug evaluation in progressive non-genetic cardiomyopathy.

Angiotensin II (Ang II) presents a critical mediator in various pathological conditions such as non-genetic cardiomyopathy. Osmotic pump infusion in rodents is a commonly used approach to model cardiomyopathy associated with Ang II. However, profound differences in electrophysiology and pharmacokinetics between rodent and human cardiomyocytes may limit predictability of animal-based experiments. This study investigates the application of an Organ-on-a-chip (OOC) system in modeling Ang II-induced progressive cardiomyopathy. The disease model is constructed to recapitulate myocardial response to Ang II in a temporal manner. The long-term tissue cultivation and non-invasive functional readouts enable monitoring of both acute and chronic cardiac responses to Ang II stimulation. Along with mapping of cytokine secretion and proteomic profiles, this model presents an opportunity to quantitatively measure the dynamic pathological changes that could not be otherwise identified in animals. Further, we present this model as a testbed to evaluate compounds that target Ang II-induced cardiac remodeling. Through assessing the effects of losartan, relaxin, and saracatinib, the drug screening data implicated multifaceted cardioprotective effects of relaxin in restoring contractile function and reducing fibrotic remodeling. Overall, this study provides a controllable platform where cardiac activities can be explicitly observed and tested over the pathological process. The facile and high-content screening can facilitate the evaluation of potential drug candidates in the pre-clinical stage.

Advances in reconstructing intestinal functionalities in vitro: From two/three dimensional-cell culture platforms to human intestine-on-a-chip.

Standard two/three dimensional (2D/3D)-cell culture platforms have facilitated the understanding of the communications between various cell types and their microenvironments. However, they are still limited in recapitulating the complex functionalities in vivo, such as tissue formation, tissue-tissue interface, and mechanical/biochemical microenvironments of tissues and organs. Intestine-on-a-chip platforms offer a new way to mimic intestinal behaviors and functionalities by constructing in vitro intestinal models in microfluidic devices. This review summarizes the advances and limitations of the state-of-the-art 2D/3D-cell culture platforms, animal models, intestine chips, and the combined multi-organ chips related with intestines. Their applications to studying intestinal functions, drug testing, and disease modeling are introduced. Different intestinal cell sources are compared in terms of gene expression abilities and the recapitulated intestinal morphologies. Among these cells, cells isolated form human intestinal tissues and derived from pluripotent stem cells appear to be more suitable for in vitro reconstruction of intestinal organs. Key challenges of current intestine-on-a-chip platforms and future directions are also discussed.

A microdevice platform for characterizing the effect of mechanical strain magnitudes on the maturation of iPSC-Cardiomyocytes.

The use of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as an in vitro model of the heart is limited by their structurally and functionally immature phenotypes. During heart development, mechanical stimuli from in vivo microenvironments are known to regulate cardiomyocyte gene expression and maturation. Accordingly, protocols for culturing iPSC-CMs have recently incorporated mechanical or electromechanical stimulation to induce cellular maturation in vitro; however, the response of iPSC-CMs to different mechanical strain magnitudes is unknown, and existing techniques lack the capability to dynamically measure changes to iPSC-CM contractility in situ as maturation progresses. We developed a microdevice platform which applies cyclical strains of varying magnitudes (5%, 10%, 15% and 20%) to a monolayer of iPSC-CMs, coincidentally measuring contractile stress during mechanical stimulation using fluorescent nanobeads embedded in the microdevice's suspended membrane. Cyclic strain was found to induce circumferential cell alignment on the actuated membranes. In situ contractility measurements revealed that cyclic stimulation gradually increased cardiomyocyte contractility during a 10-day culture period. The contractile stress of iPSC-CM monolayers was found to increase with a higher strain magnitude and plateaued at 15% strain. Cardiomyocyte contractility positively correlated with the elongation of sarcomeres and an increased expression of ß-myosin heavy chain (MYH7) in a strain magnitude-dependent manner, illustrating how mechanical stress can be optimized for the phenotypic and proteomic maturation of the cells. iPSC-CMs with improved maturity have the potential to create a more accurate heart model in vitro for applications in disease modeling and therapeutic discovery.

A CNT-PDMS wearable device for simultaneous measurement of wrist pulse pressure and cardiac electrical activity.

Simultaneous measurement of multi-physiological signals can provide effective diagnosis and therapeutic assessment of diseases. This paper reports a carbon nanotube (CNT) - Polydimethylsiloxane (PDMS) - based wearable device with piezo-resistive and voltage-sensing capabilities for simultaneously capturing wrist pulse pressure and cardiac electrical signal. The layout design of sensing elements in the device was guided by analyzing strain distribution and electric field distribution for minimizing the interference between wrist pulse and cardiac electric activity during measurement. Each device was preconditioned under the strain of 20% until the resistance change of the device reached equilibrium. After preconditioning, the relationship between the resistance change and the pressure was calibrated, which determined the device sensitivity to be 0.01 Pa-1 and the linear pressure range of the device to be 0.4 kPa to 14.0 kPa. Mechanisms of CNT-PDMS for sensing strain signal and electrical pulse signal were explored by scanning electron microscopy (SEM) imaging and equivalent circuit modeling. The device was applied to monitor the wrist pulse and ECG signals of volunteers during the recovering process after physical exercises.

