Liquid Phase Separation Process Research Articles

Surface-catalyzed liquid–liquid phase separation and amyloid-like assembly in microscale compartments

Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid.In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid–liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies.These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process.

Core-shell structure with reversible composition of Al-Bi immiscible alloy particles prepared by pulsated orifice ejection method

The effect of different step time on the liquid phase separation process and macroscopic morphology of Al-60 wt% Bi immiscible alloys were investigated by pulsated orifice ejection method (POEM). The results show that the decrease of the step time will accelerate the solidification and growth of the minor-phase globule in the liquid phase separation process, which will have a significant effect on the macroscopic morphology after solidification. The Al-60 wt% Bi core–shell structure particles with Al as the core and Bi as the shell were successfully prepared when the step time was 100 μs. In the same experiment, Al-60 wt% Bi core–shell structure particles with Bi as the core and Al as the shell at a step time of 50 μs were successfully prepared. The same particle size and the same overheat of Al-60wt.%Bi particles with reversed core and shell composition were achieved under containerless solidification conditions.

FUS fibrillation occurs through a nucleation-based process below the critical concentration required for liquid–liquid phase separation

FUS is an RNA-binding protein involved in familiar forms of ALS and FTLD that also assembles into fibrillar cytoplasmic aggregates in some neurodegenerative diseases without genetic causes. The self-adhesive prion-like domain in FUS generates reversible condensates via the liquid–liquid phase separation process (LLPS) whose maturation can lead to the formation of insoluble fibrillar aggregates in vitro, consistent with the appearance of cytoplasmic inclusions in ageing neurons. Using a single-molecule imaging approach, we reveal that FUS can assemble into nanofibrils at concentrations in the nanomolar range. These results suggest that the formation of fibrillar aggregates of FUS could occur in the cytoplasm at low concentrations of FUS, below the critical ones required to trigger the liquid-like condensate formation. Such nanofibrils may serve as seeds for the formation of pathological inclusions. Interestingly, the fibrillation of FUS at low concentrations is inhibited by its binding to mRNA or after the phosphorylation of its prion-like domain, in agreement with previous models.

Protein-RNA interactions: from mass spectrometry to drug discovery.

Proteins and RNAs are fundamental parts of biological systems, and their interactions affect many essential cellular processes. Therefore, it is crucial to understand at a molecular and at a systems level how proteins and RNAs form complexes and mutually affect their functions. In the present mini-review, we will first provide an overview of different mass spectrometry (MS)-based methods to study the RNA-binding proteome (RBPome), most of which are based on photochemical cross-linking. As we will show, some of these methods are also able to provide higher-resolution information about binding sites, which are important for the structural characterisation of protein-RNA interactions. In addition, classical structural biology techniques such as nuclear magnetic resonance (NMR) spectroscopy and biophysical methods such as electron paramagnetic resonance (EPR) spectroscopy and fluorescence-based methods contribute to a detailed understanding of the interactions between these two classes of biomolecules. We will discuss the relevance of such interactions in the context of the formation of membrane-less organelles (MLOs) by liquid-liquid phase separation (LLPS) processes and their emerging importance as targets for drug discovery.

Bio‐inspired functional coacervates

AbstractMany functional coacervates have been identified in biological systems, which have attracted widespread interest. Coacervation is a liquid–liquid phase separation (LLPS) process in which a macromolecule‐enriched liquid phase is formed together with a macromolecule‐depleted phase. Bio‐inspired coacervates possess excellent features such as underwater delivery, low interface energy, shear thinning, and excellent biocompatibility. They also serve as good delivery platforms for different types of molecules. In this review, we briefly discuss some important extracellular coacervate systems, including mussel adhesives, sandcastle worm glue, squid beak, and tropoelastin. We then provide an overview of the recent development of bio‐inspired functional coacervates for various biomedical applications, including medical adhesives, drug delivery, and tissue engineering. Bio‐inspired functional coacervates offer a promising material platform for developing new materials for biomedical applications.

