Learning hydrodynamic equations for active matter from particle simulations and experiments.

Recent advances in high-resolution imaging techniques and particle-based simulation methods have enabled the precise microscopic characterization of collective dynamics in various biological and engineered active matter systems. In parallel, data-driven algorithms for learning interpretable continuum models have shown promising potential for the recovery of underlying partial differential equations (PDEs) from continuum simulation data. By contrast, learning macroscopic hydrodynamic equations for active matter directly from experiments or particle simulations remains a major challenge, especially when continuum models are not known a priori or analytic coarse graining fails, as often is the case for nondilute and heterogeneous systems. Here, we present a framework that leverages spectral basis representations and sparse regression algorithms to discover PDE models from microscopic simulation and experimental data, while incorporating the relevant physical symmetries. We illustrate the practical potential through a range of applications, from a chiral active particle model mimicking nonidentical swimming cells to recent microroller experiments and schooling fish. In all these cases, our scheme learns hydrodynamic equations that reproduce the self-organized collective dynamics observed in the simulations and experiments. This inference framework makes it possible to measure a large number of hydrodynamic parameters in parallel and directly from video data.

Shared biophysical mechanisms determine early biofilm architecture development across different bacterial species.

Bacterial biofilms are among the most abundant multicellular structures on Earth and play essential roles in a wide range of ecological, medical, and industrial processes. However, general principles that govern the emergence of biofilm architecture across different species remain unknown. Here, we combine experiments, simulations, and statistical analysis to identify shared biophysical mechanisms that determine early biofilm architecture development at the single-cell level, for the species Vibrio cholerae, Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa grown as microcolonies in flow chambers. Our data-driven analysis reveals that despite the many molecular differences between these species, the biofilm architecture differences can be described by only 2 control parameters: cellular aspect ratio and cell density. Further experiments using single-species mutants for which the cell aspect ratio and the cell density are systematically varied, and mechanistic simulations show that tuning these 2 control parameters reproduces biofilm architectures of different species. Altogether, our results show that biofilm microcolony architecture is determined by mechanical cell-cell interactions, which are conserved across different species.

Topological Metric Detects Hidden Order in Disordered Media.

Recent advances in microscopy techniques make it possible to study the growth, dynamics, and response of complex biophysical systems at single-cell resolution, from bacterial communities to tissues and organoids. In contrast to ordered crystals, it is less obvious how one can reliably distinguish two amorphous yet structurally different cellular materials. Here, we introduce a topological earth mover's (TEM) distance between disordered structures that compares local graph neighborhoods of the microscopic cell-centroid networks. Leveraging structural information contained in the neighborhood motif distributions, the TEM metric allows an interpretable reconstruction of equilibrium and nonequilibrium phase spaces and embedded pathways from static system snapshots alone. Applied to cell-resolution imaging data, the framework recovers time ordering without prior knowledge about the underlying dynamics, revealing that fly wing development solves a topological optimal transport problem. Extending our topological analysis to bacterial swarms, we find a universal neighborhood size distribution consistent with a Tracy-Widom law.

Restricted Proteolysis and LC-MS/MS To Evaluate the Orientation of Surface-Immobilized Antibodies.

The molecular orientation of antibodies immobilized on solid surfaces plays a significant role in the sensitivity of immunoassays and efficiency of protein isolation using antibody-decorated nanoparticles. Optimally, nearly all antibody binding sites should be available to bind. Here we report for the first time an LC-MS/MS approach to probe antibody orientation directly, utilizing sterically restricted proteolysis. Trypsin-decorated magnetic beads (MBs, 1.5 µm) were much larger than average antibody-free areas (55 × 55 nm) of oriented antibodies on MBs, restricting proteolysis to mainly Fab regions. Randomly attached antibodies on MB surfaces served as controls. The tryptic-hydrolyzed peptides were quantified using LC-MS/MS peptide analysis as markers for average positions of Fc and Fab of antibodies on the beads. Different patterns of digestion rates were found due to proteolysis of the oriented and nonoriented antibodies on MBs. For oriented antibodies, the peptides from outer Fab regions gave a much higher digestion rate than those from Fc regions, while for randomly immobilized antibodies digestion rates for Fab and Fc peptides were similar. This novel approach is a useful and convenient tool to characterize antibody orientation for immunoassays and other applications. The relative degree of orientation can be assessed using a metric Ro denoting amount of Fab marker peptides found divided by Fc + Fab marker peptides × 100%. Oriented antibodies on the MBs also provided more efficient antigen capture compared to randomly immobilized antibodies.

