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There is an increasing understanding that condensation is a crucial intermediate step in the assembly of biological materials and for a multitude of cellular processes. To apply and to understand these mechanisms, in vitro biophysical characterisation techniques are central. The formation and biophysical properties of protein condensates depend on a multitude of factors, such as protein concentration, pH, temperature, salt concentration, and presence of other biomolecules as well as protein purification and storage conditions. Here we show how critical the procedures for preparing protein samples for in vitro studies are. We compare two purification methods of the recombinant spider silk protein CBM-AQ12-CBM and study the effect of background molecules, such as DNA, on the formation and properties of the condensates. We characterize the condensates using aggregation induced emitters (AIEs), coalescence studies, and micropipette aspiration. The condensated sample containing background molecules exhibit a lower threshold concentration for condensate formation accompanied by a lower surface tension and longer coalescence time when compared to the pure protein condensates. Furthermore, the partitioning of small AIEs is enhanced in the presence of background molecules. Our results highlight that the purification method and remaining background molecules strongly affect the biophysical properties of spider silk condensates. Using the acquired knowledge about spider silk protein purification we derive guidelines for reproducible condensate formation that will foster the use of spider silk proteins as adhesives or carriers for biomedical applications.

Near-infrared (NIR) fluorophores with pH-responsive properties suggest merits in biological analyses. This work establishes a general and effective method to obtain pH-responsive NIR emissive gold nanoclusters by introducing aliphatic tertiary amine (TA) groups into the ligands. Computational study suggests that the pH-responsive NIR emission is associated with electronic structure change upon protonation and deprotonation of TA groups. Photo-induced electron transfer between deprotonated TA groups and the surface Au-S motifs of gold nanoclusters can disrupt the radiative transitions and thereby decrease the photoluminescence intensity in basic environments (pH=7-11). By contrast, protonated TA groups curb the electron transfer and restore the photoluminescence intensity in acidic environments (pH=4-7). The pH-responsive NIR-emitting gold nanoclusters serve as a specific and sensitive probe for the lysosomes in the cells, offering non-invasive emissions without interferences from intracellular autofluorescence., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Gas vesicles used as contrast agents for noninvasive ultrasound imaging must be formulated to be stable, and their mechanical properties must be assessed. We report here the formation of perfluoro- n -butane microbubbles coated with surface-active proteins that are produced by filamentous fungi (hydrophobin HFBI from Trichoderma reesei ). Using pendant drop and pipette aspiration techniques, we show that these giant gas vesicles behave like glassy polymersomes, and we discover novel gas extraction regimes. We develop a model to analyze the micropipette aspiration of these compressible gas vesicles and compare them to incompressible liquid-filled vesicles. We introduce a sealing parameter to characterize the leakage of gas under aspiration through the pores of the protein coating. Utilizing this model, we can determine the elastic dilatation modulus, surface viscosity, and porosity of the membrane. These results demonstrate the engineering potential of protein-coated bubbles for echogenic and therapeutic applications and extend the use of the pipette aspiration technique to compressible and porous systems.

Recombinant expression of proteins destined to form biological materials often results in poor production yields or loss of their function due to premature aggregation. Recently, liquid-liquid phase separation has been proposed as a mechanism to control protein solubility during expression and accumulation in the cytoplasm. Here, we investigate this process in vivo during the recombinant overexpression of the mimetic spider silk mini-spidroin NT2RepCT in Escherichia coli . The protein forms intracellular liquid-like condensates that shift to a solid-like state triggered by a decrease in their microenvironmental pH. These features are also maintained in the purified sample in vitro both in the presence of a molecular crowding agent mimicking the bacterial intracellular environment, and during a biomimetic extrusion process leading to fiber formation. Overall, we demonstrate that characterization of protein condensates inside E. coli could be used as a basis for selecting proteins for both materials applications and their fundamental structure-function studies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

β-1,3-Glucans are ubiquitously observed in various biological systems with diverse physio-ecological functions, yet their underlying assembly mechanism and multiscale complexation in vitro remains poorly understood. Here, we provide for the first-time evidence of unidentified β-1,3-glucan supramolecular complexation into intricate hierarchical architectures over several length scales. We mediated these unique assemblies using a recombinantly produced β-1,3-glucan phosphorylase ( Ta 1,3BGP) by fine-tuning solution conditions during particle nucleation and growth. We report a synthesis of interconnected parallel hexagonal lamellae composed of 8 nm thick sheets of highly expanded paracrystals. The architecture consists of β-1,3-glucan triple-helices with considerable inter-intra hydrogen bonding within, as well as in between adjacent triple-helices. The results extend our understanding of β-1,3-glucan molecular organization and shed light on different aspects of the crystallization processes of biomolecules into structures unseen by nature. The presented versatile synthesis yields new materials for diverse medical and industrial applications.