Engineering of Metal–Organic Frameworks/Gelatin Hydrogel Composites Mediated by the Coacervation Process for the Capture of Acetic Acid

A novel strategy is proposed to synthesize metal–organic framework (MOF)–gelatin bionanocomposites by taking profit of the thermo-reversible character of gelatin and the liquid–liquid phase separation process, that is, coacervation. This enables the formation of bionanocomposites based on a series of chemically stable Zr4+ dicarboxylate MOFs (UiO-66 and MOF-801) differing by their hydrophilic–hydrophobic balance and their chemical functionality. Bionanocomposites with homogeneous and uniform distribution of MOF particles in the gelatin matrix as well as a high MOF loading (up to 90%) without compromising their porosity were prepared as a result of an excellent physico-chemical matching between MOFs and gelatin. Finally, this series of bionanocomposites were shaped into films or monoliths, and they have shown high performance for the selective adsorption of acetic acid in the presence of humidity. These composites can be regarded as highly efficient adsorbents for cultural heritage preservation.

A Census of Human Methionine-Rich Prion-like Domain-Containing Proteins.

Methionine-rich prion-like proteins can regulate liquid–liquid phase separation processes in response to stresses. To date, however, very few proteins have been identified as methionine-rich prion-like. Herein, we have performed a computational survey of the human proteome to search for methionine-rich prion-like domains. We present a census of 51 manually curated methionine-rich prion-like proteins. Our results show that these proteins tend to be modular in nature, with molecular sizes significantly greater than those we would expect due to random sampling effects. These proteins also exhibit a remarkably high degree of spatial compaction when compared to average human proteins, even when protein size is accounted for. Computational evidence suggests that such a high degree of compactness might be due to the aggregation of methionine residues, pointing to a potential redox regulation of compactness. Gene ontology and network analyses, performed to shed light on the biological processes in which these proteins might participate, indicate that methionine-rich and non-methionine-rich prion-like proteins share gene ontology terms related to the regulation of transcription and translation but, more interestingly, these analyses also reveal that proteins from the methionine-rich group tend to share more gene ontology terms among them than they do with their non-methionine-rich prion-like counterparts.

Preparation of polyamide 12 powder for additive manufacturing applications via thermally induced phase separation

Abstract Spherical polyamide 12 (PA12) powder for selective laser sintering (SLS) was prepared by thermally induced phase separation (TIPS) method. It was authenticated that the mixed solvent can regulate the liquid–liquid phase separation (LLPS) process by changing the ratio of diluent to non-diluent. The polymer droplets mainly coalesced in the solution, and then the crystal nucleus of PA12 was formed in the droplets. Finally, high crystallinity PA12 powder was precipitated. The morphology, particle size distribution, thermal properties, the change of crystal structure, and powder spreading performances of the obtained powder were characterized. The powder had a narrow particle size distribution, an average particle size of 55.2 μm, and a broad sintering window of 29°C. The results exhibited that the powders prepared by TIPS had excellent sintering properties, and TIPS method provided more choices for SLS technology.

Precipitation of cesium lead halide perovskite nanocrystals in glasses based on liquid phase separation

AbstractIncorporation of cesium lead halide perovskite nanocrystals (CsPbX3 PNCs, X = Cl, Br, I) into glasses can significantly improve their stabilities and extend their application areas. Precipitation of CsPbX3 PNCs in glasses is mainly based on thermal treatment; however, the formation of mechanism is not clarified. Here, several in situ methods are employed to illustrate the precipitation mechanism of CsPbX3 PNCs. It is found that precipitation of CsPbX3 PNCs in glasses is based on the liquid phase separation in solid amorphous matrix, followed by crystallization in the supercooled state. Liquid phase separation process determines the composition of CsPbX3 PNCs, and cooling process has strong effect on the crystallinity and quality of CsPbX3 PNCs. These results clarify the precipitation mechanism of CsPbX3 PNCs in glasses and provide important guideline for the development of CsPbX3 PNCs embedded glasses for opto‐electronic applications.