Flow-Induced Symmetry Breaking in Growing Bacterial Biofilms.

Bacterial biofilms represent a major form of microbial life on Earth and serve as a model active nematic system, in which activity results from growth of the rod-shaped bacterial cells. In their natural environments, ranging from human organs to industrial pipelines, biofilms have evolved to grow robustly under significant fluid shear. Despite intense practical and theoretical interest, it is unclear how strong fluid flow alters the local and global architectures of biofilms. Here, we combine highly time-resolved single-cell live imaging with 3D multiscale modeling to investigate the mechanisms by which flow affects the dynamics of all individual cells in growing biofilms. Our experiments and cell-based simulations reveal three quantitatively different growth phases in strong external flow and the transitions between them. In the initial stages of biofilm development, flow induces a downstream gradient in cell orientation, causing asymmetrical dropletlike biofilm shapes. In the later developmental stages, when the majority of cells are sheltered from the flow by the surrounding extracellular matrix, buckling-induced cell verticalization in the biofilm core restores radially symmetric biofilm growth, in agreement with predictions of a 3D continuum model.

Mushrooms use convectively created airflows to disperse their spores.

Thousands of basidiomycete fungal species rely on mushroom spores to spread across landscapes. It has long been thought that spores depend on favorable winds for dispersal--that active control of spore dispersal by the parent fungus is limited to an impulse delivered to the spores to carry them clear of the gill surface. Here we show that evaporative cooling of the air surrounding the pileus creates convective airflows capable of carrying spores at speeds of centimeters per second. Convective cells can transport spores from gaps that may be only 1 cm high and lift spores 10 cm or more into the air. This work reveals how mushrooms tolerate and even benefit from crowding and explains their high water needs.

Defining individual size in the model filamentous fungus Neurospora crassa.

It is challenging to apply the tenets of individuality to filamentous fungi: a fungal mycelium can contain millions of genetically diverse but totipotent nuclei, each capable of founding new mycelia. Moreover, a single mycelium can potentially stretch over kilometres, and it is unlikely that its distant parts share resources or have the same fitness. Here, we directly measure how a single mycelium of the model ascomycete Neurospora crassa is patterned into reproductive units (RUs), meaning subpopulations of nuclei that propagate together as spores, and function as reproductive individuals. The density of RUs is sensitive to the geometry of growth; we detected 50-fold smaller RUs when mycelia had expanding frontiers than when they were constrained to grow in one direction only. RUs fragmented further when the mycelial network was perturbed. In mycelia with expanding frontiers, RU composition was strongly influenced by the distribution of genotypes early in development. Our results provide a concept of fungal individuality that is directly connected to reproductive potential, and therefore to theories of how fungal individuals adapt and evolve over time. Our data show that the size of reproductive individuals is a dynamic and environment-dependent property, even within apparently totally connected fungal mycelia.

Microfluidic array for simultaneous detection of DNA oxidation and DNA-adduct damage.

