Cell tumbling enhances stem cell differentiation in hydrogels via nuclear mechanotransduction.

Cells can deform their local niche in three dimensions via whole-cell movements such as spreading, migration or volume expansion. These behaviours, occurring over hours to days, influence long-term cell fates including differentiation. Here we report a whole-cell movement that occurs in sliding hydrogels at the minutes timescale, termed cell tumbling, characterized by three-dimensional cell dynamics and hydrogel deformation elicited by heightened seconds-to-minutes-scale cytoskeletal and nuclear activity. Studies inhibiting or promoting the cell tumbling of mesenchymal stem cells show that this behaviour enhances differentiation into chondrocytes. Further, it is associated with a decrease in global chromatin accessibility, which is required for enhanced differentiation. Cell tumbling also occurs during differentiation into other lineages and its differentiation-enhancing effects are validated in various hydrogel platforms. Our results establish that cell tumbling is an additional regulator of stem cell differentiation, mediated by rapid niche deformation and nuclear mechanotransduction.

The lysogenic filamentous Pseudomonas bacteriophage phage Pf slows mucociliary transport.

Pseudomonas aeruginosa is a major pulmonary pathogen causing chronic pulmonary infections in people with cystic fibrosis (CF). The P. aeruginosa filamentous and lysogenic bacteriophage, Pf phage, is abundant in the airways of many people with CF and has been associated with poor outcomes in a cross-sectional cohort study. Previous studies have identified roles for Pf phage in biofilm formation, specifically forming higher-order birefringent, liquid crystals when in contact with other biopolymers in biofilms. Liquid crystalline biofilms are more adherent and viscous than those without liquid crystals. A key feature of biofilms is to enhance bacterial adherence and resist physical clearance. The effect of Pf phage on mucociliary transport is unknown. We found that primary CF and non-CF nasal epithelial cells cultured at air-liquid interface treated with Pf phage exhibit liquid crystalline structures in the overlying mucus. On these cell cultures, Pf phage entangles cilia but does not affect ciliary beat frequency. In both these in vitro cell cultures and in an ex vivo porcine trachea model, introduction of Pf phage decreases mucociliary transport velocity. Pf phage also blocks the rescue of mucociliary transport by CF transmembrane conductance regulator modulators in CF cultures. Thus, Pf phage may contribute to the pathogenesis of P. aeruginosa-associated CF lung disease via induction of liquid crystalline characteristics to airway secretions, leading to impaired mucociliary transport. Targeting Pf phage may be useful in treatment CF as well as other settings of chronic P. aeruginosa infections.

Air-liquid intestinal cell culture allows in situ rheological characterization of intestinal mucus.

Intestinal health heavily depends on establishing a mucus layer within the gut with physical properties that strike a balance between being sufficiently elastic to keep out harmful pathogens yet viscous enough to flow and turnover the contents being digested. Studies investigating dysfunction of the mucus layer in the intestines are largely confined to animal models, which require invasive procedures to collect the mucus fluid. In this work, we develop a nondestructive method to study intestinal mucus. We use an air-liquid interface culture of primary human intestinal epithelial cells that exposes their apical surface to allow in situ analysis of the mucus layer. Mucus collection is not only invasive but also disrupts the mucus microstructure, which plays a crucial role in the interaction between mucus and the gut microbiome. Therefore, we leverage a noninvasive rheology technique that probes the mechanical properties of the mucus without removal from the culture. Finally, to demonstrate biomedical uses for this cell culture system, we characterize the biochemical and biophysical properties of intestinal mucus due to addition of the cytokine IL-13 to recapitulate the gut environment of Nippostrongylus brasiliensis infection.

Rheological Characterization and Theoretical Modeling Establish Molecular Design Rules for Tailored Dynamically Associating Polymers.

Dynamically associating polymers have long been of interest due to their highly tunable viscoelastic behavior. Many applications leverage this tunability to create materials that have specific rheological properties, but designing such materials is an arduous, iterative process. Current models for dynamically associating polymers are phenomenological, assuming a structure for the relationship between association kinetics and network relaxation. We present the Brachiation model, a molecular-level theory of a polymer network with dynamic associations that is rooted in experimentally controllable design parameters, replacing the iterative experimental process with a predictive model for how experimental modifications to the polymer will impact rheological behavior. We synthesize hyaluronic acid chains modified with supramolecular host-guest motifs to serve as a prototypical dynamic network exhibiting tunable physical properties through control of polymer concentration and association rates. We use dynamic light scattering microrheology to measure the linear viscoelasticity of these polymers across six decades in frequency and fit our theory parameters to the measured data. The parameters are then altered by a magnitude corresponding to changes made to the experimental parameters and used to obtain new rheological predictions that match the experimental results well, demonstrating the ability for this theory to inform the design process of dynamically associating polymeric materials.

Bacteriophage and Bacterial Susceptibility, Resistance, and Tolerance to Antibiotics.