Small Molecules Modulate Liquid‐to‐Solid Transitions in Phase‐Separated Tau Condensates

AbstractIn Tau protein condensates formed by the Liquid–Liquid Phase Separation (LLPS) process, liquid‐to‐solid transitions lead to the formation of fibrils implicated in Alzheimer's disease. Here, by tracking two contacting Tau‐rich droplets using a simple and nonintrusive video microscopy, we found that the halftime of the liquid‐to‐solid transition in the Tau condensate is affected by the Hofmeister series according to the solvation energy of anions. After dissecting functional groups of physiologically relevant small molecules using a multivariate approach, we found that charged groups facilitate the liquid‐to‐solid transition in a manner similar to the Hofmeister effect, whereas hydrophobic alkyl chains and aromatic rings inhibit the transition. Our results not only elucidate the driving force of the liquid‐to‐solid transition in Tau condensates, but also provide guidelines to design small molecules to modulate this important transition for many biological functions for the first time.

Pluripotency transcription factors at the focus: the phase separation paradigm in stem cells

The transcription factors (TFs) OCT4, SOX2 and NANOG are key players of the gene regulatory network of pluripotent stem cells. Evidence accumulated in recent years shows that even small imbalances in the expression levels or relative concentrations of these TFs affect both, the maintenance of pluripotency and cell fate decisions. In addition, many components of the transcriptional machinery including RNA polymerases, cofactors and TFs such as those required for pluripotency, do not distribute homogeneously in the nucleus but concentrate in multiple foci influencing the delivery of these molecules to their DNA-targets. How cells control strict levels of available pluripotency TFs in this heterogeneous space and the biological role of these foci remain elusive. In recent years, a wealth of evidence led to propose that many of the nuclear compartments are formed through a liquid-liquid phase separation process. This new paradigm early penetrated the stem cells field since many key players of the pluripotency circuitry seem to phase-separate. Overall, the formation of liquid compartments may modulate the kinetics of biochemical reactions and consequently regulate many nuclear processes. Here, we review the state-of-the-art knowledge of compartmentalization in the cell nucleus and the relevance of this process for transcriptional regulation, particularly in pluripotent stem cells. We also highlight the recent advances and new ideas in the field showing how compartmentalization may affect pluripotency preservation and cell fate decisions.

Insight into membraneless organelles and their associated proteins: Drivers, Clients and Regulators.

In recent years, attention has been devoted to proteins forming immiscible liquid phases within the liquid intracellular medium, commonly referred to as membraneless organelles (MLO). These organelles enable the spatiotemporal associations of cellular components that exchange dynamically with the cellular milieu. The dysregulation of these liquid-liquid phase separation processes (LLPS) may cause various diseases including neurodegenerative pathologies and cancer, among others. Until very recently, databases containing information on proteins forming MLOs, as well as tools and resources facilitating their analysis, were missing. This has recently changed with the publication of 4 databases that focus on different types of experiments, sets of proteins, inclusion criteria, and levels of annotation or curation. In this study we integrate and analyze the information across these databases, complement their records, and produce a consolidated set of proteins that enables the investigation of the LLPS phenomenon. To gain insight into the features that characterize different types of MLOs and the roles of their associated proteins, they were grouped into categories: High Confidence MLO associated (including Drivers and reviewed proteins), Potential Clients and Regulators, according to their annotated functions. We show that none of the databases taken alone covers the data sufficiently to enable meaningful analysis, validating our integration effort as essential for gaining better understanding of phase separation and laying the foundations for the discovery of new proteins potentially involved in this important cellular process. Lastly, we developed a server, enabling customized selections of different sets of proteins based on MLO location, database, disorder content, among other attributes (https://forti.shinyapps.io/mlos/).