Exposure to chemical pollutants and pharmaceuticals may cause health issues caused by metabolite-related toxicity. This paper reports a new microfluidic electrochemical sensor array with the ability to simultaneously detect common types of DNA damage including oxidation and nucleobase adduct formation. Sensors in the 8-electrode screen-printed carbon array were coated with thin films of metallopolymers osmium or ruthenium bipyridyl-poly(vinylpyridine) chloride (OsPVP, RuPVP) along with DNA and metabolic enzymes by layer-by-layer electrostatic assembly. After a reaction step in which test chemicals and other necessary reagents flow over the array, OsPVP selectively detects oxidized guanines on the DNA strands, and RuPVP detects DNA adduction by metabolites on nucleobases. We demonstrate array performance for test chemicals including 17ß-estradiol (E2), its metabolites 4-hydroxyestradiol (4-OHE2), 2-hydroxyestradiol (2-OHE2), catechol, 2-nitrosotoluene (2-NO-T), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 2-acetylaminofluorene (2-AAF). Results revealed DNA-adduct and oxidation damage in a single run to provide a metabolic-genotoxic chemistry screen. The array measures damage directly in unhydrolyzed DNA, and is less expensive, faster, and simpler than conventional methods to detect DNA damage. The detection limit for oxidation is 672 8-oxodG per 106 bases. Each sensor requires only 22 ng of DNA, so the mass detection limit is 15 pg (∼10 pmol) 8-oxodG.

Voltammetric microwell array for oxidized guanosine in intact ds-DNA.

Oxidative stress in humans causes damage to biomolecules by generating reactive oxygen species (ROS). DNA can be oxidatively damaged by ROS, which may lead to carcinogenesis. Here we report a microfluidic electrochemical array designed to rapidly detect oxidation in intact DNA in replicate measurements. Sensor arrays were fabricated by wet-chemistry patterning of gold compact discs. The eight-sensor array is incorporated into a 60 µL microfluidic channel connected to a pump and sample valve. The array features 7 nm thick osmium bipyridyl poly(vinylpyridine) chloride [Os(bpy)2(PVP)10Cl](+) films assembled layer-by-layer with polyions onto the gold sensors. 8-Hydroxy-7,8-hydro-2'-deoxyguanosine (8-oxodG) is selectively oxidized by [Os(bpy)2(PVP)10Cl](+) in intact ds-DNA to provide catalytic square wave voltammograms (SWV). The device is easy-to-use, fast, inexpensive, reusable, and can detect one 8-oxodG per 6600 nucleobases. The mass detection limit is 150-fold lower than a previously reported dip-and-read voltammetric sensor for oxidized DNA. Fast assays (<1 min) and moderate sample consumption (15 pmol DNA) suggest potential for research and clinical applications. Practical use is illustrated by detecting DNA oxidation from cigarette smoke and ash extracts in dispersions with NADPH and Cu(2+).

Development and Clinical Evaluation of a High-Throughput LC-MS/MS Assay for Vitamin B6 in Human Plasma and Serum.

BACKGROUND: Pyridoxal 5'-phosphate (PLP) is the primary circulatory form of vitamin B6, an essential cofactor for numerous biochemical enzymatic reactions. Conventional PLP analysis using high-performance liquid chromatography (HPLC) with fluorescence requires derivatization and long injection-to-injection time. Development of high-throughput LC-MS/MS assays is desirable. METHODS: Stable isotope labeled internal standard was added to aliquots of samples, proteins were precipitated using trichloroacetic acid, and supernatants were analyzed by multiple reaction monitoring using LC-MS/MS in positive ion mode. Analysis time for PLP was 3.0 min using single column HPLC separation and 2.4 min using alternating column regeneration (ACR). Clinical evaluation of the method included review of results (n = 102 386) from routine performance of the assay. RESULTS: The assay was linear to 500 nmol/L; limit of quantification was 5 nmol/L. Imprecision (CV) of the assay was <5%. Equivalent performance was observed for single HPLC column and ACR. In 62% of routinely analyzed patient samples, PLP concentrations were within the reference interval; higher PLP concentrations were observed in samples from males than from females. Vitamin B6 deficiency was lowest in children and highest in elderly adults. Lower PLP concentrations were observed in samples collected during winter/spring than during summer/fall. We observed lower concentrations in plasma collected in lithium heparin tubes, suggesting PLP degradation caused by the anticoagulant. CONCLUSIONS: This LC-MS/MS method allows PLP determination using simple sample preparation and short analysis time. We observed association of PLP concentrations with age, sex, and season of sample collection. Our data indicate that lithium heparin anticoagulant tubes reduce measured PLP concentration.