Label-free conduction velocity mapping and gap junction assessment of functional iPSC-Cardiomyocyte monolayers.

Cardiac conduction is an important function of the heart. To date, accurate measurement of conduction velocity (CV) in vitro is hindered by the low spatial resolution and poor signal-to-noise ratio of microelectrode arrays (MEAs), or the cytotoxicity and end-point analysis of fluorescence optical imaging. Here, we have developed a new label-free method based on defocused brightfield imaging to quantify CV by analyzing centroid displacements and contraction trajectories of each cardiomyocyte in a monolayer of human stem cell-derived cardiomyocytes (iPSC-CMs). Our data revealed that the time delay between intracellular calcium release and the initiation of cell contraction is highly consistent across cardiomyocytes; however, the duration a cell takes to reach its maximum beating magnitude varies significantly, proving that the time delay in excitation-contraction coupling is largely constant in iPSC-CMs. Standard calcium imaging of the same iPSC-CM populations (~106 cells) was conducted for comparison with our label-free method. The results confirmed that our label-free method was capable of achieving highly accurate CV mapping (17.64 ± 0.89 cm/s vs. 17.95 ± 2.29 cm/s, p-value>0.1). Additionally, our method effectively revealed various shapes in cell beating pattern. We also performed label-free CV mapping on disease-specific iPSC-CM monolayers with plakophilin-2 (PKP2) knockdown, which effectively quantified their low CV values and further validated the arrhythmogenic role of PKP2 mutation in arrhythmogenic right ventricular cardiomyopathy (ARVC) through the disruption of cardiac conduction. The label-free method offers a cytotoxic-free technique for long-term measurement of dynamic beating trajectories, beating propagation and conduction velocities of cardiomyocyte monolayers.

Electrical impedance-based contractile stress measurement of human iPSC-Cardiomyocytes.

Heart failure fundamentally results from loss of cardio myocyte contractility. Developing new methods that quantify the contractile stress of the human cardiomyocyte would facilitate the study of the molecular mechanism of heart failure and advance therapy development, to improve the current five year survival for these patients. The measurement of cellular electrical impedance measurement was recently applied to monitor cardiomyocyte beating rate and rhythm, for the study at cellular maturation, and for drug screening. However, due to the lack of a quantified relationship between the impedance signal and contractile stress, change of cardiomyocyte contractile stress cannot genuinely be quantified from impedance measurements. Here, we report the first quantitative relationship between contractile stress and impedance, which enables the accurate prediction of cardiomyocyte contractility using impedance signals. Through simultaneous measurement of beating human iPSC-cardiomyocytes using impedance spectroscopy and atomic force microscopy, a power-law relationship between impedance and contractile stress was established with a confidence level of 95%. The quantitative relationship was validated using pharmacology known to alter cardiomyocyte contractility and beating (verapamil, using clinically relevant concentrations of 0.05 µM, 0.10 µM, and 0.15 µM). The contractile stress values as measured by AFM were 9.04 ± 0.14 kPa (0.05 µM), 7.72 ± 0.11 kPa (0.10 µM) and 6.23 ± 0.17 kPa (0.15 µM), and as predicted by impedance using the derived power-law relationship were 9.39 kPa, 7.76 kPa, and 6.05 kPa with a relative error of 3.73%. Our power-law relationship is the first to describe a quantitative correlation between contractile stress and impedance, broadening the application of electrical impedance measurement for characterizing complex cardiac functions (beating rate, beating rhythm and contractile stress).

Recapitulating pancreatic tumor microenvironment through synergistic use of patient organoids and organ-on-a-chip vasculature.

Tumor progression relies heavily on the interaction between the neoplastic epithelial cells and their surrounding stromal partners. This cell cross-talk affects stromal development, and ultimately the heterogeneity impacts drug efflux and efficacy. To mimic this evolving paradigm, we have micro-engineered a three-dimensional (3D) vascularized pancreatic adenocarcinoma tissue in a tri-culture system composed of patient derived pancreatic organoids, primary human fibroblasts and endothelial cells on a perfusable InVADE platform situated in a 96-well plate. Uniquely, through synergistic engineering we combined the benefits of cellular fidelity of patient tumor derived organoids with the addressability of a plastic organ-on-a-chip platform. Validation of this platform included demonstrating the growth of pancreatic tumor organoids by monitoring the change in metabolic activity of the tissue. Investigation of tumor microenvironmental behavior highlighted the role of fibroblasts in symbiosis with patient organoid cells, resulting in a six-fold increase of collagen deposition and a corresponding increase in tissue stiffness in comparison to fibroblast free controls. The value of a perfusable vascular network was evident in drug screening, as perfusion of gemcitabine into a stiffened matrix did not show the dose-dependent effects on tumor viability as those under static conditions. These findings demonstrate the importance of studying the dynamic synergistic relationship between patient cells with stromal fibroblasts, in a 3D perfused vascular network, to accurately understand and recapitulate the tumor microenvironment.

