Quantifying cell cycle regulation by tissue crowding.

The spatiotemporal coordination and regulation of cell proliferation is fundamental in many aspects of development and tissue maintenance. Cells have the ability to adapt their division rates in response to mechanical constraints, yet we do not fully understand how cell proliferation regulation impacts cell migration phenomena. Here, we present a minimal continuum model of cell migration with cell cycle dynamics, which includes density-dependent effects and hence can account for cell proliferation regulation. By combining minimal mathematical modeling, Bayesian inference, and recent experimental data, we quantify the impact of tissue crowding across different cell cycle stages in epithelial tissue expansion experiments. Our model suggests that cells sense local density and adapt cell cycle progression in response, during G1 and the combined S/G2/M phases, providing an explicit relationship between each cell-cycle-stage duration and local tissue density, which is consistent with several experimental observations. Finally, we compare our mathematical model's predictions to different experiments studying cell cycle regulation and present a quantitative analysis on the impact of density-dependent regulation on cell migration patterns. Our work presents a systematic approach for investigating and analyzing cell cycle data, providing mechanistic insights into how individual cells regulate proliferation, based on population-based experimental measurements.

Optimal Control of Collective Electrotaxis in Epithelial Monolayers.

Epithelial monolayers are some of the best-studied models for collective cell migration due to their abundance in multicellular systems and their tractability. Experimentally, the collective migration of epithelial monolayers can be robustly steered e.g. using electric fields, via a process termed electrotaxis. Theoretically, however, the question of how to design an electric field to achieve a desired spatiotemporal movement pattern is underexplored. In this work, we construct and calibrate an ordinary differential equation model to predict the average velocity of the centre of mass of a cellular monolayer in response to stimulation with an electric field. We use this model, in conjunction with optimal control theory, to derive physically realistic optimal electric field designs to achieve a variety of aims, including maximising the total distance travelled by the monolayer, maximising the monolayer velocity, and keeping the monolayer velocity constant during stimulation. Together, this work is the first to present a unified framework for optimal control of collective monolayer electrotaxis and provides a blueprint to optimally steer collective migration using other external cues.

Overriding native cell coordination enhances external programming of collective cell migration.

As collective cell migration is essential in biological processes spanning development, healing, and cancer progression, methods to externally program cell migration are of great value. However, problems can arise if the external commands compete with strong, preexisting collective behaviors in the tissue or system. We investigate this problem by applying a potent external migratory cue-electrical stimulation and electrotaxis-to primary mouse skin monolayers where we can tune cell-cell adhesion strength to modulate endogenous collectivity. Monolayers with high cell-cell adhesion showed strong natural coordination and resisted electrotactic control, with this conflict actively damaging the leading edge of the tissue. However, reducing preexisting coordination in the tissue by specifically inhibiting E-cadherin-dependent cell-cell adhesion, either by disrupting the formation of cell-cell junctions with E-cadherin-specific antibodies or rapidly dismantling E-cadherin junctions with calcium chelators, significantly improved controllability. Finally, we applied this paradigm of weakening existing coordination to improve control and demonstrate accelerated wound closure in vitro. These results are in keeping with those from diverse, noncellular systems and confirm that endogenous collectivity should be considered as a key quantitative design variable when optimizing external control of collective migration.

Tardigrades exhibit robust interlimb coordination across walking speeds and terrains.

Tardigrades must negotiate heterogeneous, fluctuating environments and accordingly utilize locomotive strategies capable of dealing with variable terrain. We analyze the kinematics and interleg coordination of freely walking tardigrades (species: Hypsibius exemplaris). We find that tardigrade walking replicates several key features of walking in insects despite disparities in size, skeleton, and habitat. To test the effect of environmental changes on tardigrade locomotor control circuits we measure kinematics and interleg coordination during walking on two substrates of different stiffnesses. We find that the phase offset between contralateral leg pairs is flexible, while ipsilateral coordination is preserved across environmental conditions. This mirrors similar results in insects and crustaceans. We propose that these functional similarities in walking coordination between tardigrades and arthropods is either due to a generalized locomotor control circuit common to panarthropods or to independent convergence onto an optimal strategy for robust multilegged control in small animals with simple circuitry. Our results highlight the value of tardigrades as a comparative system toward understanding the mechanisms-neural and/or mechanical-underlying coordination in panarthropod locomotion.

Learning the rules of collective cell migration using deep attention networks.

