Tunable Single-Cell Extraction for Molecular Analyses.

Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level.

Encapsulated bacteria deform lipid vesicles into flagellated swimmers.

We study a synthetic system of motile Escherichia coli bacteria encapsulated inside giant lipid vesicles. Forces exerted by the bacteria on the inner side of the membrane are sufficient to extrude membrane tubes filled with one or several bacteria. We show that a physical coupling between the membrane tube and the flagella of the enclosed cells transforms the tube into an effective helical flagellum propelling the vesicle. We develop a simple theoretical model to estimate the propulsive force from the speed of the vesicles and demonstrate the good efficiency of this coupling mechanism. Together, these results point to design principles for conferring motility to synthetic cells.

Characterization and Control of the Run-and-Tumble Dynamics of Escherichia Coli.

We characterize the full spatiotemporal gait of populations of swimming Escherichia coli using renewal processes to analyze the measurements of intermediate scattering functions. This allows us to demonstrate quantitatively how the persistence length of an engineered strain can be controlled by a chemical inducer and to report a controlled transition from perpetual tumbling to smooth swimming. For wild-type E. coli, we measure simultaneously the microscopic motility parameters and the large-scale effective diffusivity, hence quantitatively bridging for the first time small-scale directed swimming and macroscopic diffusion.

Correction: Sizing multimodal suspensions with differential dynamic microscopy.

Correction for 'Sizing multimodal suspensions with differential dynamic microscopy' by Joe J. Bradley et al., Soft Matter, 2023, 19, 8179-8192, https://doi.org/10.1039/D3SM00593C.

Sizing multimodal suspensions with differential dynamic microscopy.

Differential dynamic microscopy (DDM) can be used to extract the mean particle size from videos of suspensions. However, many suspensions have multimodal particle size distributions, for which a single 'mean' is not a sufficient description. After clarifying how different particle sizes contribute to the signal in DDM, we show that standard DDM analysis can extract the mean sizes of two populations in a bimodal suspension given prior knowledge of the sample's bimodality. Further, the use of the CONTIN algorithm obviates the need for such prior knowledge. Finally, we show that by selectively analysing portions of the DDM images, we can size a trimodal suspension where the large particles would otherwise dominate the signal, again without prior knowledge of the trimodality.

A combined rheometry and imaging study of viscosity reduction in bacterial suspensions.

Suspending self-propelled "pushers" in a liquid lowers its viscosity. We study how this phenomenon depends on system size in bacterial suspensions using bulk rheometry and particle-tracking rheoimaging. Above the critical bacterial volume fraction needed to decrease the viscosity to zero, [Formula: see text], large-scale collective motion emerges in the quiescent state, and the flow becomes nonlinear. We confirm a theoretical prediction that such instability should be suppressed by confinement. Our results also show that a recent application of active liquid-crystal theory to such systems is untenable.

Metaheuristic for Optimal Dynamic K-Coloring Application on Band Sharing for Automotive Radars.

The number of vehicles equipped with radars on the road has been increasing for years and is expected to reach 50% of cars by 2030. This rapid rise in radars will likely increase the risk of harmful interference, especially since radar specifications from standardization bodies (e.g., ETSI) provide requirements in terms of maximum transmit power but do no mandate specific radar waveform parameters nor channel access scheme policies. Techniques for interference mitigation are thus becoming very important to ensure the long-term correct operation of radars and upper-layer ADAS systems that depend on them in this complex environment. In our previous work, we have shown that organizing the radar band into time-frequency resources that do not interfere with each other vastly reduces the amount of interference by facilitating band sharing. In this paper, a metaheuristic is presented to find the optimal resource sharing between radars, knowing their relative positions and thereby the line-of-sight and non-line-of-sight interference risks during a realistic scenario. The metaheuristic aims at optimally minimizing interference while minimizing the number of resource changes that radars have to make. It is a centralized approach where everything about the system is known (e.g., the past and future positions of the vehicles). This and the high computational load induce that this algorithm is not meant to be used in real-time. However, the metaheuristic approach can be extremely useful for finding near optimal solutions in simulations, allowing for the extraction of efficient patterns, or as data generation for machine learning.

Analysis of Co-Channel Coexistence Mitigation Methods Applied to IEEE 802.11p and 5G NR-V2X Sidelink.

