BATMAN: A Brain-like Approach for Tracking Maritime Activity and Nuance.

As commercial geospatial intelligence data becomes more widely available, algorithms using artificial intelligence need to be created to analyze it. Maritime traffic is annually increasing in volume, and with it the number of anomalous events that might be of interest to law enforcement agencies, governments, and militaries. This work proposes a data fusion pipeline that uses a mixture of artificial intelligence and traditional algorithms to identify ships at sea and classify their behavior. A fusion process of visual spectrum satellite imagery and automatic identification system (AIS) data was used to identify ships. Further, this fused data was further integrated with additional information about the ship's environment to help classify each ship's behavior to a meaningful degree. This type of contextual information included things such as exclusive economic zone boundaries, locations of pipelines and undersea cables, and the local weather. Behaviors such as illegal fishing, trans-shipment, and spoofing are identified by the framework using freely or cheaply accessible data from places such as Google Earth, the United States Coast Guard, etc. The pipeline is the first of its kind to go beyond the typical ship identification process to help aid analysts in identifying tangible behaviors and reducing the human workload.

A novel jamming phase diagram links tumor invasion to non-equilibrium phase separation.

It is well established that the early malignant tumor invades surrounding extracellular matrix (ECM) in a manner that depends upon material properties of constituent cells, surrounding ECM, and their interactions. Recent studies have established the capacity of the invading tumor spheroids to evolve into coexistent solid-like, fluid-like, and gas-like phases. Using breast cancer cell lines invading into engineered ECM, here we show that the spheroid interior develops spatial and temporal heterogeneities in material phase which, depending upon cell type and matrix density, ultimately result in a variety of phase separation patterns at the invasive front. Using a computational approach, we further show that these patterns are captured by a novel jamming phase diagram. We suggest that non-equilibrium phase separation based upon jamming and unjamming transitions may provide a unifying physical picture to describe cellular migratory dynamics within, and invasion from, a tumor.

Correction: Direct growth of a porous substrate on high-quality graphene via in situ phase inversion of a polymeric solution.

Correction for 'Direct growth of a porous substrate on high-quality graphene via in situ phase inversion of a polymeric solution' by Yanzhe Qin et al., Nanoscale, 2020, 12, 4953-4958, DOI: 10.1039/C9NR09693K.

In primary airway epithelial cells, the unjamming transition is distinct from the epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) and the unjamming transition (UJT) each comprises a gateway to cellular migration, plasticity and remodeling, but the extent to which these core programs are distinct, overlapping, or identical has remained undefined. Here, we triggered partial EMT (pEMT) or UJT in differentiated primary human bronchial epithelial cells. After triggering UJT, cell-cell junctions, apico-basal polarity, and barrier function remain intact, cells elongate and align into cooperative migratory packs, and mesenchymal markers of EMT remain unapparent. After triggering pEMT these and other metrics of UJT versus pEMT diverge. A computational model attributes effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, those tissues that are subject to development, injury, or disease become endowed with rich mechanisms for cellular migration, plasticity, self-repair, and regeneration.

Epithelial layer unjamming shifts energy metabolism toward glycolysis.

In development of an embryo, healing of a wound, or progression of a carcinoma, a requisite event is collective epithelial cellular migration. For example, cells at the advancing front of a wound edge tend to migrate collectively, elongate substantially, and exert tractions more forcefully compared with cells many ranks behind. With regards to energy metabolism, striking spatial gradients have recently been reported in the wounded epithelium, as well as in the tumor, but within the wounded cell layer little is known about the link between mechanical events and underlying energy metabolism. Using the advancing confluent monolayer of MDCKII cells as a model system, here we report at single cell resolution the evolving spatiotemporal fields of cell migration speeds, cell shapes, and traction forces measured simultaneously with fields of multiple indices of cellular energy metabolism. Compared with the epithelial layer that is unwounded, which is non-migratory, solid-like and jammed, the leading edge of the advancing cell layer is shown to become progressively more migratory, fluid-like, and unjammed. In doing so the cytoplasmic redox ratio becomes progressively smaller, the NADH lifetime becomes progressively shorter, and the mitochondrial membrane potential and glucose uptake become progressively larger. These observations indicate that a metabolic shift toward glycolysis accompanies collective cellular migration but show, further, that this shift occurs throughout the cell layer, even in regions where associated changes in cell shapes, traction forces, and migration velocities have yet to penetrate. In characterizing the wound healing process these morphological, mechanical, and metabolic observations, taken on a cell-by-cell basis, comprise the most comprehensive set of biophysical data yet reported. Together, these data suggest the novel hypothesis that the unjammed phase evolved to accommodate fluid-like migratory dynamics during episodes of tissue wound healing, development, and plasticity, but is more energetically expensive compared with the jammed phase, which evolved to maintain a solid-like non-migratory state that is more energetically economical.

