A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease.

Intestinal handling of dietary proteins usually prevents local inflammatory and immune responses and promotes oral tolerance. However, in ~ 1% of the world population, gluten proteins from wheat and related cereals trigger an HLA DQ2/8-restricted TH1 immune and antibody response leading to celiac disease. Prior epithelial stress and innate immune activation are essential for breaking oral tolerance to the gluten component gliadin. How gliadin subverts host intestinal mucosal defenses remains elusive. Here, we show that the α-gliadin-derived LGQQQPFPPQQPY peptide (P31-43) inhibits the function of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel pivotal for epithelial adaptation to cell-autonomous or environmental stress. P31-43 binds to, and reduces ATPase activity of, the nucleotide-binding domain-1 (NBD1) of CFTR, thus impairing CFTR function. This generates epithelial stress, tissue transglutaminase and inflammasome activation, NF-κB nuclear translocation and IL-15 production, that all can be prevented by potentiators of CFTR channel gating. The CFTR potentiator VX-770 attenuates gliadin-induced inflammation and promotes a tolerogenic response in gluten-sensitive mice and cells from celiac patients. Our results unveil a primordial role for CFTR as a central hub orchestrating gliadin activities and identify a novel therapeutic option for celiac disease.

Compressional stress stiffening & softening of soft hydrogels - how to avoid artefacts in their rheological characterisation.

Hydrogels have been successfully employed as analogues of the extracellular matrix to study biological processes such as cells' migration, growth, adhesion and differentiation. These are governed by many factors, including the mechanical properties of hydrogels; yet, a one-to-one correlation between the viscoelastic properties of gels and cell fate is still missing from literature. In this work we provide experimental evidence supporting a possible explanation for the persistence of this knowledge gap. In particular, we have employed common tissues' surrogates such as polyacrylamide and agarose gels to elucidate a potential pitfall occurring when performing rheological characterisations of soft-materials. The issue is related to (i) the normal force applied to the samples prior to performing the rheological measurements, which may easily drive the outcomes of the investigation outside the materials' linear viscoelastic regime, especially when tests are performed with (ii) geometrical tools having unbefitting dimensions (i.e., too small). We corroborate that biomimetic hydrogels can show either compressional stress softening or stiffening, and we provide a simple solution to quench these undesired phenomena, which would likely lead to potentially misleading conclusions if they were not mitigated by a good practice in performing rheological measurements, as elucidated in this work.

Effect of surfactant concentration on diffusion and microstructure in water-in-oil emulsions studied by low-field benchtop NMR and optical microscopy.

Emulsions are ubiquitous in many consumer products, including food, cosmetics and pharmaceuticals. Whilst their macroscopic characterisation is well-established, understanding their microscopic behaviour is very challenging. In our previous work we investigated oil-in-water emulsions by studying the effect of water on structuring and dynamics of such systems. In the present work, we investigate the effect of surfactant concentration on microstructure and diffusion within the water-in-oil emulsion system by using low-field pulsed-field gradient (PFG) NMR studies carried out with a benchtop NMR instrument, in conjunction with optical imaging. The results reveal that at high surfactant concentration the formation of smaller droplets gives rise to a third component in the PFG NMR attenuation plot, which is mostly attributed to restricted diffusion near the droplet boundaries. In addition, structuring effects due to increase in surfactant concentration at the boundaries could also contribute to further slowing down water diffusion at the boundaries. As the surfactant concentration decreases, the average droplet size becomes larger and both restriction and structuring effects at the droplet boundaries become less significant, as suggested by the PFG NMR plot, whereby the presence of a third diffusion component becomes less pronounced.

Correction: Post-liquefaction normospermic human semen behaves as a weak-gel viscoelastic fluid.

Correction for 'Post-liquefaction normospermic human semen behaves as a weak-gel viscoelastic fluid' by Giovanna Tomaiuolo et al., Soft Matter, 2023, https://doi.org/10.1039/d3sm00443k.

Post-liquefaction normospermic human semen behaves as a weak-gel viscoelastic fluid.

The rheological behavior of human semen is overlooked and essentially unexplored in the scientific literature. Here, we provide the first quantitative experimental evidence that post-liquafaction normospermic human semen behaves as a viscoelastic fluid and the shear moduli can be scaled according to the weak-gel model.

Flow-induced gelation of microfiber suspensions.