Collective, coordinated cellular motions underpin key processes in all multicellular organisms, yet it has been difficult to simultaneously express the 'rules' behind these motions in clear, interpretable forms that effectively capture high-dimensional cell-cell interaction dynamics in a manner that is intuitive to the researcher. Here we apply deep attention networks to analyze several canonical living tissues systems and present the underlying collective migration rules for each tissue type using only cell migration trajectory data. We use these networks to learn the behaviors of key tissue types with distinct collective behaviors-epithelial, endothelial, and metastatic breast cancer cells-and show how the results complement traditional biophysical approaches. In particular, we present attention maps indicating the relative influence of neighboring cells to the learned turning decisions of a 'focal cell'-the primary cell of interest in a collective setting. Colloquially, we refer to this learned relative influence as 'attention', as it serves as a proxy for the physical parameters modifying the focal cell's future motion as a function of each neighbor cell. These attention networks reveal distinct patterns of influence and attention unique to each model tissue. Endothelial cells exhibit tightly focused attention on their immediate forward-most neighbors, while cells in more expansile epithelial tissues are more broadly influenced by neighbors in a relatively large forward sector. Attention maps of ensembles of more mesenchymal, metastatic cells reveal completely symmetric attention patterns, indicating the lack of any particular coordination or direction of interest. Moreover, we show how attention networks are capable of detecting and learning how these rules change based on biophysical context, such as location within the tissue and cellular crowding. That these results require only cellular trajectories and no modeling assumptions highlights the potential of attention networks for providing further biological insights into complex cellular systems.

Practical fluorescence reconstruction microscopy for large samples and low-magnification imaging.

Fluorescence reconstruction microscopy (FRM) describes a class of techniques where transmitted light images are passed into a convolutional neural network that then outputs predicted epifluorescence images. This approach enables many benefits including reduced phototoxicity, freeing up of fluorescence channels, simplified sample preparation, and the ability to re-process legacy data for new insights. However, FRM can be complex to implement, and current FRM benchmarks are abstractions that are difficult to relate to how valuable or trustworthy a reconstruction is. Here, we relate the conventional benchmarks and demonstrations to practical and familiar cell biology analyses to demonstrate that FRM should be judged in context. We further demonstrate that it performs remarkably well even with lower-magnification microscopy data, as are often collected in screening and high content imaging. Specifically, we present promising results for nuclei, cell-cell junctions, and fine feature reconstruction; provide data-driven experimental design guidelines; and provide researcher-friendly code, complete sample data, and a researcher manual to enable more widespread adoption of FRM.

Epithelial self-healing is recapitulated by a 3D biomimetic E-cadherin junction.

Epithelial monolayers undergo self-healing when wounded. During healing, cells collectively migrate into the wound site, and the converging tissue fronts collide and form a stable interface. To heal, migrating tissues must form cell-cell adhesions and reorganize from the front-rear polarity characteristic of cell migration to the apical-basal polarity of an epithelium. However, identifying the "stop signal" that induces colliding tissues to cease migrating and heal remains an open question. Epithelial cells form integrin-based adhesions to the basal extracellular matrix (ECM) and E-cadherin-mediated cell-cell adhesions on the orthogonal, lateral surfaces between cells. Current biological tools have been unable to probe this multicellular 3D interface to determine the stop signal. We addressed this problem by developing a unique biointerface that mimicked the 3D organization of epithelial cell adhesions. This "minimal tissue mimic" (MTM) comprised a basal ECM substrate and a vertical surface coated with purified extracellular domain of E-cadherin, and was designed for collision with the healing edge of an epithelial monolayer. Three-dimensional imaging showed that adhesions formed between cells, and the E-cadherin-coated MTM resembled the morphology and dynamics of native epithelial cell-cell junctions and induced the same polarity transition that occurs during epithelial self-healing. These results indicate that E-cadherin presented in the proper 3D context constitutes a minimum essential stop signal to induce self-healing. That the Ecad:Fc MTM stably integrated into an epithelial tissue and reduced migration at the interface suggests that this biointerface is a complimentary approach to existing tissue-material interfaces.

Tail-assisted pitch control in lizards, robots and dinosaurs.