Direct communication between vehicles and surrounding objects, called vehicle-to-everything (V2X), is ready for the market and promises to raise the level of safety and comfort while driving. To this aim, specific bands have been reserved in some countries worldwide and different wireless technologies have been developed; however, these are not interoperable. Recently, the issue of co-channel coexistence has been raised, leading the European Telecommunications Standards Institute (ETSI) to propose a number of solutions, called mitigation methods, for the coexistence of the IEEE 802.11p based ITS-G5 and the 3GPP fourth generation (4G) long term evolution (LTE)-V2X sidelink. In this work, several of the envisioned alternatives are investigated when adapted to the coexistence of the IEEE 802.11p with its enhancement IEEE 802.11bd and the latest 3GPP standards, i.e., the fifth generation (5G) new radio (NR)-V2X. The results, obtained through an open-source simulator that is shared with the research community for the evaluation of additional proposals, show that the methods called A and C, which require modifications to the standards, improve the transmission range of one or both systems without affecting the other, at least in low-density scenarios.

Run-to-Tumble Variability Controls the Surface Residence Times of E. coli Bacteria.

Motile bacteria are known to accumulate at surfaces, eventually leading to changes in bacterial motility and biofilm formation. We use a novel two-color, three-dimensional Lagrangian tracking technique to follow simultaneously the body and the flagella of a wild-type Escherichia coli. We observe long surface residence times and surface escape corresponding mostly to immediately antecedent tumbling. A motility model accounting for a large behavioral variability in run-time duration reproduces all experimental findings and gives new insights into surface trapping efficiency.

Probing the dynamics of turbid colloidal suspensions using differential dynamic microscopy.

Few techniques can reliably measure the dynamics of colloidal suspensions or other soft materials over a wide range of turbidities. Here we systematically investigate the capability of Differential Dynamic Microscopy (DDM) to characterise particle dynamics in turbid colloidal suspensions based on brightfield optical microscopy. We measure the Intermediate Scattering Function (ISF) of polystyrene microspheres suspended in water over a range of concentrations, turbidities, and up to 4 orders of magnitude in time-scales. These DDM results are compared to data obtained from both Dynamic Light Scattering (DLS) and Two-colour Dynamic Light Scattering (TCDLS). The latter allows for suppression of multiple scattering for moderately turbid suspensions. We find that DDM can obtain reliable diffusion coefficients at up to 10 and 1000 times higher particle concentrations than TCDLS and standard DLS, respectively. Additionally, we investigate the roles of the four length-scales relevant when imaging a suspension: the sample thickness L, the imaging depth z, the imaging depth of field DoF, and the photon mean free path . More detailed experiments and analysis reveal the appearance of a short-time process as turbidity is increased, which we associate with multiple scattering events within the imaging depth of the field. The long-time process corresponds to the particle dynamics from which particle-size can be estimated in the case of non-interacting particles. Finally, we provide a simple theoretical framework, ms-DDM, for turbid samples, which accounts for multiple scattering.

A Methodology for Abstracting the Physical Layer of Direct V2X Communications Technologies.

Recent advancements in vehicle-to-everything (V2X) communications have greatly increased the flexibility of the physical (PHY) and medium access control (MAC) layers. This increases the complexity when investigating the system from a network perspective to evaluate the performance of the supported applications. Such flexibility, in fact, needs to be taken into account through a cross-layer approach, which might lead to challenging evaluation processes. As an accurate simulation of the signals appears unfeasible, a typical solution is to rely on simple models for incorporating the PHY layer of the supported technologies based on off-line measurements or accurate link-level simulations. Such data are, however, limited to a subset of possible configurations, and extending them to others is costly when not even impossible. The goal of this paper is to develop a new approach for modeling the PHY layer of V2X communications that can be extended to a wide range of configurations without leading to extensive measurement or simulation campaigns at the link layer. In particular, given a scenario and starting from results in terms of the packet error rate (PER) vs. signal-to-interference-plus-noise ratio (SINR) related to a subset of possible configurations, we first approximated the curves with step functions characterized by a given SINR threshold, and we then derived one parameter, called implementation loss, that was used to obtain the SINR threshold and evaluate the network performance under any configuration in the same scenario. The proposed methodology, leading to a good trade-off among the complexity, generality, and accuracy of the performance evaluation process, was validated through extensive simulations with both IEEE 802.11p and LTE-V2X sidelink technologies in various scenarios. The results first show that the curves can be effectively approximated by using an SINR threshold, with a value corresponding to 0.5 PER, and then demonstrate that the network-level outputs derived from the proposed approach are very close to those obtained with complete curves, despite not being restricted to a few possible configurations.

Hook length of the bacterial flagellum is optimized for maximal stability of the flagellar bundle.