Irradiation Induces Epithelial Cell Unjamming.

The healthy and mature epithelial layer is ordinarily quiescent, non-migratory, solid-like, and jammed. However, in a variety of circumstances the layer transitions to a phase that is dynamic, migratory, fluid-like and unjammed. This has been demonstrated in the developing embryo, the developing avian airway, the epithelial layer reconstituted in vitro from asthmatic donors, wounding, and exposure to mechanical stress. Here we examine the extent to which ionizing radiation might similarly provoke epithelial layer unjamming. We exposed primary human bronchial epithelial (HBE) cells maintained in air-liquid interface (ALI) to sub-therapeutic doses (1 Gy) of ionizing radiation (IR). We first assessed: (1) DNA damage by measuring p-H2AX, (2) the integrity of the epithelial layer by measuring transepithelial electrical resistance (TEER), and (3) the extent of epithelial cell differentiation by detecting markers of differentiated airway epithelial cells. As expected, IR exposure induced DNA damage but, surprisingly, disrupted neither normal differentiation nor the integrity of the epithelial cell layer. We then measured cell shape and cellular migration to determine the extent of the unjamming transition (UJT). IR caused cell shape elongation and increased cellular motility, both of which are hallmarks of the UJT as previously confirmed. To understand the mechanism of IR-induced UJT, we inhibited TGF-ß receptor activity, and found that migratory responses were attenuated. Together, these observations show that IR can provoke epithelial layer unjamming in a TGF-ß receptor-dependent manner.

Direct growth of a porous substrate on high-quality graphene via in situ phase inversion of a polymeric solution.

The key for graphene applications is the successful transfer of graphene from a growth metal substrate to a substrate for application without compromising its high quality. However, state-of-the-art polymethyl methacrylate (PMMA) assisted transfer methods introduce wrinkles, folds and cracks, which are exacerbated for porous substrates. Here we report a novel in situ technique to transfer graphene onto a porous substrate which resolves these issues. Using phase-inversion a porous substrate is grown onto a graphene film with strong adhesion that perfectly matches graphene's topography, and the growth metal substrate is subsequently etched away. We achieve 63 cm2 high-quality single-layered graphene with almost 100% coverage over the pores of the substrate and pore ratios up to 35%. Our study resolves the three main challenges of transferring graphene to porous substrates, which are matching the topographies between the graphene and the porous substrate, achieving high pore ratios and minimizing the stresses on the suspended graphene; this approach may therefore serve as a general guide for attaching graphene or other 2D materials to scaffold structures.

Unjamming and collective migration in MCF10A breast cancer cell lines.