We study the fusion of homogeneous cell aggregates and of hybrid aggregates combining cells and microparticles. In all cases, we find that the contact area does not vary linearly over time, as observed for liquid drops, but rather it follows a power law in t 2/3 . This result is interpreted by generalizing the fusion model of soft viscoelastic solid balls to viscoelastic liquid balls, akin to jelly pearls. We also explore the asymmetric fusion between a homogeneous aggregate and a hybrid aggregate. This latter experiment allows the determination of the self-diffusion coefficient of the cells in a tissue by following the spatial distribution of internalized particles in the cells.

High-sensitivity troponin has become an essential emergency biomarker for diagnosing or ruling out an ACS. The establishment of a point of care biology related to the reorganization and fusion of laboratories raise a question about transferability of results between techniques. In this study, we propose to compare the bioclinical performances of high-sensitivity troponin measured by two different techniques on laboratory immunoanalyzer (Siemens Advia Centaur XPT) and on point of care device (Mitsubishi Pathfast). The assay of high-sensitivity troponin (n = 90 patients), according to our study, show consistent clinical results with both method.

In time of SARS-Cov2 pandemic, neurologists need to be vigilant for cerebrovascular complications of Covid-19. We present a case of bilateral occipito-temporal infarction revealed by a sudden cortical blindness with haemorrhagic transformation after intravenous thrombolysis in a diabetic patient infected by Covid-19. Differential diagnoses are discussed in front of this unusual presentation and evolution., Competing Interests: Declaration of Competing Interest The authors report no disclosures. Informed consent for publication has been signed by the wife of the patient., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ferrofluids exhibit a unique combination of liquid properties and strong magnetic response, which leads to a rich variety of interesting functional properties. Here, the magnetic-field-induced splitting of ferrofluid droplets immersed in an immiscible liquid is presented, and related fascinating dynamics and applications are discussed. A magnetic field created by a permanent magnet induces instability on a mother droplet, which divides into two daughter droplets in less than 0.1 s. During the splitting process, the droplet undergoes a Plateau-Rayleigh-like instability, which is investigated using high-speed imaging. The dynamics of the resulting satellite droplet formation is shown to depend on the roughness of the supporting surface. Further increasing the field results in additional splitting events and self-assembly of microdroplet populations, which can be magnetically actuated. The effects of magnetization and interfacial tension are systematically investigated by varying magnetic nanoparticles and surfactant concentrations, and a variety of outcomes from labyrinthine patterns to discrete droplets are observed. As the splitting process depends on interfacial tension, the droplet splitting can be used as a measure for interfacial tension as low as 0.1 mN m -1 . Finally, a population-based digital microfluidics concept based on the self-assembled microdroplets is presented., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recent developments suggest that the phase transition of natural and synthetic biomacromolecules represents an important and ubiquitous mechanism underlying structural assemblies toward the fabrication of high-performance materials. Such a transition results in the formation of condensed liquid droplets, described as condensates or coacervates. Being able to effectively control the assembly of such entities is essential for tuning the quality and their functionality. Here we describe how self-coacervation of genetically engineered spidroin-inspired proteins can be preceded by a wide range of kosmotropic salts. We studied the kinetics and mechanisms of coacervation in different conditions, from direct observation of initial phase separation to the early stage of nucleation/growth and fusion into large fluid assemblies. We found that coacervation induced by kosmotropic salts follows the classical nucleation theory and critically relies on precursor clusters of few weak-interacting protein monomers. Depending on solution conditions and the strength of the supramolecular interaction as a function of time, coacervates with a continuum of physiochemical properties were observed. We observed similar characteristics in other protein-based coacervates, which include having a spherical-ellipsoid shape in solution, an interconnected bicontinuous network, surface adhesion, and wetting properties. Finally, we demonstrated the use of salt-induced self-coacervates of spidroin-inspired protein as a cellulosic binder in dried condition., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The generation of highly reactive oxygen species (ROS) at room temperature for application in organic synthesis and wastewater treatment represents a great challenge of the current chemical industry. In fact, the development of biodegradable scaffolds to support ROS-generating active sites is an important prerequisite for the production of environmentally benign catalysts. Herein, the electrostatic cocrystallization of a cationic phthalocyanine (Pc) and negatively charged tobacco mosaic virus (TMV) is described, together with the capacity of the resulting crystals to photogenerate ROS. To this end, a novel peripherally crowded zinc Pc (1) is synthesized. With 16 positive charges, this photosensitizer shows no aqueous aggregation, and is able to act as a molecular glue in the unidimensional assembly of TMV. A step-wise decrease of ionic strength in mixtures of both components results in exceptionally long fibers, constituted by hexagonally bundled viruses thoroughly characterized by electron and confocal microscopy. The fibers are able to produce ROS in a proof-of-concept microfluidic device, where they are immobilized and irradiated in several cycles, showing a resilient performance. The bottom-up approach also enables the light-triggered disassembly of fibers after use. This work represents an important example of a biohybrid material with projected application in light-mediated heterogeneous catalysis., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Controlling the propagation of primary tumors is fundamental to avoiding the epithelial to mesenchymal transition process leading to the dissemination and seeding of tumor cells throughout the body. Here we demonstrate that nanoparticles (NPs) limit the propagation of cell aggregates of CT26 murine carcinoma cells used as tumor models. The spreading behavior of these aggregates incubated with NPs is studied on fibronectin-coated substrates. The cells spread with the formation of a cell monolayer, the precursor film, around the aggregate. We study the effect of NPs added either during or after the formation of aggregates. We demonstrate that, in both cases, the spreading of the cell monolayer is slowed down in the presence of NPs and occurs only above a threshold concentration that depends on the size and surface chemistry of the NPs. The density of cells in the precursor films, measured by confocal microscopy, shows that the NPs stick cells together. The mechanism of slowdown is explained by the increase in cell-cell interactions due to the NPs adsorbed on the membrane of the cells. The present results demonstrate that NPs can modulate the collective migration of cells; therefore, they may have important implications for cancer treatment.

