Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140 ms.

Genetic requirements for cell division in a genomically minimal cell.

Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell.

Conceptual and Experimental Tools to Understand Spatial Effects and Transport Phenomena in Nonlinear Biochemical Networks Illustrated with Patchy Switching.

Many biochemical systems are spatially heterogeneous and exhibit nonlinear behaviors, such as state switching in response to small changes in the local concentration of diffusible molecules. Systems as varied as blood clotting, intracellular calcium signaling, and tissue inflammation are all heavily influenced by the balance of rates of reaction and mass transport phenomena including flow and diffusion. Transport of signaling molecules is also affected by geometry and chemoselective confinement via matrix binding. In this review, we use a phenomenon referred to as patchy switching to illustrate the interplay of nonlinearities, transport phenomena, and spatial effects. Patchy switching describes a change in the state of a network when the local concentration of a diffusible molecule surpasses a critical threshold. Using patchy switching as an example, we describe conceptual tools from nonlinear dynamics and chemical engineering that make testable predictions and provide a unifying description of the myriad possible experimental observations. We describe experimental microfluidic and biochemical tools emerging to test conceptual predictions by controlling transport phenomena and spatial distribution of diffusible signals, and we highlight the unmet need for in vivo tools.

Single-Cell Tracing Dissects Regulation of Maintenance and Inheritance of Transcriptional Reinduction Memory.

Transcriptional memory of gene expression enables adaptation to repeated stimuli across many organisms. However, the regulation and heritability of transcriptional memory in single cells and through divisions remains poorly understood. Here, we combined microfluidics with single-cell live imaging to monitor Saccharomyces cerevisiae galactokinase 1 (GAL1) expression over multiple generations. By applying pedigree analysis, we dissected and quantified the maintenance and inheritance of transcriptional reinduction memory in individual cells through multiple divisions. We systematically screened for loss- and gain-of-memory knockouts to identify memory regulators in thousands of single cells. We identified new loss-of-memory mutants, which affect memory inheritance into progeny. We also unveiled a gain-of-memory mutant, elp6Δ, and suggest that this new phenotype can be mediated through decreased histone occupancy at the GAL1 promoter. Our work uncovers principles of maintenance and inheritance of gene expression states and their regulators at the single-cell level.

Regulation of branched versus linear Arp2/3-generated actin filaments.

Activation of the Arp2/3 complex by VCA-motif-bearing actin nucleation-promoting factors results in the formation of "daughter" actin filaments branching off the sides of pre-existing "mother" filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates "linear" actin filaments. Uncovering the similarities and differences between these two mechanisms is fundamental to understanding how actin cytoskeleton dynamics are regulated. Here, analysis of individual filaments reveals that, unexpectedly, the VCA motifs of WASP, N-WASP, and WASH destabilize existing branches, as well as SPIN90-Arp2/3 at linear filament ends. Furthermore, branch stabilizer cortactin and destabilizer GMF each have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the ends of linear filaments is not destabilized by piconewton forces and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to the regulatory proteins we have tested, albeit with some differences, but significantly differ in their responses to aging and mechanical stress. These kinetic differences likely reflect the small conformational differences recently reported between Arp2/3 in branch junctions and linear filaments and suggest that their turnover in cells may be differently regulated.

Comparative Analysis of Droplet-Based Ultra-High-Throughput Single-Cell RNA-Seq Systems.

Since its establishment in 2009, single-cell RNA sequencing (RNA-seq) has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. Although most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, with the same cell line and bioinformatics pipeline, we developed directly comparable datasets for each of three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. Although each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.

Substrate geometry affects population dynamics in a bacterial biofilm.