Bacteriophages, viruses that infect and replicate within bacteria, impact bacterial responses to antibiotics in complex ways. Recent studies using lytic bacteriophages to treat bacterial infections (phage therapy) demonstrate that phages can promote susceptibility to chemical antibiotics and that phage/antibiotic synergy is possible. However, both lytic and lysogenic bacteriophages can contribute to antimicrobial resistance. In particular, some phages mediate the horizontal transfer of antibiotic resistance genes between bacteria via transduction and other mechanisms. In addition, chronic infection filamentous phages can promote antimicrobial tolerance, the ability of bacteria to persist in the face of antibiotics. In particular, filamentous phages serve as structural elements in bacterial biofilms and prevent the penetration of antibiotics. Over time, these contributions to antibiotic tolerance favor the selection of resistance clones. Here, we review recent insights into bacteriophage contributions to antibiotic susceptibility, resistance, and tolerance. We discuss the mechanisms involved in these effects and address their impact on bacterial fitness.

Biochemical, biophysical, and immunological characterization of respiratory secretions in severe SARS-CoV-2 infections.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e., resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percentages of solids and protein content were greatly elevated in COVID-19 compared with heathy control samples and closely resembled levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) were major components of respiratory secretions in COVID-19 and were likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors, with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observed increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor-stimulated gene-6 staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicated that increases in HA and DNA in COVID-19 respiratory secretion samples correlated with enhanced inflammatory burden and suggested that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Biochemical, Biophysical, and Immunological Characterization of Respiratory Secretions in Severe SARS-CoV-2 (COVID-19) Infections.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We find the percent solids and protein content are greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibit heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observe increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factorâ€"stimulated gene-6 (TSG6) staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicate that increases in HA and DNA in COVID-19 respiratory secretion samples correlate with enhanced inflammatory burden and suggest that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids.

Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin-like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue-derived, epithelial-only intestinal organoids. HELP enables the encapsulation of dissociated patient-derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal-derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin-ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G' ≈ 1 kPa), slower stress relaxation rate (t1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10-3-1 × 10-3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal-derived products or synthetic polyethylene glycol for potential clinical translation.

Microrheology reveals simultaneous cell-mediated matrix stiffening and fluidization that underlie breast cancer invasion.

Living tissues embody a unique class of hybrid materials in which active and thermal forces are inextricably linked. Mechanical characterization of tissues demands descriptors that respect this hybrid nature. In this work, we develop a microrheology-based force spectrum analysis (FSA) technique to dissect the active and passive fluctuations of the extracellular matrix (ECM) in three-dimensional (3D) cell culture models. In two different stromal models and a 3D breast cancer spheroid model, our FSA reveals emergent hybrid dynamics that involve both high-frequency stress stiffening and low-frequency fluidization of the ECM. We show that this is a general consequence of nonlinear coupling between active forces and the frequency-dependent viscoelasticity of stress-stiffening networks. In 3D breast cancer spheroids, this dual active stiffening and fluidization is tightly connected with invasion. Our results suggest a mechanism whereby breast cancer cells reconcile the seemingly contradictory requirements for both tension and malleability in the ECM during invasion.

Dynamic light scattering microrheology for soft and living materials.

We present a method for using dynamic light scattering in the single-scattering limit to measure the viscoelastic moduli of soft materials. This microrheology technique only requires a small sample volume of 12 µL to measure up to six decades in time of rheological behavior. We demonstrate the use of dynamic light scattering microrheology (DLSµR) on a variety of soft materials, including dilute polymer solutions, covalently-crosslinked polymer gels, and active, biological fluids. In this work, we detail the procedure for applying the technique to new materials and discuss the critical considerations for implementing the technique, including a custom analysis script for analyzing data output. We focus on the advantages of applying DLSµR to biologically relevant materials: breast cancer cells encapsulated in a collagen gel and cystic fibrosis sputum. DLSµR is an easy, efficient, and economical rheological technique that can guide the design of new polymeric materials and facilitate the understanding of the underlying physics governing behavior of naturally derived materials.

Biochemical and Biophysical Characterization of Respiratory Secretions in Severe SARS-CoV-2 (COVID-19) Infections.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percent solids and protein content are all greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis (CF), a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. These results highlight the dramatic biophysical properties of COVID-19 respiratory secretions and suggest that DNA and hyaluronan may be viable therapeutic targets in COVID-19 infection.

Brachiation of a polymer chain in the presence of a dynamic network.

The viscoelastic behavior of a physically crosslinked gel involves a spectrum of molecular relaxation processes, which at the single-chain level involve the chain undergoing transient hand-to-hand motion through the network. We develop a self-consistent theory for describing transiently associating polymer solutions that captures these complex dynamics. A single polymer chain transiently binds to a viscoelastic background that represents the polymer network formed by surrounding polymer chains. The viscoelastic background is described in the equation of motion as a memory kernel, which is self-consistently determined based on the predicted rheological behavior from the chain itself. The solution to the memory kernel is translated into rheological predictions of the complex modulus over a wide range of frequencies to capture the time-dependent behavior of a physical gel. Using the loss tangent predictions, a phase diagram is shown for the sol-gel transition of polymers with dynamic association affinities. This theory provides a predictive, molecular-level framework for the design of associating gels and supramolecular assemblies with targeted rheological properties.