The flow behavior of fiber suspensions has been studied extensively, especially in the limit of dilute concentrations and rigid fibers; at the other extreme, however, where the suspensions are concentrated and the fibers are highly flexible, much less is understood about the flow properties. We use a microfluidic method to produce uniform concentrated suspensions of high aspect ratio, flexible microfibers, and we demonstrate the shear thickening and gelling behavior of such microfiber suspensions, which, to the best of our knowledge, has not been reported previously. By rheological means, we show that flowing the suspension triggers the irreversible formation of topological entanglements of the fibers resulting in an entangled water-filled network. This phenomenon suggests that flexible fiber suspensions can be exploited to produce a new family of flow-induced gelled materials, such as porous hydrogels. A significant consequence of these flow properties is that the microfiber suspension is injectable through a needle, from which it can be extruded directly as a hydrogel without any chemical reactions or further treatments. Additionally, we show that this fiber hydrogel is a soft, viscoelastic, yield-stress material.

Antibiofilm Properties of Temporin-L on Pseudomonas fluorescens in Static and In-Flow Conditions.

Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation of this structure represents the most important adaptive mechanism that leads to antibacterial resistance, and therefore, closely connected to pathogenicity. Antimicrobial peptides (AMPs) could represent attractive candidates for the design of new antibiotics because of their specific characteristics. AMPs show a broad activity spectrum, a relative selectivity towards their targets (microbial membranes), the ability to act on both proliferative and quiescent cells, a rapid mechanism of action, and above all, a low propensity for developing resistance. This article investigates the effect at subMIC concentrations of Temporin-L (TL) on biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, showing that TL displays antibiofilm properties. Biofilm formation in static conditions was analyzed by the Crystal Violet assay. Investigation of biofilms in dynamic conditions was performed in a commercial microfluidic device consisting of a microflow chamber to simulate real flow conditions in the human body. Biofilm morphology was examined using Confocal Laser Scanning Microscopy and quantified via image analysis. The investigation of TL effects on P. fluorescens showed that when subMIC concentrations of this peptide were added during bacterial growth, TL exerted antibiofilm activity, impairing biofilm formation both in static and dynamic conditions. Moreover, TL also affects mature biofilm as confocal microscopy analyses showed that a large portion of preformed biofilm architecture was clearly perturbed by the peptide addition with a significative decrease of all the biofilm surface properties and the overall biomass. Finally, in these conditions, TL did not affect bacterial cells as the live/dead cell ratio remained unchanged without any increase in damaged cells, confirming an actual antibiofilm activity of the peptide.

Dissolution of concentrated surfactant solutions: from microscopy imaging to rheological measurements through numerical simulations.

Concentrated aqueous solutions of surfactants, often referred to as pastes, experience complex phase and rheology changes upon dissolution in water, which is a typical step in the production of liquid detergents. During the dilution process, depending on water content, surfactant molecules can arrange in different morphologies, such as lamellar or cubic and hexagonal structures. These phases are characterized by different physico-chemical properties, such as viscosity or diffusivity, which lead to non-simple transport mechanisms during the dissolution process. In this work, we investigate the dissolution of concentrated Sodium Lauryl Ether Sulfate (SLES) pastes in water under quiescent conditions by coupling different experimental techniques. A thorough rheological characterization of the system showed non-monotonic changes of several orders of magnitude in its viscosity and viscoelastic moduli as a function of water content. Time-lapse microscopy allowed us to image the dynamic evolution of the phase changes as water penetrated in a disk-shaped sample (with the same geometry used in rheological tests). Numerical simulation, based on a simple diffusion-based multi-parameter model is shown to describe satisfactorily SLES dissolution data.

Confined flow behaviour of droplets in microcapillary flow.

The problem of droplets flowing in a micritions is relevant in several applications including flow in porous media. When the flow in the capillary is laminar with negligible gravity effects, droplet velocity and deformation depend upon three independent parameters: the droplet size relative to the capillary radius [Formula: see text][Formula: see text], which is a measure of confinement, the viscosity ratio [Formula: see text] between the droplet and the continuous phase and the capillary number Ca which measures the ratio of viscous to capillary forces. Although droplet microconfined flow behaviour in capillaries has been widely investigated by theoretical models, experimental results are still scarce. Here, an experimental campaign focused on the flow behaviour of axisymmetric confined droplets flowing in a microcapillary is carried out. Our experimental results were obtained by using a water in soybean oil emulsion with a low viscosity ratio and the effect of the aforementioned three parameters, [Formula: see text], [Formula: see text] and Ca, on droplet motion was investigated. Moreover, our experimental results are compared with numerical solutions available in the literature.

Measuring Interfacial Tension of Emulsions in Situ by Microfluidics.

Interfacial tension is a key parameter affecting industrially relevant properties of emulsions, such as morphology and stability. Although several methods are available to measure interfacial tension, they are based on generation of droplets starting from separate emulsion components and cannot directly probe the interfacial tension of an emulsion as such. Here, a novel microfluidic tensiometry device to measure interfacial tension of a water-in-oil emulsion in situ as a function of surfactant concentration is presented. In our approach, interfacial tension is obtained from a quantitative analysis of the deformation of individual emulsion droplets under steady state shear flow in microfluidic channels. The technique is validated by comparing the results with experimental data obtained by the pendant drop method in a broad range of interfacial tension values. A very good agreement is found, and an estimate of the surfactant critical micellar concentration (CMC) is also obtained. The proposed microfluidic setup can be used even at high surfactant concentrations, where the measurement is made more challenging by sample viscoelasticity, thus providing a powerful tool to determine the interfacial tension of complex systems in an extended concentration range. The technique could be also used for in-line monitoring of emulsion processing.