Biofilms inhabit a range of environments, such as dental plaques or soil micropores, often characterized by noneven surfaces. However, the impact of surface irregularities on the population dynamics of biofilms remains elusive, as most experiments are conducted on flat surfaces. Here, we show that the shape of the surface on which a biofilm grows influences genetic drift and selection within the biofilm. We culture Escherichia coli biofilms in microwells with a corrugated bottom surface and observe the emergence of clonal sectors whose size corresponds to that of the corrugations, despite no physical barrier separating different areas of the biofilm. The sectors are remarkably stable and do not invade each other; we attribute this stability to the characteristics of the velocity field within the biofilm, which hinders mixing and clonal expansion. A microscopically detailed computer model fully reproduces these findings and highlights the role of mechanical interactions such as adhesion and friction in microbial evolution. The model also predicts clonal expansion to be limited even for clones with a significant growth advantage-a finding which we confirm experimentally using a mixture of antibiotic-sensitive and antibiotic-resistant mutants in the presence of sublethal concentrations of the antibiotic rifampicin. The strong suppression of selection contrasts sharply with the behavior seen in range expansion experiments in bacterial colonies grown on agar. Our results show that biofilm population dynamics can be affected by patterning the surface and demonstrate how a better understanding of the physics of bacterial growth can be used to control microbial evolution.

Directed evolution of material-producing microorganisms.

Nature is home to a variety of microorganisms that create materials under environmentally friendly conditions. While this offers an attractive approach for sustainable manufacturing, the production of materials by native microorganisms is usually slow and synthetic biology tools to engineer faster microorganisms are only available when prior knowledge of genotype-phenotype links is available. Here, we utilize a high-throughput directed evolution platform to enhance the fitness of whole microorganisms under selection pressure and identify genetic pathways to enhance the material production capabilities of native species. Using Komagataeibacter sucrofermentans as a model cellulose-producing microorganism, we show that our droplet-based microfluidic platform enables the directed evolution of these bacteria toward a small number of cellulose overproducers from an initial pool of 40,000 random mutants. Sequencing of the evolved strains reveals an unexpected link between the cellulose-forming ability of the bacteria and a gene encoding a protease complex responsible for protein turnover in the cell. The ability to enhance the fitness of microorganisms toward a specific phenotype and to unravel genotype-phenotype links makes this high-throughput directed evolution platform a promising tool for the development of new strains for the sustainable manufacturing of materials.

Flexible liquid-diode microtubes from multimodal microfluidics.

Directional transport of liquids is of great importance in energy saving, chemical/biomedical engineering, and microfluidics applications. Despite considerable progress in engineering different open surfaces to achieve liquid manipulation, the realization of diode-like liquid transport in enclosed spaces is still challenging. Here, a flexible diode microtube is presented for directional liquid transport within confined spaces using pulsed microfluidics. The microtubes exhibit sophisticated microstructures on the inner wall, replicated from a precisely controlled flow configuration in the microfluidic channel. Under the effect of asymmetric pinning and unbalanced Laplace pressure, such microtubes enable directional liquid transport in closed channels. More importantly, by integrating in situ flow lithography with the microfluidic system, segmented liquid diodes are fabricated as assembly units for the construction of fluidic-electronic circuits that perform logic operations. These results demonstrate the capacity of the present liquid-diode microtubes for flexible, directional, and programmable liquid transport. We believe that it can open an avenue for designing advanced fluidic circuit-based devices toward versatile practical applications.

Fluid inertia controls mineral precipitation and clogging in pore to network-scale flows.

Mineral precipitation caused by fluid mixing presents complex control and predictability challenges in a variety of natural and engineering processes, including carbon mineralization, geothermal energy, and microfluidics. Precipitation dynamics, particularly under the influence of fluid flow, remain poorly understood. Combining microfluidic experiments and three-dimensional reactive transport simulations, we demonstrate that fluid inertia controls mineral precipitation and clogging at flow intersections, even in laminar flows. We observe distinct precipitation regimes as a function of Reynolds number (Re). At low Reynolds numbers (Re < 10), precipitates form a thin, dense layer along the mixing interface, which shuts precipitation off, while at high Reynolds numbers (Re > 50), strong three-dimensional flows significantly enhance precipitation over the entire intersection, resulting in rapid clogging. When injection rates from two inlets are uneven, flow symmetry-breaking leads to unexpected flow bifurcation phenomena, which result in enhanced concurrent precipitation in both downstream channels. Finally, we extend our findings to rough channel networks and demonstrate that the identified inertial effects on precipitation at the intersection scale are also present and even more dramatic at the network scale. This study sheds light on the fundamental mechanisms underlying mixing-induced mineral precipitation and provides a framework for designing and optimizing processes involving mineral precipitation.

