Phase Separation Characteristics Research Articles

Temperature-dependent intracellular crystallization of firefly luciferase in mammalian cells is suppressed by D-luciferin and stabilizing inhibitors

Firefly luciferase (Fluc) from Photinus pyralis is one of the most widely used reporter proteins in biomedical research. Despite its widespread use, Fluc's protein phase transition behaviors and phase separation characteristics have not received much attention. Current research uncovers Fluc's intrinsic property to phase separate in mammalian cells upon a simple cell culture temperature change. Specifically, Fluc spontaneously produced needle-shaped crystal-like inclusion bodies upon temperature shift to the hypothermic temperatures ranging from 25 °C to 31 °C. The crystal-like inclusion bodies were not associated with or surrounded by membranous organelles and were likely built from the cytosolic pool of Fluc. Furthermore, the crystal-like inclusion formation was suppressed when cells were cultured in the presence of D-luciferin and its synthetic analog, as well as the benzothiazole family of so-called stabilizing inhibitors. These two classes of compounds inhibited intracellular Fluc crystallization by different modes of action as they had contrasting effects on steady-state luciferase protein accumulation levels. This study suggests that, under substrate insufficient conditions, the excess Fluc phase separates into a crystal-like state that can modulate intracellular soluble enzyme availability and protein turnover rate.

YTHDF3 phase separation regulates HSPA13-dependent clear cell renal cell carcinoma development and immune evasion.

Insufficient understanding about the immune evasion mechanism leads to the inability in predicting current immunotherapy effects in clear cell renal cell carcinoma (ccRCC) and sensitizing ccRCC to immunotherapy. RNA binding proteins (RBPs) can promote tumor progression and immune evasion. However, research on RBPs, particularly m6A reader YTHDF3, in ccRCC development and immune evasion is limited. In this study, we found that YTHDF3 level was downregulated in ccRCC and was an independent prognostic biomarker for ccRCC. Decreased YTHDF3 expression was correlated with the malignancy, immune evasion, and poor response to anti-programmed death ligand 1 (PD-L1)/CTLA-4 in ccRCC. YTHDF3 overexpression restrained ccRCC cell malignancy, PD-L1 expression, CD8+ T cell infiltration and activities invivo, indicating its inhibitory role in ccRCC development and immune evasion. Mechanistically, YTHDF3 WT was found to have phase separation characteristics and suppress ccRCC malignancy and immune evasion. Whereas YTHDF3 mutant, which disrupted phase separation, abolished its function. YTHDF3 enhanced the degradation of its target mRNA HSPA13 by phase separation and recruiting DDX6, resulting in the downregulation of the downstream immune checkpoint PD-L1. HSPA13 overexpression restored ccRCC malignancy and immune evasion suppressed by YTHDF3 overexpression. In all, our results identify a new model of YTHDF3 in regulating ccRCC progression and immune evasion through phase separation.

Liquid-liquid phase separation of microtubule-binding proteins in the regulation of spindle assembly.

Cell division is a highly regulated process essential for the accurate segregation of chromosomes. Central to this process is the assembly of a bipolar mitotic spindle, a highly dynamic microtubule (MT)-based structure responsible for chromosome movement. The nucleation and dynamics of MTs are intricately regulated by MT-binding proteins. Over the recent years, various MT-binding proteins have been reported to undergo liquid-liquid phase separation, forming either single- or multi-component condensates on MTs. Herein, we provide a comprehensive summary of the phase separation characteristics of these proteins. We underscore their critical roles in MT nucleation, spindle assembly and kinetochore-MT attachment during the cell division process. Furthermore, we discuss the current challenges and various remaining unsolved problems, highlights the ongoing research efforts aimed at a deeper understanding of the role of the phase separation process during spindle assembly and orientation. Our review aims to contribute to the collective knowledge in this area and stimulate further investigations that will enhance our comprehension of the intricate mechanisms governing cell division.

Enhancement of Phase Stability and Thermoelectric Performance of Meta‐Stable AgSbTe2 by Thermal Cycling Process

AbstractThermoelectric materials are crucial components in thermoelectric devices employed for environmentally friendly solid‐state cooling and waste heat recovery, demanding not only high performance but also superior thermal or phase stability. The high‐performance thermoelectric material AgSbTe2 encounters challenges when utilized in thermoelectric modules due to the precipitation of Ag2Te, resulting in both thermally unstable characteristics and diminished performance. In this study, a thermal cycling process is employed to enhance the thermal stability and thermoelectric performance of AgSbTe2. Through thermal cycling, secondary phases of AgSbTe2 are made uniform, and the undesired Ag2Te is substituted with Sb2Te3 using an optimized thermal cycling process. As a result, the thermal stability of AgSbTe2 is enhanced due to its meta‐stable state, with <5% variance in thermoelectric figure of merit (ZT) measurements, and the ZT value is raised from 0.8 to 1.7 at 643 K through the optimized thermal cycling. The findings indicate that thermal cycling is an effective strategy for enhancing the thermal stability and thermoelectric performance of AgSbTe2, presenting a novel approach for achieving uniform secondary phases in materials with phase separation or phase change characteristics.