In 1969, a palaeontologist proposed that theropod dinosaurs used their tails as dynamic stabilizers during rapid or irregular movements, contributing to their depiction as active and agile predators. Since then the inertia of swinging appendages has been implicated in stabilizing human walking, aiding acrobatic manoeuvres by primates and rodents, and enabling cats to balance on branches. Recent studies on geckos suggest that active tail stabilization occurs during climbing, righting and gliding. By contrast, studies on the effect of lizard tail loss show evidence of a decrease, an increase or no change in performance. Application of a control-theoretic framework could advance our general understanding of inertial appendage use in locomotion. Here we report that lizards control the swing of their tails in a measured manner to redirect angular momentum from their bodies to their tails, stabilizing body attitude in the sagittal plane. We video-recorded Red-Headed Agama lizards (Agama agama) leaping towards a vertical surface by first vaulting onto an obstacle with variable traction to induce a range of perturbations in body angular momentum. To examine a known controlled tail response, we built a lizard-sized robot with an active tail that used sensory feedback to stabilize pitch as it drove off a ramp. Our dynamics model revealed that a body swinging its tail experienced less rotation than a body with a rigid tail, a passively compliant tail or no tail. To compare a range of tails, we calculated tail effectiveness as the amount of tailless body rotation a tail could stabilize. A model Velociraptor mongoliensis supported the initial tail stabilization hypothesis, showing as it did a greater tail effectiveness than the Agama lizards. Leaping lizards show that inertial control of body attitude can advance our understanding of appendage evolution and provide biological inspiration for the next generation of manoeuvrable search-and-rescue robots.

Galvanotactic control of collective cell migration in epithelial monolayers.

Many normal and pathological biological processes involve the migration of epithelial cell sheets. This arises from complex emergent behaviour resulting from the interplay between cellular signalling networks and the forces that physically couple the cells. Here, we demonstrate that collective migration of an epithelium can be interactively guided by applying electric fields that bias the underlying signalling networks. We show that complex, spatiotemporal cues are locally interpreted by the epithelium, resulting in rapid, coordinated responses such as a collective U-turn, divergent migration, and unchecked migration against an obstacle. We observed that the degree of external control depends on the size and shape of the cell population, and on the existence of physical coupling between cells. Together, our results offer design and engineering principles for the rational manipulation of the collective behaviour and material properties of a tissue.

A review of the effect of omega-3 polyunsaturated fatty acids on blood triacylglycerol levels in normolipidemic and borderline hyperlipidemic individuals.

Circulating levels of triacylglycerol (TG) is a recognized risk factor for developing cardiovascular disease, a leading cause of death worldwide. The Institute of Medicine and the American Heart Association both recommend the consumption of n-3 polyunsaturated fatty acids (PUFA), specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), to reduce serum TG in hyperlipidemic individuals. Additionally, a number of systematic reviews have shown that individuals with any degree of dyslipidemia, elevated serum TG and/or cholesterol, may benefit from a 20-30% reduction in serum TG after consuming n-3 PUFA derived from marine sources. Given that individuals with serum lipid levels ranging from healthy to borderline dyslipidemic constitute a large portion of the population, the focus of this review was to assess the potential for n-3 PUFA consumption to reduce serum TG in such individuals. A total of 1341 studies were retrieved and 38 clinical intervention studies, assessing 2270 individuals, were identified for inclusion in the current review. In summary, a 9-26% reduction in circulating TG was demonstrated in studies where ≥ 4 g/day of n-3 PUFA were consumed from either marine or EPA/DHA-enriched food sources, while a 4-51% reduction was found in studies where 1-5 g/day of EPA and/or DHA was consumed through supplements. Overall, this review summarizes the current evidence with regards to the beneficial effect of n-3 PUFA on circulating TG levels in normolipidemic to borderline hyperlipidemic, otherwise healthy, individuals. Thus demonstrating that n-3 PUFA may play an important role in the maintenance of cardiovascular health and disease prevention.

Building Biomaterials to Mimic 3D Cell-Cell Junctions.

Cell-cell interactions typically occur in a 3D context that is distinct from conventional 2D cell-substrate interactions in a Petri dish. Here, we describe a benchtop method to combine a 2D extracellular matrix surface with a 3D, vertical boundary functionalized with the extracellular domain of E-cadherin. The methodology is suitable for any biology laboratory without requiring advanced microfabrication equipment or training. Overall, this cell-mimetic interface uniquely recapitulates key aspects of cell-cell adhesion and can serve as a versatile, reductionist technique to study general cell-cell interactions in a 3D context.

E-cadherin biomaterials reprogram collective cell migration and cell cycling by forcing homeostatic conditions.