The sulfobetaine (SB) moiety, which comprises a quaternary ammonium group linked to a negatively charged sulfonate ester, is known to impart nonfouling properties to interfaces coated with polysulfobetaines or grafted with SB-polymeric brushes. Increasingly, evidence emerges that the SB group is, overall, a better antifouling group than the phosphorylcholine (PC) moiety extensively used in the past. We report here the synthesis of a series of SB-modified chitosans (CH-SB) carrying between 20 and 40 mol % SB per monosaccharide unit. Chitosan (CH) itself is a naturally derived copolymer of glucosamine and N-acetyl-glucosamine linked with a β-1,4 bond. Analysis by quartz crystal microbalance with dissipation (QCM-D) indicates that CH-SB films (thickness ∼ 20 nm) resist adsorption of bovine serum albumin (BSA) with increasing efficiency as the SB content of the polymer augments (surface coverage ∼ 15 μg cm -2 for films of CH with 40 mol % SB). The cell adhesivity of CH-SB films coated on glass was assessed by determining the spreading dynamics of CT26 cell aggregates. When placed on chitosan films, known to be cell-adhesive, the CT26 cell aggregates spread by forming a cell monolayer around them. The spreading of CT26 cell aggregates on zwitterion-modified chitosans films is thwarted remarkably. In the cases of CH-SB30 and CH-SB40 films, only a few isolated cells escape from the aggregates. The extent of aggregate spreading, quantified based on the theory of liquid wetting, provides a simple in vitro assay of the nonfouling properties of substrates toward specific cell lines. This assay can be adopted to test and compare the fouling characteristics of substrates very different from the chemical viewpoint.

Despite extensive knowledge on the mechanisms that drive single-cell migration, those governing the migration of cell clusters, as occurring during embryonic development and cancer metastasis, remain poorly understood. Here, we investigate the collective migration of cell on adhesive gels with variable rigidity, using 3D cellular aggregates as a model system. After initial adhesion to the substrate, aggregates spread by expanding outward a cell monolayer, whose dynamics is optimal in a narrow range of rigidities. Fast expansion gives rise to the accumulation of mechanical tension that leads to the rupture of cell-cell contacts and the nucleation of holes within the monolayer, which becomes unstable and undergoes dewetting like a liquid film. This leads to a symmetry breaking and causes the entire aggregate to move as a single entity. Varying the substrate rigidity modulates the extent of dewetting and induces different modes of aggregate motion: "giant keratocytes," where the lamellipodium is a cell monolayer that expands at the front and retracts at the back; "penguins," characterized by bipedal locomotion; and "running spheroids," for nonspreading aggregates. We characterize these diverse modes of collective migration by quantifying the flows and forces that drive them, and we unveil the fundamental physical principles that govern these behaviors, which underscore the biological predisposition of living material to migrate, independent of length scale.