Most bacteria swim in liquid environments by rotating one or several flagella. The long external filament of the flagellum is connected to a membrane-embedded basal body by a flexible universal joint, the hook, which allows the transmission of motor torque to the filament. The length of the hook is controlled on a nanometer scale by a sophisticated molecular ruler mechanism. However, why its length is stringently controlled has remained elusive. We engineered and studied a diverse set of hook-length variants of Salmonella enterica. Measurements of plate-assay motility, single-cell swimming speed, and directional persistence in quasi-2D and population-averaged swimming speed and body angular velocity in 3D revealed that the motility performance is optimal around the wild-type hook length. We conclude that too-short hooks may be too stiff to function as a junction and too-long hooks may buckle and create instability in the flagellar bundle. Accordingly, peritrichously flagellated bacteria move most efficiently as the distance travelled per body rotation is maximal and body wobbling is minimized. Thus, our results suggest that the molecular ruler mechanism evolved to control flagellar hook growth to the optimal length consistent with efficient bundle formation. The hook-length control mechanism is therefore a prime example of how bacteria evolved elegant but robust mechanisms to maximize their fitness under specific environmental constraints.

Characterising shear-induced dynamics in flowing complex fluids using differential dynamic microscopy.

Microscopic dynamics reveal the origin of the bulk rheological response in complex fluids. In model systems particle motion can be tracked, but for industrially relevant samples this is often impossible. Here we adapt differential dynamic microscopy (DDM) to study flowing highly-concentrated samples without particle resolution. By combining an investigation of oscillatory flow, using a novel "echo-DDM" analysis, and steady shear, through flow-DDM, we characterise the yielding of a silicone oil emulsion on both the microscopic and bulk level. Through measuring the rate of shear-induced droplet rearrangements and the flow velocity, the transition from a solid-like to liquid-like state is shown to occur in two steps: with droplet mobilisation marking the limit of linear visco-elasticity, followed by the development of shear localisation and macroscopic yielding. Using this suite of techniques, such insight could be developed for a wide variety of challenging complex fluids.

Particle sizing for flowing colloidal suspensions using flow-differential dynamic microscopy.

Particle size is a key variable in understanding the behaviour of the particulate products that underpin much of our modern lives. Typically obtained from suspensions at rest, measuring the particle size under flowing conditions would enable advances for in-line testing during manufacture and high-throughput testing during development. However, samples are often turbid, multiply scattering light and preventing the direct use of common sizing techniques. Differential dynamic microscopy (DDM) is a powerful technique for analysing video microscopy of such samples, measuring diffusion and hence particle size without the need to resolve individual particles while free of substantial user input. However, when applying DDM to a flowing sample, diffusive dynamics are rapidly dominated by flow effects, preventing particle sizing. Here, we develop "flow-DDM", a novel analysis scheme that combines optimised imaging conditions, a drift-velocity correction and modelling of the impact of flow. Flow-DDM allows a decoupling of flow from diffusive motion that facilitates successful particle size measurements at flow speeds an order of magnitude higher than for DDM. We demonstrate the generality of the technique by applying flow-DDM to two separate microscopy methods and flow geometries.

Osmotaxis in Escherichia coli through changes in motor speed.

Bacterial motility, and in particular repulsion or attraction toward specific chemicals, has been a subject of investigation for over 100 years, resulting in detailed understanding of bacterial chemotaxis and the corresponding sensory network in many bacterial species. For Escherichia coli most of the current understanding comes from the experiments with low levels of chemotactically active ligands. However, chemotactically inactive chemical species at concentrations found in the human gastrointestinal tract produce significant changes in E. coli's osmotic pressure and have been shown to lead to taxis. To understand how these nonspecific physical signals influence motility, we look at the response of individual bacterial flagellar motors under stepwise changes in external osmolarity. We combine these measurements with a population swimming assay under the same conditions. Unlike for chemotactic response, a long-term increase in swimming/motor speeds is observed, and in the motor rotational bias, both of which scale with the osmotic shock magnitude. We discuss how the speed changes we observe can lead to steady-state bacterial accumulation.

Coiled Coil Affinity-Based Systems for the Controlled Release of Biofunctionalized Gold Nanoparticles from Alginate Hydrogels.

Affinity-based systems represent a promising solution to control the delivery of therapeutics using hydrogels. Here, we report a hybrid system that is based on the peptidic E/K coiled coil affinity pair to mediate the release of gold nanoparticles (NPs) from alginate scaffolds. On one hand, the gold NPs were functionalized with the Ecoil-tagged epidermal growth factor (EGF). The bioactivity of the grafted EGF and the bioavailability of the Ecoil moiety were confirmed by EGF receptor phosphorylation assays and by capturing the functionalized NPs on a Kcoil-derivatized surface. On the other hand, alginate chains were modified with azido-homoalanine Kcoil (Aha-Kcoil) by azide-alkyne click chemistry. The hybrid system was formed by dispersing NPs functionalized with the Ecoil-tagged EGF in alginate hydrogels containing either 0, 10, or 20% of Kcoil-modified alginate (Alg-Kcoil). With 20% of Alg-Kcoil, the release of Ecoil-functionalized NPs was reduced by half when compared to the release of NPs without Ecoil, whereas little to no differences were noticed with either 0 or 10% of Alg-Kcoil. Differential dynamic microscopy was used to determine the diffusion coefficient of the NPs, and the results showed a decrease in the diffusion coefficient of Ecoil-functionalized NPs, when compared to bare PEGylated NPs. Altogether, our data demonstrated that the E/K coiled coil system can control the release of NPs in a high Kcoil content alginate gel, opening diverse applications in drug delivery.