Spatiotemporally controlled microvortices provide advanced microfluidic components.

Microvortices are emerging components that impart functionality to microchannels by exploiting inertia effects such as high shear stress, effective fluid diffusion, and large pressure loss. Exploring the dynamic generation of vortices further expands the scope of microfluidic applications, including cell stimulation, fluid mixing, and transport. Despite the crucial role of vortices' development within sub-millisecond timescales, previous studies in microfluidics did not explore the modulation of the Reynolds number (Re) in the range of several hundred. In this study, we modulated high-speed flows (54 < [Formula: see text] < 456) within sub-millisecond timescales using a piezo-driven on-chip membrane pump. By applying this method to microchannels with asymmetric geometries, we successfully controlled the spatiotemporal development of vortices, adjusting their behavior in response to oscillatory flow directions. These different vortices induced different pressure losses, imparting the microchannels with direction-dependent flow resistance, mimicking a diode-like behavior. Through precise control of vortex development, we managed to regulate this direction-dependent resistance, enabling the rectification of oscillatory flow resembling a diode and the ability to switch its rectification direction. This component facilitated bidirectional flow control without the need for mechanical valves. Moreover, we demonstrated its application in microfluidic cell pipetting, enabling the isolation of single cells. Consequently, based on modulating high-speed flow, our approach offers precise control over the spatiotemporal development of vortices in microstructures, thereby introducing innovative microfluidic functionalities.

Cancer-on-a-chip model shows that the adenomatous polyposis coli mutation impairs T cell engagement and killing of cancer spheroids.

Evaluating the ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells is crucial, for instance, to predict the efficiency of cell therapy in personalized medicine. However, the destruction of a tumor by CTLs involves CTL migration in the extra-tumoral environment, accumulation on the tumor, antigen recognition, and cooperation in killing the cancer cells. Therefore, identifying the limiting steps in this complex process requires spatio-temporal measurements of different cellular events over long periods. Here, we use a cancer-on-a-chip platform to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL migration and cytotoxicity against 3D tumor spheroids. The APC mutated CTLs are found to have a reduced ability to destroy tumor spheroids compared with control cells, even though APC mutants migrate in the extra-tumoral space and accumulate on the spheroids as efficiently as control cells. Once in contact with the tumor however, mutated CTLs display reduced engagement with the cancer cells, as measured by a metric that distinguishes different modes of CTL migration. Realigning the CTL trajectories around localized killing cascades reveals that all CTLs transition to high engagement in the 2 h preceding the cascades, which confirms that the low engagement is the cause of reduced cytotoxicity. Beyond the study of APC mutations, this platform offers a robust way to compare cytotoxic cell efficiency of even closely related cell types, by relying on a multiscale cytometry approach to disentangle complex interactions and to identify the steps that limit the tumor destruction.

High-throughput measurement of elastic moduli of microfibers by rope coiling.

There are many fields where it is of interest to measure the elastic moduli of tiny fragile fibers, such as filamentous bacteria, actin filaments, DNA, carbon nanotubes, and functional microfibers. The elastic modulus is typically deduced from a sophisticated tensile test under a microscope, but the throughput is low and limited by the time-consuming and skill-intensive sample loading/unloading. Here, we demonstrate a simple microfluidic method enabling the high-throughput measurement of the elastic moduli of microfibers by rope coiling using a localized compression, where sample loading/unloading are not needed between consecutive measurements. The rope coiling phenomenon occurs spontaneously when a microfiber flows from a small channel into a wide channel. The elastic modulus is determined by measuring either the buckling length or the coiling radius. The throughput of this method, currently 3,300 fibers per hour, is a thousand times higher than that of a tensile tester. We demonstrate the feasibility of the method by testing a nonuniform fiber with axially varying elastic modulus. We also demonstrate its capability for in situ inline measurement in a microfluidic production line. We envisage that high-throughput measurements may facilitate potential applications such as screening or sorting by mechanical properties and real-time control during production of microfibers.