Liquid-liquid phase separation of biomacromolecules plays a critical role in many of their functions, both as cellular components and in structural assembly. Phase separation is also a key mechanism in the assembly of engineered recombinant proteins for the general aim to build new materials with unique structures and properties. Here the phase separation process of an engineered protein with a block-architecture was studied. As a central block, we used a modified spider silk sequence, predicted to be unstructured. In each terminus, folded globular blocks were used. We studied the kinetics and mechanisms of phase formation and analyzed the evolving structures and their viscoelastic properties. Individual droplets were studied with a micropipette technique, showing both how properties vary between individual drops and explaining overall bulk rheological properties. A very low surface energy allowed easy deformation of droplets and led to efficient infiltration into cellulosic fiber networks. Based on these findings, we demonstrated an efficient use of the phase-separated material as an adhesive for cellulose. We also conclude that the condensed state is metastable, showing an ensemble of properties in individual droplets and that an understanding of protein phase behavior will lead to developing a wider use of proteins as structural polymers., Competing Interests: The authors declare no competing financial interest.

We study spreading on soft substrates of cellular aggregates using CT26 cells that produce an extracellular matrix (ECM). Compared to our previous work on the spreading of S180 cellular aggregates, which did not secrete ECMs, we found that the spreading velocity of the precursor film is also maximal for intermediate rigidities, but new striking features show up. First, we observed a cascade of liquid-gas-liquid (L/G/L) transitions of the precursor film as the substrate rigidity is decreased. We attribute the L/G transition to a decrease of cell/cell adhesion resulting from the weakening of the cell/substrate adhesion. We attribute the reentrant liquid phase (G/L) observed on soft substrates to the slow spreading of the aggregates on ultra-soft substrates, which gives time to the cells to secrete more ECM proteins and stick together. Second, a nematic order appears in the cohesive (liquid) states of the precursor film, attributed to the gradient of cell's velocities.

We study the spreading of cell aggregates deposited on adhesive substrates decorated with microparticles (MPs). A cell monolayer expands around the aggregate. The cells on the periphery of the monolayer take up the MPs, clearing the substrate as they progress and forming an aureole of cells filled with MPs. We study the dynamics of spreading and determine the width of the aureole and the level of MP internalization in cells as a function of MP size, composition, and density. From the radius and width of the aureole, we quantify the volume fraction of MPs within the cell, which leads to an easy, fast, and inexpensive measurement of the cell - particle internalization.

We report a one-pot synthesis of water dispersible fluorescent silica nanoparticles (NPs) functionalized with terminal amine groups, starting from silicon tetrabromide (SiBr 4 ) and aminopropyltriethoxy silane (APTES). The NPs range from 1 to 2 nm in diameter, and exhibit an intense blue emission with a quantum yield (QY) of around 34% in water. They were characterized using XRD, XPS, TEM and FTIR spectroscopy for structural analysis. A tentative mechanism explaining the origin of the NPs emission in the blue region is presented based on the distinctive features of their low temperature photoluminescence (PL), photoluminescence excitation (PLE) spectrum and time correlated single photon counting lifetime decay profiles. The outstanding PL QY and photostability of the NPs, together with their water dispersibility and biocompatibility, constitute a unique set of properties among existing silica NPs and enable the application of the NPs in various fields.

We present the case of an Addison's disease revealed by a serious hyponatremia. The serum concentration of ACTH and 21-hydroxylase antibodies were increased and lead to the diagnosis. The cortisol blood level was lowered but required to take into account the stress induced by the hospitalisation of the patient. Addison's disease is characterized by the destruction of the adrenal cortex. Autoimmune adrenalitis is the main cause of adrenal insufficiency. Treatment involves normalisation of sodium concentration and corticosteroids replacement. With a good patient compliance, the survival rate of Addisonian patient is similar to that of the normal population. Management of patient requires vigilance because of the occurrence of others autoimmunes diseases during patient life.