Electron irradiation-induced elements depletion and macroscopic changes in ternary borosilicate glasses: Mechanism and composition dependence

Glass, utilized for immobilizing high-level radioactive waste, experiences a decrease in mechanical properties and an increased risk of radioactive release after being exposed to β-radiation. A series of ternary borosilicate glasses were irradiated with 10 MeV electrons to doses ranging from 1 × 104 to 1 × 107 Gy. Nano-indentation, X-ray photoelectron spectroscopy, Time-of-Flight secondary ion mass spectrometry and Fourier-Transform infrared spectroscopy were used to characterize the glasses. After irradiation, the hardness of the glass decreased dramatically; the depletion of sodium, boron, oxygen and the enrichment of hydrogen, carbon in the glass surface were observed. The diffusion coefficients and depletion depths at different doses were fitted, both related to the composition of glass. With the absorbed dose increasing, Q0 ∼ Q2 transformed to Q3 and Q4, BO3 and BO4 decreased, molecular water and hydroxyl appeared. The formation of the depletion zone caused the reduction in hardness induced by irradiation. The glass showed phase separation characteristics. The mechanism and detailed process of element depletion were discussed. The composition dependence of depletion was explained based on the energy barrier model. The diffusion coefficient of elements depends on the activation energy of alkali.

Effects of the heterogeneous hydration of alkaline-earth aluminosilicate glasses on phase evolution in hardened blended cement paste

Supplementary cementitious materials (SCMs) affect the hydration process and long-term properties of cementitious systems. Since a large percentage of SCMs belong to alkaline-earth aluminosilicate glasses that possess a phase-separated structure, further research into their hydration characteristics is essential. This study investigated the effects of the hydration characteristics of synthetic glasses with various phase-separated structure on the phase evolution of cementitious systems by combining simulations with experiments. The results showed that with the increasing degree of phase separation, the hydration of synthetic glass transitions from homogeneous to heterogeneous hydration. The phase evolution of hardened paste, particularly for calcium hydroxide, is influenced by the difference in hydration rates among each separated glassy phase. Compared to homogeneous hydration, the rapid hydration of the Ca-rich phase reduced the consumption of calcium hydroxide, while the rapid hydration of the Si-rich phase accelerated calcium hydroxide consumption. For glasses exhibiting phase separation characteristics, the consistency between thermodynamic simulations employing a heterogeneous hydration model and experimental results exhibits a notable enhancement.

Effect of TiB2 on the second Bi-rich phase in immiscible Al-Bi alloy

Immiscible alloys are easy to form segregation due to their liquid phase separation characteristics. Refractory solid particles have been proved can inhibit the segregation of the second phase in this type of alloy during solidification process by either promoting nucleation or preventing the coagulation of the second phase. However, it is not clear that whether the solid particle could provide a steric hindrance effect after serving as nucleation particles. In the present work, the refining mechanism of TiB2 solid particles on the second Bi-rich phase in Al92Bi8 alloy was disclosed in both the terms of heterogeneous nucleation and steric hindrance effect. The results showed that TiB2 particles have remarkable refining effect on the second Bi-rich phase in Al92Bi8 alloy. TiB2 particles first act as nucleation particles to promote nucleation of the liquid Bi-rich phase, then migrate to and self-assemble on the interface between the liquid Bi-rich phase and the Al matrix to reduce the system interfacial energy, furtherly providing steric hindrance effect to prevent the collision and coagulation of the liquid Bi-rich phase.

Superconductivity of Li doped BSCCO mesoscopic fiber

Mesoscopic Li doped BSCCO fibres are synthesized by the electrospinning technology and the sequent sintering treatment. With the assistance of high resolution energy-dispersive x-ray spectroscopy detector in the Aberration-corrected transmission electron microscopy, we discovered hidden CuO grains and separated Bi-2212 phases, which is proved by the existence of fishtail effect in the magnetization loops. The electric measurement is fulfilled via four probe method with the help of focus ion beam operation. A non-trivial resistance behaviour appears, which can be attributed to its polycrystalline and superconducting phase separation features.