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

Targeted whole-genome recovery of single viral species in a complex environmental sample.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. We present an experimental method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A unique gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. We demonstrate this method's efficacy by spiking two known viral genomes, Simian virus 40 (SV40, 5,243 bp) and Human Adenovirus 5 (HAd5, 35,938 bp), into a sewage sample with a final abundance in the droplets of around 0.1% and 0.015%, respectively. We achieve 100% recovery of the complete sequence of the spiked-in SV40 genome with uniform coverage distribution. For the larger HAd5 genome, we cover approximately 99.4% of its sequence. Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables single-genome whole-genome amplification and targeting characterizations of rare viral species and will facilitate our ability to access the mutational profile in single-virus genomes and contribute to an improved understanding of viral ecology.

High-throughput quantification of red blood cell deformability and oxygen saturation to probe mechanisms of sickle cell disease.

The complex, systemic pathology of sickle cell disease is driven by multiple mechanisms including red blood cells (RBCs) stiffened by polymerized fibers of deoxygenated sickle hemoglobin. A critical step toward understanding the pathologic role of polymer-containing RBCs is quantifying the biophysical changes in these cells in physiologically relevant oxygen environments. We have developed a microfluidic platform capable of simultaneously measuring single RBC deformability and oxygen saturation under controlled oxygen and shear stress. We found that RBCs with detectable amounts of polymer have decreased oxygen affinity and decreased deformability. Surprisingly, the deformability of the polymer-containing cells is oxygen-independent, while the fraction of these cells increases as oxygen decreases. We also find that some fraction of these cells is present at most physiologic oxygen tensions, suggesting a role for these cells in the systemic pathologies. Additionally, the ability to measure these pathological cells should provide clearer targets for evaluating therapies.

Triggering interfacial instabilities during forced imbibition by adjusting the aspect ratio in depth-variable microfluidic porous media.

We present a comprehensive description of the aspect ratio impact on interfacial instability in porous media where a wetting liquid displaces a nonwetting fluid. Building on microfluidic experiments, we evidence imbibition scenarios yielding interfacial instabilities and macroscopic morphologies under different depth confinements, which were controlled by aspect ratio and capillary number. We report a phenomenon whereby a smaller aspect ratio of depth-variable microfluidic porous media and lower capillary number trigger interfacial instability during forced imbibition; otherwise, a larger aspect ratio of uniform-depth microfluidic porous media and higher capillary number will suppress the interfacial instability, which seemingly ignored or contradicts conventional expectations with compact and faceted growth during imbibition. Pore-scale theoretical analytical models, numerical simulations, as well as microfluidic experiments were combined for characteristics of microscopic interfacial dynamics and macroscopic displacement results as a function of aspect ratio, depth variation, and capillary number. Our results present a complete dynamic view of the imbibition process over a full range of regimes from interfacial stabilization to destabilization. We predict the mode of imbibition in porous media based on pore-scale interfacial behavior, which fits well with microfluidic experiments. The study provides insights into the role of aspect ratio in controlling interfacial instabilities in microfluidic porous media. The finding provides design or prediction principles for engineered porous media, such as microfluidic devices, membranes, fabric, exchange columns, and even soil and rocks concerning their desired immiscible imbibition behavior.

Shear rate sensitizes bacterial pathogens to H2O2 stress.