The effect of flow on viscoelastic emulsion microstructure.

Emulsions made of oil, water and surfactants are widespread soft materials with complex structures depending on composition and temperature. Emulsion phase behavior at rest has been widely investigated but flow-induced effects, which are very relevant in many applications, can still be further explored towards improved emulsion microstructural design. In this work, we use low energy emulsification processing to create small-sized emulsions. In a previous report, we showed the emulsion morphology development and the effect of flow on the microstructure of a highly viscoelastic attractive emulsion which result in a concentrated nanoemulsion after viscoelastic droplet filaments are disrupted. Here, we show that upon stopping the flow, the filaments slowly buckle, recoil and finally form clusters of randomly flocculated droplets. We thus obtain two completely different emulsion morphologies simply induced by the action of flow, where in both cases attractive interactions play a key role. The emulsion high interfacial area represents a valuable feature for several applications such as upstream operations, microreaction media and drug delivery.

A novel approach to quantify the wound closure dynamic.

The Wound Healing (WH) assay is widely used to investigate cell migration in vitro, in order to reach a better understanding of many physiological and pathological phenomena. Several experimental factors, such as uneven cell density among different samples, can affect the reproducibility and reliability of this assay, leading to a discrepancy in the wound closure kinetics among data sets corresponding to the same cell sample. We observed a linear relationship between the wound closure velocity and cell density, and suggested a novel methodological approach, based on transport phenomena concepts, to overcome this source of error on the analysis of the Wound Healing assay. In particular, we propose a simple scaling of the experimental data, based on the interpretation of the wound closure as a diffusion-reaction process. We applied our methodology to the MDA-MB-231 breast cancer cells, whose motility was perturbed by silencing or over-expressing genes involved in the control of cell migration. Our methodological approach leads to a significant improvement in the reproducibility and reliability in the in vitro WH assay.

Swelling-induced structural changes and microparticle uptake of gelatin gels probed by NMR and CLSM.

Gelatin gels are increasingly involved in many industrial applications due to several advantages including cost efficiency and biocompatibility. Generally, their production requires the use of aqueous solvents, which cause significant swelling, due to the ability of solvent molecules to penetrate through the gel microstructure and increase its volume. Since swelling mechanisms and their effect on the gel structure are not fully understood, further investigations are required. In this work, we combine macroscopic measurements of the swelling ratio (SR) with Nuclear Magnetic Resonance (NMR) and Confocal Laser Scanning Microscopy (CLSM) to investigate changes in the gelatin structure as a function of both polymer concentration and swelling time. SR values increase as a function of time until a maximum is reached and then show a slight drop for all the gelatin concentrations after 24 h swelling time, probably due to a network relaxation process. NMR allows determination of mass transport and molecular dynamics of water inside the gelatin pores, while CLSM is used to visualize the penetration of tracers (polystyrene microbeads) with a diameter much larger than the gel pores. Structural parameters, such as average pore size and tortuosity, are estimated. In particular, the pore size decreases for higher polymer concentration and increases during swelling, until reaching a maximum, and then dropping at longer times. The penetration of tracers provides evidence of the heterogeneity of the gel structure and shows that single microcarriers can be loaded in gelatin gels upon swelling.

Flow-induced nanostructuring of gelled emulsions.

Although the phase behavior of emulsions has been thoroughly investigated, the effect of flow on emulsion morphology, which is relevant for many applications, is far from being fully elucidated. Here, we investigate an emulsion based on two common nonionic surfactants in a range of water concentration where complex and diverse microstructures are found at rest, such as multilamellar and bicontinuous phases. In spite of such complexity, once subjected to shear flow, all the emulsions investigated are characterized by thinning filaments which eventually break up into a concentrated suspension of micro-sized water-based droplets dispersed in a continuous oil phase. The so-formed droplets tend to align in string-like structures. The emulsions exhibit a yield stress, whose value can be estimated by the plug-core velocity profiles in pressure-driven capillary flow, thus providing evidence of weakly attractive interdroplet interactions. The latter are consistent with droplet clustering and percolation observed at rest. These results can also be relevant to the flow behavior of other liquid-liquid systems, such as polymer blends, where the flow-induced microstructure is under debate as well.

Comparison between fibroblast wound healing and cell random migration assays in vitro.