Stiffness and ATP recovery of stored red blood cells in serum.

In transfusion medicine, there has been a decades-long debate about whether the age of stored red blood cells (RBCs) is a factor in transfusion efficacy. Existing clinical studies investigating whether older RBCs cause worse clinical outcomes have provided conflicting information: some have shown that older blood is less effective, while others have shown no such difference. The controversial results could have been biased by the vastly different conditions of the patients involved in the clinical studies; however, another source of inconsistency is a lack of understanding of how well and quickly stored RBCs can recover their key parameters, such as stiffness and ATP concentration, after transfusion. In this work, we quantitatively studied the stiffness and ATP recovery of stored RBCs in 37 °C human serum. The results showed that in 37 °C human serum, stored RBCs are able to recover their stiffness and ATP concentration to varying extents depending on how long they have been stored. Fresher RBCs (1-3 weeks old) were found to have a significantly higher capacity for stiffness and ATP recovery in human serum than older RBCs (4-6 weeks old). For instance, for 1-week-old RBCs, although the shear modulus before recovery was 1.6 times that of fresh RBCs, 97% of the cells recovered in human serum to have 1.1 times the shear modulus of fresh RBCs, and the ATP concentration of 1-week-old RBCs after recovery showed no difference from that of fresh RBCs. However, for 6-week-old RBCs, only ~70% of the RBCs showed stiffness recovery in human serum; their shear modulus after recovery was still 2.1 times that of fresh RBCs; and their ATP concentration after recovery was 25% lower than that of fresh RBCs. Our experiments also revealed that the processes of stiffness recovery and ATP recovery took place on the scale of tens of minutes. We hope that this study will trigger the next steps of comprehensively characterizing the recovery behaviors of stored RBCs (e.g., recovery of normal 2,3-DPG [2,3-Diphosphoglycerate]and SNO [S-nitrosation] levels) and quantifying the in vivo recovery of stored RBCs in transfusion medicine.

A Feedforward Mechanism Mediated by Mechanosensitive Ion Channel PIEZO1 and Tissue Mechanics Promotes Glioma Aggression.

Alteration of tissue mechanical properties is a physical hallmark of solid tumors including gliomas. How tumor cells sense and regulate tissue mechanics is largely unknown. Here, we show that mechanosensitive ion channel Piezo regulates mitosis and tissue stiffness of Drosophila gliomas, but not non-transformed brains. PIEZO1 is overexpressed in aggressive human gliomas and its expression inversely correlates with patient survival. Deleting PIEZO1 suppresses the growth of glioblastoma stem cells, inhibits tumor development, and prolongs mouse survival. Focal mechanical force activates prominent PIEZO1-dependent currents from glioma cell processes, but not soma. PIEZO1 localizes at focal adhesions to activate integrin-FAK signaling, regulate extracellular matrix, and reinforce tissue stiffening. In turn, a stiffer mechanical microenvironment elevates PIEZO1 expression to promote glioma aggression. Therefore, glioma cells are mechanosensory in a PIEZO1-dependent manner, and targeting PIEZO1 represents a strategy to break the reciprocal, disease-aggravating feedforward circuit between tumor cell mechanotransduction and the aberrant tissue mechanics. VIDEO ABSTRACT.

Microdevice Platform for Continuous Measurement of Contractility, Beating Rate, and Beating Rhythm of Human-Induced Pluripotent Stem Cell-Cardiomyocytes inside a Controlled Incubator Environment.

The heart completes a complex set of tasks, including the initiation or propagation of an electrical signal with regularity (proper heart rate and rhythm) and generating sufficient force of contraction (contractility). Probing mechanisms of heart diseases and quantifying drug efficacies demand a platform that is capable of continuous operation inside a cell incubator for long-term measurement of cardiomyocyte (CM) monolayers. Here, we report a microdevice array that is capable of performing continuous, long-term (14 days) measurement of contractility, beating rate, and beating rhythm in a monolayer of human-induced pluripotent stem cell-CMs (hiPSC-CMs). The device consists of a deformable membrane with embedded carbon nanotube (CNT)-based strain sensors. Contraction of the hiPSC-CMs seeded on the membrane induces electrical resistance change of the CNT strain sensor. Continuously reading the sensor signals revealed that hiPSC-CMs started to beat from day 2 and plateaued on day 5. Average contractile stress generated by a monolayer of hiPSC-CMs was determined to be 2.34 ± 0.041 kPa with a beating rate of 1.17 ± 0.068 Hz. The device arrays were also used to perform comprehensive measurement of the beating rate, rhythm, and contractility of the hiPSC-CMs and quantify the cell responses to different concentrations of agonists and antagonists, which altered the average contractile stress to the range of 1.15 ± 0.13 to 3.96 ± 0.53 kPa. The continuous measurement capability of the device arrays also enabled the generation of Poincaré plots for revealing subtle changes in the beating rhythm of hiPSC-CMs under different drug treatments.