Dynamic optical rectification and delivery of active particles.

We use moving light patterns to control the motion of Escherichia coli bacteria whose motility is photo-activated. Varying the pattern speed controls the magnitude and direction of the bacterial flux, and therefore the accumulation of cells in up- and down-stream reservoirs. We validate our results with two-dimensional simulations and a 1-dimensional analytic model, and use these to explore parameter space. We find that cell accumulation is controlled by a competition between directed flux and undirected, stochastic transport. Our results point to a number of design principles for using moving light patterns and light-activated micro-swimmers in a range of practical applications.

Probing the Spatiotemporal Dynamics of Catalytic Janus Particles with Single-Particle Tracking and Differential Dynamic Microscopy.

We demonstrate differential dynamic microscopy and particle tracking for the characterization of the spatiotemporal behavior of active Janus colloids in terms of the intermediate scattering function (ISF). We provide an analytical solution for the ISF of the paradigmatic active Brownian particle model and find striking agreement with experimental results from the smallest length scales, where translational diffusion and self-propulsion dominate, up to the largest ones, which probe effective diffusion due to rotational Brownian motion. At intermediate length scales, characteristic oscillations resolve the crossover between directed motion to orientational relaxation and allow us to discriminate active Brownian motion from other reorientation processes, e.g., run-and-tumble motion. A direct comparison to theoretical predictions reliably yields the rotational and translational diffusion coefficients of the particles, the mean and width of their speed distribution, and the temporal evolution of these parameters.

Stakeholder engagement in diabetes self-management: patient preference for peer support and other insights.

Background: Self-management support (SMS) for patients with diabetes can improve adherence to treatment, mitigate disease-related distress, and improve health outcomes. Translating this evidence into real-world practice is needed, as it is not clear which SMS models are acceptable to patients, and feasible and sustainable for primary care practices. Objective: To use the Boot Camp Translation (BCT) method to engage patient, practice, community resource and research stakeholders in translation of evidence about SMS and diabetes distress into mutually acceptable care models and to inform patient-centred outcomes research (PCOR). Participants: Twenty-seven diabetes care stakeholders, including patients and providers from a local network of federally qualified health centres participated. Methods: Stakeholders met in-person and by conference call over the course of 8 months. Subject matter experts provided education on the diabetes SMS evidence. Facilitators engaged the group in discussions about barriers to self-management and opportunities for improving delivery of SMS. Key Results: BCT participants identified lack of social support, personal resources, trust, knowledge and confidence as barriers to diabetes self-management. Intervention opportunities emphasized peer support, use of multidisciplinary care teams and centralized systems for sharing information about community and practice resources. BCT informed new services and a PCOR study proposal. Conclusions: Patients and family engaged in diabetes care research value peer support, group visits, and multidisciplinary care teams as key features of SMS models. SMS should be tailored to an individual patient's health literacy. BCT can be used to engage multiple stakeholders in translation of evidence into practice and to inform PCOR.

Flagellated bacterial motility in polymer solutions.

It is widely believed that the swimming speed, v, of many flagellated bacteria is a nonmonotonic function of the concentration, c, of high-molecular-weight linear polymers in aqueous solution, showing peaked v(c) curves. Pores in the polymer solution were suggested as the explanation. Quantifying this picture led to a theory that predicted peaked v(c) curves. Using high-throughput methods for characterizing motility, we measured v and the angular frequency of cell body rotation, Ω, of motile Escherichia coli as a function of polymer concentration in polyvinylpyrrolidone (PVP) and Ficoll solutions of different molecular weights. We find that nonmonotonic v(c) curves are typically due to low-molecular-weight impurities. After purification by dialysis, the measured v(c) and Ω(c) relations for all but the highest-molecular-weight PVP can be described in detail by Newtonian hydrodynamics. There is clear evidence for non-Newtonian effects in the highest-molecular-weight PVP solution. Calculations suggest that this is due to the fast-rotating flagella seeing a lower viscosity than the cell body, so that flagella can be seen as nano-rheometers for probing the non-Newtonian behavior of high polymer solutions on a molecular scale.