Breaking the trade-off between selectivity and permeability of nanocomposite membrane modified by UIO66@PDA through nonsolvent thermally induced phase separation method

Developing polymer membranes with high permeability, fouling resistance, and rejection capability is a challenging task that requires the use of novel materials to enhance these properties. In this study, composite ultrafiltration membranes were fabricated by incorporating two types of metal–organic frameworks (MOFs), UIO66 and UIO66@PDA, into polyvinylidene fluoride (PVDF) using the nonsolvent thermally induced phase separation (NTIPS) method. The results demonstrated that the NTIPS method combined the characteristics of nonsolvent-induced phase separation (NIPS) and thermally induced phase separation (TIPS) to fabricate asymmetric membranes with a dense ultra-thin top layer and a bicontinuous network structure. Moreover, the introduction of 1.0 wt% UIO66@PDA into PVDF membranes yielded notable improvements in mechanical strength, thermal stability, and porosity. Concurrently, this led to a water flux rise from 182.5 to 464.7 L·m−2·h−1, and enhanced BSA and HA rejection to 96% and 83%, respectively. Additionally, there was a significant improvement in the membrane's antifouling capacity, achieving a water flux recovery ratio of 93%. This was facilitated by the increased hydrophilicity of UIO66@PDA, which attracted more water molecules and reduced membrane fouling. This work offers a solution to overcome the permeability-selectivity trade-off in membrane separation by hydrophilic modification of hydrophobic membranes, while also addressing fouling issues.

Phase separation of intrinsically disordered FG-Nups is driven by highly dynamic FG motifs

The intrinsically disordered FG-Nups in the central channel of the nuclear pore complex (NPC) form a selective permeability barrier, allowing small molecules to traverse by passive diffusion, while large molecules can only translocate with the help of nuclear transport receptors. The exact phase state of the permeability barrier remains elusive. In vitro experiments have shown that some FG-Nups can undergo phase separation into condensates that display NPC-like permeability barrier properties. Here, we use molecular dynamics simulations at amino acid resolution to study the phase separation characteristics of each of the disordered FG-Nups of the yeast NPC. We find that GLFG-Nups undergo phase separation and reveal that the FG motifs act as highly dynamic hydrophobic stickers that are essential for the formation of FG-Nup condensates featuring droplet-spanning percolated networks. Additionally, we study phase separation in an FG-Nup mixture that resembles the NPC stoichiometry and observe that an NPC condensate is formed containing multiple GLFG-Nups. We find that the phase separation of this NPC condensate is also driven by FG-FG interactions, similar to the homotypic FG-Nup condensates. Based on the observed phase separation behavior, the different FG-Nups of the yeast NPC can be divided into two classes: The FG-Nups (mostly GLFG-type) located in the central channel of the NPC form a highly dynamic percolated network formed by many short-lived FG-FG interactions, while the peripheral FG-Nups (mostly FxFG-type) at the entry and exit of the NPC channel likely form an entropic brush.

Physical Concept to Explain the Regulation of Lipid Membrane Phase Separation under Isothermal Conditions.

Lateral phase separation within lipid bilayer membranes has attracted considerable attention in the fields of biophysics and cell biology. Living cells organize laterally segregated compartments, such as raft domains in an ordered phase, and regulate their dynamic structures under isothermal conditions to promote cellular functions. Model membrane systems with minimum components are powerful tools for investigating the basic phenomena of membrane phase separation. With the use of such model systems, several physicochemical characteristics of phase separation have been revealed. This review focuses on the isothermal triggering of membrane phase separation from a physical point of view. We consider the free energy of the membrane that describes lateral phase separation and explain the experimental results of model membranes to regulate domain formation under isothermal conditions. Three possible regulation factors are discussed: electrostatic interactions, chemical reactions and membrane tension. These findings may contribute to a better understanding of membrane lateral organization within living cells that function under isothermal conditions and could be useful for the development of artificial cell engineering.