In spite of the great promises that the development of nanotechnologies can offer, concerns regarding potential adverse health effects of occupational exposure to nanoparticle (NP) is raised. We recently identified metal oxide NP in lung tissue sections of welders, located inside macrophages infiltrated in fibrous regions. This suggests a role of these NP in the lung alterations observed in welders. We therefore designed a study aimed to investigate the pulmonary effects, in mice, of repeated exposure to NP administered at occupationally relevant doses. We therefore chose four metal oxide NPs representative of those found in the welder's lungs: Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 and CrOOH. These NPs were administered weekly for up to 3 months at two different doses: 5 μg, chosen as occupationally relevant to welding activity, and 50 μg, chosen as occupationally relevant to the context of an NP-manufacturing facility. Our results show that 3 month-repeated exposures to 5 μg NP induced limited pulmonary effects, characterized by the development of a mild peribronchiolar fibrosis observed for MnFe 2 O 4 and CrOOH NP only. This fibrotic event was further extended in terms of intensity and localization after the repeated administration of 50 μg NP: all but Fe 2 O 3 NP induced the development of peribronchiolar, perivascular and alveolar fibrosis, together with an interstitial inflammation. Our data demonstrate for the first time a potential risk for respiratory health posed by repeated exposure to NP at occupationally relevant doses. Given these results, the development of occupational exposure limits (OELs) specifically dedicated to NP exposure might therefore be an important issue to address.

We present direct evidence that nanoparticles (NPs) can stick together cells that are inherently non-adhesive. Using cadherin-depleted S180 murine cells lines, which exhibit very low cell-cell adhesion, we show that NPs can assemble dispersed single cells into large cohesive aggregates. The dynamics of aggregation, which is controlled by diffusion and collision, can be described as a second-order kinetic law characterized by a rate of collision that depends on the size, concentration, and surface chemistry of the NPs. We model the cell-cell adhesion induced by the "nanostickers" using a three-state dynamical model, where the NPs are free, adsorbed on the cell membrane or internalized by the cells. We define a "sticking efficiency parameter" to compare NPs and look for the most efficient type of NP. We find that 20 nm carboxylated polystyrene NPs are more efficient nanostickers than 20 nm silica NPs which were reported to induce fast wound healing and to glue soft tissues. Nanostickers, by increasing the cohesion of tissues and tumors, may have important applications for tissue engineering and cancer treatment.

Functional near-IR (NIR) emitting nanoparticles (NPs) adapted for two-photon excitation fluorescence cell imaging were obtained starting from octadecyl-terminated silicon nanocrystals (ncSi-OD) of narrow photoluminescence (PL) spectra having no long emission tails, continuously tunable over the 700-1000 nm window, PL quantum yields exceeding 30%, and PL lifetimes of 300 μs or longer. These NPs, consisting of a Pluronic F127 shell and a core made up of assembled ncSi-OD kept apart by an octadecyl (OD) layer, were readily internalized into the cytosol, but not the nucleus, of NIH3T3 cells and were non-toxic. Asymmetrical field-flow fractionation (AF4) analysis was carried out to determine the size of the NPs in water. HiLyte Fluor 750 amine was linked via an amide link to NPs prepared with Pluronic-F127-COOH, as a first demonstration of functional NIR-emitting water dispersible ncSi-based nanoparticles.

Membrane tubes are commonly extruded from cells and vesicles when a point-like force is applied on the membrane. We report here the unexpected formation of membrane tubes from lymph node cancer prostate (LNCaP) cell aggregates in the absence of external applied forces. The spreading of LNCaP aggregates deposited on adhesive glass substrates coated with fibronectin is very limited because cell-cell adhesion is stronger than cell-substrate adhesion. Some cells on the aggregate periphery are very motile and try to escape from the aggregate, leading to the formation of membrane tubes. Tethered networks and exchange of cargos between cells were observed as well. Growth of the tubes is followed by either tube retraction or tube rupture. Hence, even very cohesive cells are successful in escaping aggregates, which may lead to epithelial mesenchymal transition and tumor metastasis. We interpret the dynamics of formation and retraction of tubes in the framework of membrane mechanics.

This study describes chitosan-phosphorylcholine (CH-PC) films able to support the formation of cell aggregates (spheroids), which are important for tissue engineering and pharmacological studies. The surface topography, charge, thickness, and rheology of CH-PC thin films were characterized by AFM, zeta-potential measurements, SPR spectroscopy, and QCM-D measurements. The CH-PC films are highly hydrated gels, independently of the level of PC incorporation (15-40 mol-% PC/glucosamine units). QCM-D studies established that the amount of fibrinogen adsorbed on CH-PC films decreased with increasing PC content. CH-PC surfaces underwent a transition from moderately cell-adhesive (CH-PC15) to non-adhesive (CH-PC40). Optical micrographs of HUVEC and MCF-7 cell lines cultured on CH-PC surfaces showed that they form spheroids on CH-PC25 and CH-PC40 films., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)