Cells regularly experience fluid flow in natural systems. However, most experimental systems rely on batch cell culture and fail to consider the effect of flow-driven dynamics on cell physiology. Using microfluidics and single-cell imaging, we discover that the interplay of physical shear rate (a measure of fluid flow) and chemical stress trigger a transcriptional response in the human pathogen Pseudomonas aeruginosa. In batch cell culture, cells protect themselves by quickly scavenging the ubiquitous chemical stressor hydrogen peroxide (H2O2) from the media. In microfluidic conditions, we observe that cell scavenging generates spatial gradients of H2O2. High shear rates replenish H2O2, abolish gradients, and generate a stress response. Combining mathematical simulations and biophysical experiments, we find that flow triggers an effect like "wind-chill" that sensitizes cells to H2O2 concentrations 100 to 1,000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H2O2 concentration required to generate a transcriptional response closely match their respective values in the human bloodstream. Thus, our results explain a long-standing discrepancy between H2O2 levels in experimental and host environments. Finally, we demonstrate that the shear rate and H2O2 concentration found in the human bloodstream trigger gene expression in the blood-relevant human pathogen Staphylococcus aureus, suggesting that flow sensitizes bacteria to chemical stress in natural environments.

Rapid formation of bioaggregates and morphology transition to biofilm streamers induced by pore-throat flows.

Bioaggregates are condensed porous materials comprising microbes, organic and inorganic matters, and water. They are commonly found in natural and engineered porous media and often cause clogging. Despite their importance, the formation mechanism of bioaggregates in porous media systems is largely unknown. Through microfluidic experiments and direct numerical simulations of fluid flow, we show that the rapid bioaggregation is driven by the interplay of the viscoelastic nature of biomass and hydrodynamic conditions at pore throats. At an early stage, unique flow structures around a pore throat promote the biomass attachment at the throat. Then, the attached biomass fluidizes when the shear stress at the partially clogged pore throat reaches a critical value. After the fluidization, the biomass is displaced and accumulated in the expansion region of throats forming bioaggregates. We further find that such criticality in shear stress triggers morphological changes in bioaggregates from rounded- to streamer-like shapes. This knowledge was used to control the clogging of throats by tuning the flow conditions: When the shear stress at the throat exceeded the critical value, clogging was prevented. The bioaggregation process did not depend on the detailed pore-throat geometry, as we reproduced the same dynamics in various pore-throat geometries. This study demonstrates that pore-throat structures, which are ubiquitous in porous media systems, induce bioaggregation and can lead to abrupt disruptions in flow.

Molecular responses during bacterial filamentation reveal inhibition methods of drug-resistant bacteria.

Bacterial antimicrobial resistance (AMR) is among the most significant challenges to current human society. Exposing bacteria to antibiotics can activate their self-saving responses, e.g., filamentation, leading to the development of bacterial AMR. Understanding the molecular changes during the self-saving responses can reveal new inhibition methods of drug-resistant bacteria. Herein, we used an online microfluidics mass spectrometry system for real-time characterization of metabolic changes of bacteria during filamentation under the stimulus of antibiotics. Significant pathways, e.g., nucleotide metabolism and coenzyme A biosynthesis, correlated to the filamentation of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) were identified. A cyclic dinucleotide, c-di-GMP, which is derived from nucleotide metabolism and reported closely related to bacterial resistance and tolerance, was observed significantly up-regulated during the bacterial filamentation. By using a chemical inhibitor, ebselen, to inhibit diguanylate cyclases which catalyzes the synthesis of c-di-GMP, the minimum inhibitory concentration of ceftriaxone against ESBL-E. coli was significantly decreased. This inhibitory effect was also verified with other ESBL-E. coli strains and other beta-lactam antibiotics, i.e., ampicillin. A mutant strain of ESBL-E. coli by knocking out the dgcM gene was used to demonstrate that the inhibition of the antibiotic resistance to beta-lactams by ebselen was mediated through the inhibition of the diguanylate cyclase DgcM and the modulation of c-di-GMP levels. Our study uncovers the molecular changes during bacterial filamentation and proposes a method to inhibit antibiotic-resistant bacteria by combining traditional antibiotics and chemical inhibitors against the enzymes involved in bacterial self-saving responses.