Development of a three-stage, cross-current air sparged mixer-settler for the purification of degraded PUREX solvent

ABSTRACT Tri-butyl phosphate (TBP) diluted with in n-dodecane (n-DD) is used as the solvent in PUREX process. Solvent degradation due to radiolysis and hydrolysis during the process is one of the major problems prevailing in reprocessing plants. This paper presents an overview of the design, fabrication, and performance evaluation of a three-stage, continuous, cross-current air sparged mixer-settler developed for purifying the degraded PUREX solvent using hydrazine carbonate. The developmental program consisted of various stages, starting with small-scale batch experiments to investigate the solvent treatment kinetics and phase separation characteristics, followed by design fabrication and performance evaluation of three-stage air sparged mixer-settler. The study on solvent treatment kinetics in a 1.6 L batch air sparged mixer using 1.5 M hydrazine carbonate showed that the concentration of di-butyl phosphate (DBP) in the simulated degraded solvent could be reduced from 8,000 mg L−1 to less than 40 mg L−1 within 180 seconds. Performance evaluation of the three-stage air sparged mixer settler had shown that the DBP concentration in the simulated degraded organic phase could be brought down from 8,000 mg L−1 to less than 15 mg L−1, and the equipment could process up to 60 L h−1 throughput with negligible phase entrainment.

Systematic Design of a Graphene Ink Formulation for Aerosol Jet Printing.

Aerosol jet printing is a noncontact, digital, additive manufacturing technique compatible with a wide variety of functional materials. Although promising, development of new materials and devices using this technique remains hindered by limited rational ink formulation, with most recent studies focused on device demonstration rather than foundational process science. In the present work, a systematic approach to formulating a polymer-stabilized graphene ink is reported, which considers the effect of solvent composition on dispersion, rheology, wetting, drying, and phase separation characteristics that drive process outcomes. It was found that a four-component solvent mixture composed of isobutyl acetate, diglyme, dihydrolevoglucosenone, and glycerol supported efficient ink atomization and controlled in-line drying to reduce overspray and wetting instabilities while maintaining high resolution and electrical conductivity, thus overcoming a trade-off in deposition rate and resolution common to aerosol jet printing. Biochemical sensors were printed for amperometric detection of the pesticide parathion, exhibiting a detection limit of 732 nM and a sensitivity of 34 nA μM-1, demonstrating the viability of this graphene ink for fabricating functional electronic devices.

Incorporation of 53BP1 into phase-separated bodies in cancer cells during aberrant mitosis.

53BP1 (also known as TP53BP1) is a key mediator of the non-homologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid-liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. This article has an associated First Person interview with the first author of the paper.

Protein Phase Separation: New Insights into Carcinogenesis

Phase separation is now acknowledged as an essential biologic mechanism wherein distinct activated molecules assemble into a different phase from the surrounding constituents of a cell. Condensates formed by phase separation play an essential role in the life activities of various organisms under normal physiological conditions, including the advanced structure and regulation of chromatin, autophagic degradation of incorrectly folded or unneeded proteins, and regulation of the actin cytoskeleton. During malignant transformation, abnormally altered condensate assemblies are often associated with the abnormal activation of oncogenes or inactivation of tumor suppressors, resulting in the promotion of the carcinogenic process. Thus, understanding the role of phase separation in various biological evolutionary processes will provide new ideas for the development of drugs targeting specific condensates, which is expected to be an effective cancer therapy strategy. However, the relationship between phase separation and cancer has not been fully elucidated. In this review, we mainly summarize the main processes and characteristics of phase separation and the main methods for detecting phase separation. In addition, we summarize the cancer proteins and signaling pathways involved in phase separation and discuss their promising future applications in addressing the unmet clinical therapeutic needs of people with cancer. Finally, we explain the means of targeted phase separation and cancer treatment.

Aquivion-based anion exchange membranes: Synthesis optimization via dispersant agents and reaction time

Alkaline membrane water electrolysis fed by renewable energy is a promising technology to produce “green” hydrogen for a variety of applications. Thanks to their unique characteristics of chemical, thermal and electrochemical stability, perfluorinated polymers are proposed as an alternative to hydrocarbon-based anion exchange membranes (AEMs) for applications in water electrolysers. A simple two-step functionalization reaction to introduce quaternary ammonium groups onto Aquivion® perfuorinated backbone is reported using a low toxicity dispersant. The most appropriate dispersant (Novec 7500) and reaction parameters (5 °C for 2 hrs) are selected. The complete conversion of the precursors into a quaternary ammonium salt is confirmed by a solid-state NMR study. Physico-chemical properties, thermal behavior and anion conductivity of the formed AEM are investigated. The phase separation characteristics of this perfluorinated polymer allow to reach a membrane ion mobility (μeff) 1.99 10−4 cm2 V−1 s−1, one order of magnitude higher than the FAA3-50 commercial membrane, meaning an enhanced ion dissociation. A very limited degradation of functional groups is demonstrated after immersion in alkaline solution, with only 2 % of ion exchange capacity (IEC) reduction against 14 % of the FAA3-50 commercial membrane. An electrolysis current density of 0.9 A cm−2 at 2.2 V and 90 °C, is achieved showing very promising applications for green hydrogen production.