Dynamic behaviors of minor droplets and the role of bubbles in phase-separating Al[sbnd]Bi immiscible alloy

Minor droplets growth and solute segregation have their peculiarities in phase-separating immiscible alloys and can take complex pathways during solidification. The dynamic behaviors of liquid phase separation and surface enrichment in solidifying Al-10 wt% Bi immiscible alloys were revealed by synchrotron X-ray imaging technique. The large-sized Bi droplets would precipitate from Al-matrix melt directly and almost all of the droplets possess different existing states. The distribution of Bi droplets, resulting from the heterogeneous nucleation and repeatedly nucleation, varies with the growth time during cooling. And the growth dynamics of Bi droplets is revealed to be inconsistent with the Gaussian model. We also found that hydrogen bubbles can prevent the occurrence of the liquid phase separation process.

Green Synthesis of Metal-Organic Framework Bacterial Cellulose Nanocomposites for Separation Applications.

Metal organic frameworks (MOFs) are porous crystalline materials that can be designed to act as selective adsorbents. Due to their high porosity they can possess very high adsorption capacities. However, overcoming the brittleness of these crystalline materials is a challenge for many industrial applications. In order to make use of MOFs for large-scale liquid phase separation processes they can be immobilized on solid supports. For this purpose, nanocellulose can be considered as a promising supporting material due to its high flexibility and biocompatibility. In this study a novel flexible nanocellulose MOF composite material was synthesised in aqueous media by a novel and straightforward in situ one-pot green method. The material consisted of MOF particles of the type MIL-100(Fe) (from Material Institute de Lavoisier, containing Fe(III) 1,3,5-benzenetricarboxylate) immobilized onto bacterial cellulose (BC) nanofibers. The novel nanocomposite material was applied to efficiently separate arsenic and Rhodamine B from aqueous solution, achieving adsorption capacities of 4.81, and 2.77 mg g−1, respectively. The adsorption process could be well modelled by the nonlinear pseudo-second-order fitting.

Ultrasonic cavitation and acoustic streaming effects during liquid phase separation and dynamic solidification of ternary Al–Sn–Si immiscible alloy

Ultrasonic melt treatment is an environmentally friendly and no-polluting external field technology and has great application potential in materials fabrication. Power ultrasound with different intensities was introduced into the liquid phase separation and solidification process of ternary Al-45%Sn-5%Si immiscible alloy to explore the effects of ultrasound on the formation of macro- and microstructure. It was found that the previously apparent macrosegregation caused by the large density difference between immiscible liquid (Al) and (Sn) phases was gradually reduced and even suppressed with the increase of ultrasonic amplitude. Meanwhile, the ultrasonic waves also significantly modified monotectic structures, within which (Al) phase transformed from coarse dendrites to refined equiaxed grains, and (Si) phase evolved from long strips to small blocks. The introduction of ultrasound weakened the texture of solidified structure, whereas the growth orientation relationship between monotectic (Al) and (Si) phases remained unchanged. The theoretical analysis and calculation results indicated that ultrasonic cavitation and acoustic streaming were the dominant effects to modulate the macro- and microstructural evolution. This would improve the exploiting and utilization of immiscible alloys by ultrasonic processing technology.

Experimental study of a pilot membrane desalination system: The effects of transmembrane pressure

Currently, the field of desalination is launched among the domains that are progressing significantly in the cycle of innovation and technological development on a global scale. In particular, the reverse osmosis (RO) desalination technique which is a technique of separation by dense membrane whose driving force is a pressure gradient. This process can be defined as a liquid-phase separation process (seawater or brackish water) by permeation through a highly innovative semi-permeable membrane with a very high freshwater surface productivity by stopping the molecules and the ions of salt and other residues and let water molecules pass through the application of transmembrane pressure which varies depending on the polarization layer of the membrane and the temperature of the system feed solution (Lilane et al., 2020) [1]. The pilot used in this experimental study has the objective of determining the resistance of the membrane, and of studying the effect of the transmembrane pressure on the productive mode of the system in the case of two types of supply solutions (pure and saline water).