Each cell comprising an intact, healthy, confluent epithelial layer ordinarily remains sedentary, firmly adherent to and caged by its neighbors, and thus defines an elemental constituent of a solid-like cellular collective [1,2]. After malignant transformation, however, the cellular collective can become fluid-like and migratory, as evidenced by collective motions that arise in characteristic swirls, strands, ducts, sheets, or clusters [3,4]. To transition from a solid-like to a fluid-like phase and thereafter to migrate collectively, it has been recently argued that cells comprising the disordered but confluent epithelial collective can undergo changes of cell shape so as to overcome geometric constraints attributable to the newly discovered phenomenon of cell jamming and the associated unjamming transition (UJT) [1,2,5-9]. Relevance of the jamming concept to carcinoma cells lines of graded degrees of invasive potential has never been investigated, however. Using classical in vitro cultures of six breast cancer model systems, here we investigate structural and dynamical signatures of cell jamming, and the relationship between them [1,2,10,11]. In order of roughly increasing invasive potential as previously reported, model systems examined included MCF10A, MCF10A.Vector; MCF10A.14-3-3ζ; MCF10.ErbB2, MCF10AT; and MCF10CA1a [12-15]. Migratory speed depended on the particular cell line. Unsurprisingly, for example, the MCF10CA1a cell line exhibited much faster migratory speed relative to the others. But unexpectedly, across different cell lines higher speeds were associated with enhanced size of cooperative cell packs in a manner reminiscent of a peloton [9]. Nevertheless, within each of the cell lines evaluated, cell shape and shape variability from cell-to-cell conformed with predicted structural signatures of cell layer unjamming [1]. Moreover, both structure and migratory dynamics were compatible with previous theoretical descriptions of the cell jamming mechanism [2,10,11,16,17]. As such, these findings demonstrate the richness of the cell jamming mechanism, which is now seen to apply across these cancer cell lines but remains poorly understood.

The tumor suppressor p53 can promote collective cellular migration.

Loss of function of the tumor suppressor p53 is known to increase the rate of migration of cells transiting the narrow pores of the traditional Boyden chamber assay. Here by contrast we investigate how p53 impacts the rate of cellular migration within a 2D confluent cell layer and a 3D collagen-embedded multicellular spheroid. We use two human carcinoma cell lines, the bladder carcinoma EJ and the colorectal carcinoma HCT116. In the confluent 2-D cell layer, for both EJ and HCT cells the migratory speeds and effective diffusion coefficients for the p53 null cells were significantly smaller than in p53-expressing cells. Compared to p53 expressers, p53-null cells exhibited more organized cortical actin rings together with reduced front-rear cell polarity. Furthermore, loss of p53 caused cells to exert smaller traction forces upon their substrates, and reduced formation of cryptic lamellipodia. In the 3D multicellular spheroid, loss of p53 consistently reduced collective cellular migration into surrounding collagen matrix. As regards the role of p53 in cellular migration, extrapolation from the Boyden chamber assay to other cellular microenvironments is seen to be fraught even in terms of the sign of the effect. Together, these paradoxical results show that the effects of p53 on cellular migration are context-dependent.

Single-step assembly of asymmetric vesicles.

Asymmetric vesicles are membranes in which amphiphiles are asymmetrically distributed between each membrane leaflet. This asymmetry dictates chemical and physical properties of these vesicles, enabling their use as more realistic models of biological cell membranes, which also are asymmetric, and improves their potential for drug delivery and cosmetic applications. However, their fabrication is difficult as the self-assembly of amphiphiles always leads to symmetric vesicles. Here, we report the use of water-in-oil-in-oil-in-water triple emulsion drops to direct the assembly of the two leaflets to form asymmetric vesicles. Different compositions of amphiphiles are dissolved in each of the two oil shells of the triple emulsion; the amphiphiles diffuse to the interfaces and adsorb differentially at each of the two oil/water interfaces of the triple emulsion. These middle oil phases dewet from the innermost water cores of the triple emulsion drops, leading to the formation of membranes with degrees of asymmetry up to 70%. The triple emulsion drops are fabricated using capillary microfluidics, enabling production of highly monodisperse drops at rates as high as 300 Hz. Vesicles produced by this method can very efficiently encapsulate many different ingredients; this further enhances the utility of asymmetric vesicles as artificial cells, bioreactors and delivery vehicles.

Geometric constraints during epithelial jamming.