Extraordinary Solidification Mechanism of Liquid Alloys Under Acoustic Levitation State.

The acoustic levitation of various materials can be realized by highly intensive ultrasound, which provides a free surface and containerless state for materials processing under space simulation conditions. The nonlinear effects such as acoustic radiation pressure, acoustic streaming, and ultrasonic cavitation open up special access to modulate the fluid dynamics and solidification mechanisms of liquid materials. Here, the physical characteristics of liquid flow, undercooling capability, phase separation, and crystal nucleation and growth within acoustically levitated droplets are explored comprehensively to reveal the extraordinary solidification kinetics of liquid alloys. The sectorial shape oscillations of the 2nd to 10th order modes accompanying internal potential flow are observed for water droplets with modulated ultrasound amplitudes, while the enhanced ultrasound intensity promotes ice nucleation and thus reduces water undercooling. The migration of Sn-rich globules during phase separation of immiscible Al-Cu-Sn alloy is dominated by the droplet deformation and rotation related to acoustic levitation. The high undercooling states of liquid Ag-Cu-Ge and Ni-Sn alloys during acoustic levitation result in the refinement of (Ag) dendrites and the formation of anomalous (Ni+Ni3 Sn) eutectics. The ultrasound-liquid interaction also induces surface waves during the containerless solidification of Ag-Cu and Ni-Sn eutectic alloys.

Synthesis of melamine-based crystalline porous polymers and its silver-doped composites with one-pot approach for catalytic reduction of 4-nitrophenol

Melamine-based covalent organic frameworks are of great concern due to the low cost of melamine but difficult to synthesize in traditional solvothermal methods without the help of structural isomerization, meanwhile having limited application domains because of strong hydrophobicity. Herein, we present a robust synthesis of melamine-based crystalline porous polymer MBCPP and its nano silver-doped composites with one-pot strategy for catalytic reduction of 4-nitrophenol. With silver nanoparticles doping, the porous structure of organic frameworks becomes more regular, and the catalysts present different phase separation and specific catalytic characteristics for equivalent reduction of 4-nitrophenol, revealing the catalytic mechanism of electron transfer explained by slow diffusion inside the catalysts. Moreover, this work displays a creative metal doping strategy for the combination of metal and COFs.

Assessing the Phase Separation Propensity of Proteins in Living Cells Through Optodroplet Formation.

Phase separation is emerging as a key mechanism to describe the formation of membraneless organelles in the cell. It depends on the multivalent (self-) interaction properties of the macromolecules involved and can be observed in aqueous solutions under controlled conditions in vitro with purified components. However, to experimentally demonstrate that this process indeed occurs in the complex environment of living cells remains difficult. Here, we describe an assay based on light-induced association of proteins into complexes termed optodroplets that are in the hundred nm to μm size range. The formation and dissociation of these optodroplets can be followed over time in living cells by fluorescence microscopy to evaluate the propensity of proteins to demix and to form phase-separated subcompartments. The optodroplet assay is based on the fusion of a protein of interest with the photolyase homology region (PHR) protein domain from Arabidopsis thaliana, which can undergo reversible homo-oligomerization upon illumination with blue light. Using this approach, candidate proteins and their interaction-deficient or interaction-enhanced variants can be compared to each other or to reference proteins with known phase separation features. By quantifying the resulting microscopy images, the propensity of a given protein construct to assemble into a phase-separated subcompartment can be assessed.

The Laser Deposited Nickel-Aluminum Bronze Coatings on SUS630 Stainless Steel and Its Corrosion Resistance in 3.5 wt.% NaCl Solution

In this work, three composite structures of nickel-aluminum bronze (NAB) bonded with SUS630 stainless steel with different intermediate layers were fabricated by laser deposition. The microstructure and corrosion behavior of NAB in 3.5 wt.% NaCl solution were studied. The NAB coating directly deposited on steel substrate contains a large number of Fe-rich dendrites due to the dilution by laser energy and the Cu-Fe liquid phase separation characteristics. The microstructures of NAB were independent and isolated well from the steel substrate when the nickel intermediate layer was applied. Immersion corrosion and electrochemical tests indicated that the composite structure with the nickel intermediate layer presented better corrosion resistance than direct deposited the NAB coating, especially with the NiCr alloy intermediate layer, which led to a shallower corrosion depth and formed a denser layer of protective corrosion products.