Effects of cooling process on the solid–liquid phase separation process in ultra-high-molecular-weight polyethylene/liquid paraffin blends

Ultra-high-molecular-weight polyethylene (UHMWPE) microporous membrane is generally prepared by thermally induced phase separation process. The phase separation process is closely related to the cooling process in practical production. In this paper, the phase separation temperature of UHMWPE/liquid paraffin blends was explored by the hot-stage-optical device and differential scanning calorimeter (DSC), and the result showed that the temperature was about 105–125 °C. The effects of cooling condition and crystallization ability of UHMWPE on the separation process of the blends were also investigated by DSC. The results showed that the phase separation was affected by the cooling rates rather than the initial cooling temperature. And the crystallization of UHMWPE was mainly limited by the nucleating at a low cooling rate, which could form larger porous structure. In a word, it should be inspirational for the actual production of the UHMWPE microporous membrane. The solid–liquid phase separation will occur in the cooling process of UHMWPE/LP blends, which is driven by the external temperature difference. It is significant to understand the phase separation process. In this paper, the effects of the melting properties and cooling condition on phase separation process were investigated, the S–L phase separation range is at 105–125 °C, and the speed of phase separation is mainly related to the external temperature difference and the UHMWPE content. It could be inspirational for actual production of the UHMWPE membrane with more uniform pore structure.

Improved liquid phase separation processes for generating biodegradable microspheres loaded with high concentrations of drugs for tumor embolization

ABSTRACTDrug-eluting microspheres are the most commonly used particulate embolic agents for transarterial chemoembolization of liver tumors. However, drug loading of degradable embolic particles is often insufficient. In the current study, microspheres prepared from poly(D,L-lactic-co-glycolic acid) were investigated as a biodegradable embolic agent for arterial embolization applications. Through modified solvent evaporation and liquid-induced phase separation, high drug loading (as high as 32%) and low solvent residue (as low as 0.03%) was achieved. Degradation performance, 1H NMR, and the nonsolvent’s influence on solvent residue content were assessed. These drug-loading microspheres with biodegradability hold promise for embolization therapies.

Preparation of thermo‐ and redox‐responsive branched polymers composed of three‐armed oligo(ethylene glycol)

ABSTRACTWe herein report the preparation of thermo‐ and redox‐responsive branched polymers by the condensation reaction of three‐armed oligo(ethylene glycol) (trisOEG) and cystamine (CA). The prepared branched polymers exhibited a soluble–insoluble transition at a lower critical solution temperature (LCST) and formed coacervate droplets through a liquid–liquid phase separation process. We then demonstrated control of the LCSTs of the branched polymers by varying the feed ratio of CA and the surrounding salt concentration close to body temperature. In addition, the trisOEG‐cys x polymer formed coacervate droplets above the LCST, in which hydrophobic molecules were condensed. The redox response of the branched polymers was also investigated. Interestingly, the branched polymers degraded to low‐molecular‐weight materials (i.e., trisOEG) in the presence of dithiothereitol as a reducing agent through cleavage of the disulfide bond of CA. This facile preparation of branched polymers is expected to be valuable in the context of functional biomedical materials and modifiers for materials surfaces, such as the bases for drug delivery carriers and separation materials. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018, 56, 2623–2629

Highly Porous Polymer Structures Fabricated via Rapid Precipitation from Ternary Systems

In this study, we present a versatile fabrication route for producing polymeric foams which is different from common phase inversion processes. Highly porous (up to ∼86%) polylactic acid (PLA) structures are produced via a rapid precipitation process wherein nonsolvent hexane is directly incorporated into PLA–dichloromethane solutions. Despite many advantages, this method is underutilized due to a complex correlation between thermodynamics and kinetics during solidification making it challenging to control and understand. We describe the phase separation of these systems as a three-state process which contributes to the current knowledge and understanding of the nonsolvent induced solid–liquid phase separation process. Also, we show that the shish-kebab morphologies formed in certain foams may result in an increase in their compressive modulus. The flexibility of this fabrication route allows for producing highly porous PLA structures for various applications such as acoustic and tissue engineering.