Cells attach to the world through either cell-extracellular matrix adhesion or cell-cell adhesion, and traditional biomaterials imitate the matrix for integrin-based adhesion. However, materials incorporating cadherin proteins that mimic cell-cell adhesion offer an alternative to program cell behavior and integrate into living tissues. We investigated how cadherin substrates affect collective cell migration and cell cycling in epithelia. Our approach involved biomaterials with matrix proteins on one-half and E-cadherin proteins on the other, forming a "Janus" interface across which we grew a single sheet of cells. Tissue regions over the matrix side exhibited normal collective dynamics, but an abrupt behavior shift occurred across the Janus boundary onto the E-cadherin side, where cells attached to the substrate via E-cadherin adhesions, resulting in stalled migration and slowing of the cell cycle. E-cadherin surfaces disrupted long-range mechanical coordination and nearly doubled the length of the G0/G1 phase of the cell cycle, linked to the lack of integrin focal adhesions on the E-cadherin surface.

Parameter identifiability and model selection for partial differential equation models of cell invasion.

When employing mechanistic models to study biological phenomena, practical parameter identifiability is important for making accurate predictions across wide ranges of unseen scenarios, as well as for understanding the underlying mechanisms. In this work, we use a profile-likelihood approach to investigate parameter identifiability for four extensions of the Fisher-Kolmogorov-Petrovsky-Piskunov (Fisher-KPP) model, given experimental data from a cell invasion assay. We show that more complicated models tend to be less identifiable, with parameter estimates being more sensitive to subtle differences in experimental procedures, and that they require more data to be practically identifiable. As a result, we suggest that parameter identifiability should be considered alongside goodness-of-fit and model complexity as criteria for model selection.

Bioelectric stimulation controls tissue shape and size.

Epithelial tissues sheath organs and electro-mechanically regulate ion and water transport to regulate development, homeostasis, and hydrostatic organ pressure. Here, we demonstrate how external electrical stimulation allows us to control these processes in living tissues. Specifically, we electrically stimulate hollow, 3D kidneyoids and gut organoids and find that physiological-strength electrical stimulation of ∼ 5 - 10 V/cm powerfully inflates hollow tissues; a process we call electro-inflation. Electro-inflation is mediated by increased ion flux through ion channels/transporters and triggers subsequent osmotic water flow into the lumen, generating hydrostatic pressure that competes against cytoskeletal tension. Our computational studies suggest that electro-inflation is strongly driven by field-induced ion crowding on the outer surface of the tissue. Electrically stimulated tissues also break symmetry in 3D resulting from electrotaxis and affecting tissue shape. The ability of electrical cues to regulate tissue size and shape emphasizes the role and importance of the electrical micro-environment for living tissues.

Optimal control of collective electrotaxis in epithelial monolayers.

Epithelial monolayers are some of the best-studied models for collective cell migration due to their abundance in multicellular systems and their tractability. Experimentally, the collective migration of epithelial monolayers can be robustly steered e.g. using electric fields, via a process termed electrotaxis. Theoretically, however, the question of how to design an electric field to achieve a desired spatiotemporal movement pattern is underexplored. In this work, we construct and calibrate an ordinary differential equation model to predict the average velocity of the centre of mass of a cellular monolayer in response to stimulation with an electric field. We use this model, in conjunction with optimal control theory, to derive physically realistic optimal electric field designs to achieve a variety of aims, including maximising the total distance travelled by the monolayer, maximising the monolayer velocity, and keeping the monolayer velocity constant during stimulation. Together, this work is the first to present a unified framework for optimal control of collective monolayer electrotaxis and provides a blueprint to optimally steer collective migration using other external cues.

Spatial heterogeneity in collective electrotaxis: continuum modelling and applications to optimal control.

Collective electrotaxis is a phenomenon that occurs when a cellular collective, for example an epithelial monolayer, is subjected to an electric field. Biologically, it is well known that the velocity of migration during the collective electrotaxis of large epithelia exhibits significant spatial heterogeneity. In this work, we demonstrate that the heterogeneity of velocities in the electrotaxing epithelium can be accounted for by a continuum model of cue competition in different tissue regions. Having established a working model of competing migratory cues in the migrating epithelium, we develop and validate a reaction-convection-diffusion model that describes the movement of an epithelial monolayer as it undergoes electrotaxis. We use the model to predict how tissue size and geometry affect the collective migration of MDCK monolayers, and to propose several ways in which electric fields can be designed such that they give rise to a desired spatial pattern of collective migration. We conclude with two examples that demonstrate practical applications of the method in designing bespoke stimulation protocols.

Quantitative electroencephalography during rapid eye movement (REM) and non-REM sleep in combat-exposed veterans with and without post-traumatic stress disorder.