Emergence of three-dimensional order and structure in growing biofilms.

Surface-attached bacterial biofilms are self-replicating active liquid crystals and the dominant form of bacterial life on earth (1-4). In conventional liquid crystals and solid-state materials, the interaction potentials between the molecules that comprise the system determine the material properties. However, for growth-active biofilms it is unclear whether potential-based descriptions can account for the experimentally observed morphologies, and which potentials would be relevant. Here, we overcame previous limitations of single-cell imaging techniques (5,6) to reconstruct and track all individual cells inside growing three-dimensional (3D) biofilms with up to 10,000 individuals. Based on these data, we identify, constrain, and provide a microscopic basis for an effective cell-cell interaction potential, which captures and predicts the growth dynamics, emergent architecture, and local liquid crystalline order of Vibrio cholerae biofilms. Furthermore, we show how external fluid flows control the microscopic structure and 3D morphology of biofilms. Our analysis implies that local cellular order and global biofilm architecture in these active bacterial communities can arise from mechanical cell-cell interactions, which cells can modulate by regulating the production of particular matrix components. These results establish an experimentally validated foundation for improved continuum theories of active matter and thereby contribute to solving the important problem of controlling biofilm growth.

Breakdown of Vibrio cholerae biofilm architecture induced by antibiotics disrupts community barrier function.

Bacterial cells in nature are frequently exposed to changes in their chemical environment1,2. The response mechanisms of isolated cells to such stimuli have been investigated in great detail. By contrast, little is known about the emergent multicellular responses to environmental changes, such as antibiotic exposure3-7, which may hold the key to understanding the structure and functions of the most common type of bacterial communities: biofilms. Here, by monitoring all individual cells in Vibrio cholerae biofilms during exposure to antibiotics that are commonly administered for cholera infections, we found that translational inhibitors cause strong effects on cell size and shape, as well as biofilm architectural properties. We identified that single-cell-level responses result from the metabolic consequences of inhibition of protein synthesis and that the community-level responses result from an interplay of matrix composition, matrix dissociation and mechanical interactions between cells. We further observed that the antibiotic-induced changes in biofilm architecture have substantial effects on biofilm population dynamics and community assembly by enabling invasion of biofilms by bacteriophages and intruder cells of different species. These mechanistic causes and ecological consequences of biofilm exposure to antibiotics are an important step towards understanding collective bacterial responses to environmental changes, with implications for the effects of antimicrobial therapy on the ecological succession of biofilm communities.

Electrochemiluminescent Array to Detect Oxidative Damage in ds-DNA Using [Os(bpy)2(phen-benz-COOH)]2+/Nafion/Graphene Films.

Reactive oxygen species (ROS) oxidize guanosines in DNA to form 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), a biomarker for oxidative stress. Herein we describe a novel 64-microwell electrochemiluminescent (ECL) array enabling sensitive multiplexed detection of 8-oxodG in ds-DNA without hydrolysis. Films of Nafion and reduced graphene oxide containing ECL dye [Os(bpy)2(phen-benz-COOH)]2+ (OsNG, {bpy= 2,2'-bipyridine and phen-benz-COOH = (4-(1,10-phenanthrolin-6-yl) benzoic acid)}) were assembled into microwells on a pyrolytic graphite wafer to detect 8-oxodG in oligonucleotides by electrochemiluminescence (ECL). DNA oxidation by Fenton's reagent or by ROS formation during redox cycles involving NADPH, CuII, and model metabolites was monitored. UPLC-MS/MS of oxidized DNA samples were used for calibration. Detection limit for the fluidic arrays was one 8-oxodG per 670 intact nucleobases, or 0.15%. The method is sensitive enough to evaluate DNA oxidation from biologically relevant ROS-generating reactions of CuII, NADPH, and model metabolites.