As an injury heals, an embryo develops, or a carcinoma spreads, epithelial cells systematically change their shape. In each of these processes cell shape is studied extensively whereas variability of shape from cell-to-cell is regarded most often as biological noise. But where do cell shape and its variability come from? Here we report that cell shape and shape variability are mutually constrained through a relationship that is purely geometrical. That relationship is shown to govern processes as diverse as maturation of the pseudostratified bronchial epithelial layer cultured from non-asthmatic or asthmatic donors, and formation of the ventral furrow in the Drosophila embryo. Across these and other epithelial systems, shape variability collapses to a family of distributions that is common to all. That distribution, in turn, is accounted for by a mechanistic theory of cell-cell interaction showing that cell shape becomes progressively less elongated and less variable as the layer becomes progressively more jammed. These findings suggest a connection between jamming and geometry that spans living organisms and inert jammed systems, and thus transcends system details. Although molecular events are needed for any complete theory of cell shape and cell packing, observations point to the hypothesis that jamming behavior at larger scales of organization sets overriding geometrical constraints.

Evolution on the Biophysical Fitness Landscape of an RNA Virus.

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

Matrix Production and Sporulation in Bacillus subtilis Biofilms Localize to Propagating Wave Fronts.

Bacterial biofilms are surface-attached microbial communities encased in self-produced extracellular polymeric substances. Here we demonstrate that during the development of Bacillus subtilis biofilms, matrix production is localized to an annular front propagating at the periphery and sporulation to a second front at a fixed distance at the interior. We show that within these fronts, cells switch off matrix production and transition to sporulation after a set time delay of ∼100 min. Correlation analyses of fluctuations in fluorescence reporter activity reveal that the fronts emerge from a pair of gene-expression waves of matrix production and sporulation. The localized expression waves travel across cells that are immobilized in the biofilm matrix in contrast to active cell migration or horizontal colony spreading. Our results suggest that front propagation arises via a local developmental program occurring at the level of individual bacterial cells, likely driven by nutrient depletion and metabolic by-product accumulation. A single-length scale and timescale couples the spatiotemporal propagation of both fronts throughout development. As a result, gene expression patterns within the advancing fronts collapse to self-similar expression profiles. Our findings highlight the key role of the localized cellular developmental program associated with the propagating front in describing biofilm growth.

Biocompatible Amphiphilic Hydrogel-Solid Dimer Particles as Colloidal Surfactants.

Emulsions of two immiscible liquids can slowly coalesce over time when stabilized by surfactant molecules. Pickering emulsions stabilized by colloidal particles can be much more stable. Here, we fabricate biocompatible amphiphilic dimer particles using a hydrogel, a strongly hydrophilic material, and achieve large contrast in the wetting properties of the two bulbs, resulting in enhanced stabilization of emulsions. We generate monodisperse single emulsions of alginate and shellac solution in oil using a flow-focusing microfluidics device. Shellac precipitates from water and forms a solid bulb at the periphery of the droplet when the emulsion is exposed to acid. Molecular interactions result in amphiphilic dimer particles that consist of two joined bulbs: one hydrogel bulb of alginate in water and the other hydrophobic bulb of shellac. Alginate in the hydrogel compartment can be cross-linked using calcium cations to obtain stable particles. Analogous to surfactant molecules at the interface, the resultant amphiphilic particles stand at the water/oil interface with the hydrogel bulb submerged in water and the hydrophobic bulb in oil and are thus able to stabilize both water-in-oil and oil-in-water emulsions, making these amphiphilic hydrogel-solid particles ideal colloidal surfactants for various applications.

Ultrafast Nanofiltration through Large-Area Single-Layered Graphene Membranes.

Perforated single-layered graphene has demonstrated selectivity and flux that is orders of magnitude greater than state-of-the-art polymer membranes. However, only individual graphene sheets with sizes up to tens of micrometers have been successfully fabricated for pressurized permeation studies. Scaling-up and reinforcement of these atomic membranes with minimum cracks and pinholes remains a major hurdle for practical applications. We develop a large-area in situ, phase-inversion casting technique to create 63 cm2 high-quality single-layered perforated graphene membranes for ultrafast nanofiltration that can operate at pressures up to 50 bar. This result demonstrates the feasibility of our technique for creating robust large-area, high quality, single-layered graphene and its potential use as a pressurized nanofiltration membrane.