Sleep disturbances are a hallmark feature of post-traumatic stress disorder (PTSD), and associated with poor clinical outcomes. Few studies have examined sleep quantitative electroencephalography (qEEG), a technique able to detect subtle differences that polysomnography does not capture. We hypothesized that greater high-frequency qEEG would reflect 'hyperarousal' in combat veterans with PTSD (n = 16) compared to veterans without PTSD (n = 13). EEG power in traditional EEG frequency bands was computed for artifact-free sleep epochs across an entire night. Correlations were performed between qEEG and ratings of PTSD symptoms and combat exposure. The groups did not differ significantly in whole-night qEEG measures for either rapid eye movement (REM) or non-REM (NREM) sleep. Non-significant medium effect sizes suggest less REM beta (opposite to our hypothesis), less REM and NREM sigma and more NREM gamma in combat veterans with PTSD. Positive correlations were found between combat exposure and NREM beta (PTSD group only), and REM and NREM sigma (non-PTSD group only). Results did not support global hyperarousal in PTSD as indexed by increased beta qEEG activity. The correlation of sigma activity with combat exposure in those without PTSD and the non-significant trend towards less sigma activity during both REM and NREM sleep in combat veterans with PTSD suggests that differential information processing during sleep may characterize combat-exposed military veterans with and without PTSD.

A highly elastic, capacitive strain gauge based on percolating nanotube networks.

We present a highly elastic strain gauge based on capacitive sensing of parallel, carbon nanotube-based percolation electrodes separated by a dielectric elastomer. The fabrication, relying on vacuum filtration of single-walled carbon nanotubes and hydrophobic patterning of silicone, is both rapid and inexpensive. We demonstrate reliable, linear performance over thousands of cycles at up to 100% strain with less than 3% variability and the highest reported gauge factor for a device of this class (0.99). We further demonstrate use of this sensor in a robotics context to transduce joint angles.

Investigating the role of polyunsaturated fatty acids in bone development using animal models.

Incorporating n-3 polyunsaturated fatty acids (PUFA) in the diet may promote the development of a healthy skeleton and thereby reduce the risk of developing osteoporosis in later life. Studies using developing animal models suggest lowering dietary n-6 PUFA and increasing n-3 PUFA intakes, especially long chain n-3 PUFA, may be beneficial for achieving higher bone mineral content, density and stronger bones. To date, the evidence regarding the effects of α-linolenic acid (ALA) remain equivocal, in contrast to evidence from the longer chain products, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This review reports the results of investigations into n-3 PUFA supplementation on bone fatty acid composition, strength and mineral content in developing animal models as well as the mechanistic relationships of PUFA and bone, and identifies critical areas for future research. Overall, this review supports a probable role for essential (ALA) and long chain (EPA and DHA) n-3 PUFA for bone health. Understanding the role of PUFA in optimizing bone health may lead to dietary strategies that promote bone development and maintenance of a healthy skeleton.

E-cadherin biointerfaces reprogram collective cell migration and cell cycling by forcing homeostatic conditions.

Cells attach to the world around them in two ways-cell:extracellular-matrix adhesion and cell:cell adhesion-and conventional biomaterials are made to resemble the matrix to encourage integrin-based cell adhesion. However, interest is growing for cell-mimetic interfaces that mimic cell-cell interactions using cadherin proteins, as this offers a new way to program cell behavior and design synthetic implants and objects that can integrate directly into living tissues. Here, we explore how these cadherin-based materials affect collective cell behaviors, focusing specifically on collective migration and cell cycle regulation in cm-scale epithelia. We built culture substrates where half of the culture area was functionalized with matrix proteins and the contiguous half was functionalized with E-cadherin proteins, and we grew large epithelia across this 'Janus' interface. Parts of the tissues in contact with the matrix side of the Janus interface exhibited normal collective dynamics, but an abrupt shift in behaviors happened immediately across the Janus boundary onto the E-cadherin side, where cells formed hybrid E-cadherin junctions with the substrate, migration effectively froze in place, and cell-cycling significantly decreased. E-cadherin materials suppressed long-range mechanical correlations in the tissue and mechanical information reflected off the substrate interface. These effects could not be explained by conventional density, shape index, or contact inhibition explanations. E-cadherin surfaces nearly doubled the length of the G0/G1 phase of the cell cycle, which we ultimately connected to the exclusion of matrix focal adhesions induced by the E-cadherin culture surface.