Cell migration plays a key role in many biological processes, including cancer growth and invasion, embryogenesis, angiogenesis, inflammatory response, and tissue repair. In this work, we compare two well-established experimental approaches for the investigation of cell motility in vitro: the cell random migration (CRM) and the wound healing (WH) assay. In the former, extensive tracking of individual live cells trajectories by time-lapse microscopy and elaborate data processing are used to calculate two intrinsic motility parameters of the cell population under investigation, i.e. the diffusion coefficient and the persistence time. In the WH assay, a scratch is made in a confluent cell monolayer and the closure time of the exposed area is taken as an easy-to-measure, empirical estimate of cell migration. To compare WH and CRM we applied the two assays to investigate the motility of skin fibroblasts isolated from wild type and transgenic mice (TgPED) overexpressing the protein PED/PEA-15, which is highly expressed in patients with type 2 diabetes. Our main result is that the cell motility parameters derived from CRM can be also estimated from a time-resolved analysis of the WH assay, thus showing that the latter is also amenable to a quantitative analysis for the characterization of cell migration. To our knowledge this is the first quantitative comparison of these two widely used techniques.

i-Rheo-optical assay: Measuring the viscoelastic properties of multicellular spheroids.

This study introduces a novel mechanobiology assay, named "i-Rheo-optical assay", that integrates rheology with optical microscopy for analysing the viscoelastic properties of multicellular spheroids. These spheroids serve as three-dimensional models resembling tissue structures. The innovative technique enables real-time observation and quantification of morphological responses to applied stress using a cost-effective microscope coverslip for constant compression force application. By bridging a knowledge gap in biophysical research, which has predominantly focused on the elastic properties while only minimally exploring the viscoelastic nature in multicellular systems, the i-Rheo-optical assay emerges as an effective tool. It facilitates the measurement of broadband viscoelastic compressional moduli in spheroids, here derived from cancer (PANC-1) and non-tumoral (NIH/3T3) cell lines during compression tests. This approach plays a crucial role in elucidating the mechanical properties of spheroids and holds potential for identifying biomarkers to discriminate between healthy tissues and their pathological counterparts. Offering comprehensive insights into the biomechanical behaviour of biological systems, i-Rheo-optical assay marks a significant advancement in tissue engineering, cancer research, and therapeutic development.

Using optical tweezers for the characterization of polyelectrolyte solutions with very low viscoelasticity.

Recently, optical tweezing has been used to provide a method for microrheology addressed to measure the rheological properties of small volumes of samples. In this work, we corroborate this emerging field of microrheology by using these optical methods for the characterization of polyelectrolyte solutions with very low viscoelasticity. The influence of polyelectrolyte (i.e., polyacrylamide, PAM) concentration, specifically its aging, of the salt concentration is shown. The close agreement of the technique with classical bulk rheological measurements is demonstrated, illustrating the advantages of the technique.

Correction: 'Semen rheology and its relation to male infertility' (2022), by Tomaiuolo et al.

[This corrects the article DOI: 10.1098/rsfs.2022.0048.][This corrects the article DOI: 10.1098/rsfs.2022.0048.].

Collective rotational motion of freely expanding T84 epithelial cell colonies.

Coordinated rotational motion is an intriguing, yet still elusive mode of collective cell migration, which is relevant in pathological and morphogenetic processes. Most of the studies on this topic have been carried out on epithelial cells plated on micropatterned substrates, where cell motion is confined in regions of well-defined shapes coated with extracellular matrix adhesive proteins. The driver of collective rotation in such conditions has not been clearly elucidated, although it has been speculated that spatial confinement can play an essential role in triggering cell rotation. Here, we study the growth of epithelial cell colonies freely expanding (i.e. with no physical constraints) on the surface of cell culture plates and focus on collective cell rotation in such conditions, a case which has received scarce attention in the literature. One of the main findings of our work is that coordinated cell rotation spontaneously occurs in cell clusters in the free growth regime, thus implying that cell confinement is not necessary to elicit collective rotation as previously suggested. The extent of collective rotation was size and shape dependent: a highly coordinated disc-like rotation was found in small cell clusters with a round shape, while collective rotation was suppressed in large irregular cell clusters generated by merging of different clusters in the course of their growth. The angular motion was persistent in the same direction, although clockwise and anticlockwise rotations were equally likely to occur among different cell clusters. Radial cell velocity was quite low as compared to the angular velocity, in agreement with the free expansion regime where cluster growth is essentially governed by cell proliferation. A clear difference in morphology was observed between cells at the periphery and the ones in the core of the clusters, the former being more elongated and spread out as compared to the latter. Overall, our results, to our knowledge, provide the first quantitative and systematic evidence that coordinated cell rotation does not require a spatial confinement and occurs spontaneously in freely expanding epithelial cell colonies, possibly as a mechanism for the system.