Dispersing hydrophobic natural colourant ß-carotene in shellac particles for enhanced stability and tunable colour.

Colour is one of the most important visual attributes of food and is directly related to the perception of food quality. The interest in natural colourants, especially ß-carotene that not only imparts colour but also has well-documented health benefits, has triggered the research and development of different protocols designed to entrap these hydrophobic natural molecules to improve their stability against oxidation. Here, we report a versatile microfluidic approach that uses single emulsion droplets as templates to prepare microparticles loaded with natural colourants. The solution of ß-carotene and shellac in the solvent is emulsified by microfluidics into droplets. Upon solvent diffusion, ß-carotene and shellac co-precipitates, forming solid microparticles of ß-carotene dispersed in the shellac polymer matrix. We substantially improve the stability of ß-carotene that is protected from oxidation by the polymer matrix and achieve different colour appearances by loading particles with different ß-carotene concentrations. These particles demonstrate great promise for practical use in natural food colouring.

Microfluidic Fabrication of Pluronic Vesicles with Controlled Permeability.

Block copolymers with a low hydrophilic-to-lipophilic balance form membranes that are highly permeable to hydrophilic molecules. Polymersomes with this type of membrane enable the controllable release of molecules without membrane rupture. However, these polymersomes are difficult to assemble because of their low hydrophobicity. Here, we report a microfluidic approach to the production of these polymersomes using double-emulsion drops with ultrathin shells as templates. The small thickness of the middle oil phase enables the attraction of the hydrophobic blocks of the polymers adsorbed at each of the oil/water interfaces of the double emulsions; this results in the dewetting of the oil from the surface of the innermost water drops of the double emulsions and the ultimate formation of the polymersome. This approach to polymersome fabrication enables control of the vesicle size and results in the efficient encapsulation of hydrophilic ingredients that can be released through the polymer membrane without membrane rupture. We apply our approach to the fabrication of Pluronic L121 vesicles and characterize the permeability of their membranes. Furthermore, we show that membrane permeability can be tuned by blending different Pluronic polymers. Our work thus describes a route to producing Pluronic vesicles that are useful for the controlled release of hydrophilic ingredients.

Probing phenotypic growth in expanding Bacillus subtilis biofilms.

We develop an optical imaging technique for spatially and temporally tracking biofilm growth and the distribution of the main phenotypes of a Bacillus subtilis strain with a triple-fluorescent reporter for motility, matrix production, and sporulation. We develop a calibration procedure for determining the biofilm thickness from the transmission images, which is based on Beer-Lambert's law and involves cross-sectioning of biofilms. To obtain the phenotype distribution, we assume a linear relationship between the number of cells and their fluorescence and determine the best combination of calibration coefficients that matches the total number of cells for all three phenotypes and with the total number of cells from the transmission images. Based on this analysis, we resolve the composition of the biofilm in terms of motile, matrix-producing, sporulating cells and low-fluorescent materials which includes matrix and cells that are dead or have low fluorescent gene expression. We take advantage of the circular growth to make kymograph plots of all three phenotypes and the dominant phenotype in terms of radial distance and time. To visualize the nonlocal character of biofilm growth, we also make kymographs using the local colonization time. Our technique is suitable for real-time, noninvasive, quantitative studies of the growth and phenotype distribution of biofilms which are either exposed to different conditions such as biocides, nutrient depletion, dehydration, or waste accumulation.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (10(3)-10(6)). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

Rapid Assembly of Heterogeneous 3D Cell Microenvironments in a Microgel Array.

Heterogeneous 3D cell microenvironment arrays are rapidly assembled by combining surface-wettability-guided assembly and microdroplet-array-based operations. This approach enables precise control over individual shapes, sizes, chemical concentrations, cell density, and 3D spatial distribution of multiple components. This technique provides a cost-effective solution to meet the increasing demand of stem cell research, tissue engineering, and drug screening.