Numerical simulation of the Liquid-liquid phase separation and microstructure evolution of Al-In immiscible alloys during cooling

A numerical model has been developed to describe the microstructural evolution of Al In immiscible alloys through the miscibility gap. The model considers the common action of nucleation, diffusible growth, Brownian collision and motion collision between the second phase droplets. The simulation results are dynamically visualized and show that the volume fraction, distribution and size of the second phase droplets satisfactorily agree with the experimental results. So the model can be used to predict the microstructural evolution of Al In immiscible alloys during the cooling process.

Preparation of Microcapsules with Liquid Droplet Coalescence Method Followed by Phase Separation

Novel preparation method of microencapsules was developed on the basis of the liquid coalescence method followed by phase separation. Oil droplets of limonene dissolving expanded polystyrene as a shell material were forced to collide and coalesce with the Isopar oil droplets of core material in the continuous wates phase. When two kinds of oil droplets are collided and coalesced with each other, expanded polystyrene dissolved in the limonene oil may be phase-separated in the oil droplets newly formed to form the microcapsule shell, because the Isopar oil was a poor solvent for expanded polystyrene but a good solvent for the limonene oil. In the experiment, the diameter (or number) of limonene oil droplets dissolving expanded polystyrene was mainly changed, because the coalescence frequency between the droplets is strongly dependent on the number of droplets. Favorable core shell types of microcapsules with the shell thickness from 1.0 to 5.0 μm were able to be prepared under all the experimental conditions adopted here.

Multistep Desolvation as a Fundamental Principle Governing Peptide Self-Assembly Through Liquid-Liquid Phase Separation

Biomolecular self-assembly based on peptides and proteins is a general phenomenon encountered in natural and synthetic systems.Liquid–liquid phase separation(LLPS)is intimately involved in biomolecular self-assembly,yet the key factors at a molecular scale activating or modulating such a process remain largely elusive.Herein,we discovered in our experiments that multistep desolvation is fundamental to the formation and evolution of peptide-rich droplets:The first step was partial desolvation of peptides to form peptide clusters,and the second step was selective desolvation of hydrophobic groups within clusters to trigger LLPS and the formation of peptiderich droplets,followed by complete desolvation of droplets,initiating the nucleation of peptide selfassembly.Manipulation of the degree of desolvation at different stages was an effective strategy to control the self-assembly pathways and polymorphisms.This study sheds light on the molecular origin of LLPS-mediated self-assembly distinct from classical one-step self-assembly and paves the way for the precise control of supramolecular self-assembly.

Plant RNA-binding proteins:Phase separation dynamics and functional mechanisms underlying plant development and stress responses

RNA-binding proteins(RBPs)accompany RNA from synthesis to decay,mediating every aspect of RNA metabolism and impacting diverse cellular and developmental processes in eukaryotes.Many RBPs undergo phase separation along with their bound RNA to form and function in dynamic membraneless biomolecular condensates for spatiotemporal coordination or regulation of RNA metabolism.Increasing evidence suggests that phase-separating RBPs with RNA-binding domains and intrinsically disordered regions play important roles in plant development and stress adaptation.Here,we summarize the current knowledge about how dynamic partitioning of RBPs into condensates controls plant development and enables sensing of experimental changes to confer growth plasticity under stress conditions,with a focus on the dynamics and functional mechanisms of RBP-rich nuclear condensates and cytoplasmic granules in mediating RNA metabolism.We also discuss roles of multiple factors,such as environmental signals,protein modifications,and N6-methyladenosine RNA methylation,in modulating the phase separation behaviors of RBPs,and highlight the prospects and challenges for future research on phase-separating RBPs in crops.

LncFASA promotes cancer ferroptosis via modulating PRDX1 phase separation

Ferroptosis, a unique type of non-apoptotic cell death resulting from iron-dependent lipid peroxidation, has a potential physiological function in tumor suppression, but its underlying mechanisms have not been fully elucidated. Here, we report that the long non-coding RNA(lncRNA) LncFASA increases the susceptibility of triple-negative breast cancer(TNBC) to ferroptosis. As a tumor suppressor, LncFASA drives the formation of droplets containing peroxiredoxin1(PRDX1), a member of the peroxidase family, resulting in the accumulation of lipid peroxidation via the SLC7A11-GPX4 axis. Mechanistically, LncFASA directly binds to the Ahpc-TSA domain of PRDX1, inhibiting its peroxidase activity by driving liquid-liquid phase separation, which disrupts intracellular ROS homeostasis. Notably, high LncFASA expression indicates favorable overall survival in individuals with breast cancer, and LncFASA impairs the growth of breast xenograft tumors by modulating ferroptosis. Together, our findings illustrate the crucial role of this lncRNA in ferroptosis-mediated cancer development and provide new insights into therapeutic strategies for breast cancer.

Disruption of PABPN1 phase separation by SNRPD2 drives colorectal cancer cell proliferation and migration through promoting alternative polyadenylation of CTNNBIP1

Generally shortened 3′UTR due to alternative polyadenylation(APA)is widely observed in cancer,but its regulation mechanisms for cancer are not well characterized.Here,with profiling of APA in colorectal cancer tissues and poly(A)signal editing,we firstly identified that the shortened 3′UTR of CTNNIBP1 in colorectal cancer promotes cell proliferation and migration.We found that liquid-liquid phase separation(LLPS)of PABPN1 is reduced albeit with higher expression in cancer,and the reduction of LLPS leads to the shortened 3′UTR of CTNNBIP1and promotes cell proliferation and migration.Notably,the splicing factor SNRPD2 upregulated in colorectal cancer,can interact with glutamic-proline(EP)domain of PABPN1,and then disrupt LLPS of PABPN1,which attenuates the repression effect of PABPN1 on the proximal poly(A)sites.Our results firstly reveal a new regulation mechanism of APA by disruption of LLPS of PABPN1,suggesting that regulation of APA by interfering LLPS of 3′end processing factor may have the potential as a new way for the treatment of cancer.

The pathogenic mechanism of TAR DNA-binding protein 43(TDP-43)in amyotrophic lateral sclerosis

The onset of amyotrophic lateral sclerosis is usually characterized by focal death of both upper and/or lower motor neurons occurring in the motor cortex,basal ganglia,brainstem,and spinal cord,and commonly involves the muscles of the upper and/or lower extremities,and the muscles of the bulbar and/or respiratory regions.However,as the disease progresses,it affects the adjacent body regions,leading to generalized muscle weakness,occasionally along with memory,cognitive,behavioral,and language impairments;respiratory dysfunction occurs at the final stage of the disease.The disease has a complicated pathophysiology and currently,only riluzole,edaravone,and phenylbutyrate/taurursodiol are licensed to treat amyotrophic lateral sclerosis in many industrialized countries.The TAR DNA-binding protein 43 inclusions are observed in 97%of those diagnosed with amyotrophic lateral sclerosis.This review provides a preliminary overview of the potential effects of TAR DNAbinding protein 43 in the pathogenesis of amyotrophic lateral sclerosis,including the abnormalities in nucleoplasmic transport,RNA function,post-translational modification,liquid-liquid phase separation,stress granules,mitochondrial dysfunction,oxidative stress,axonal transport,protein quality control system,and non-cellular autonomous functions(e.g.,glial cell functions and prion-like propagation).

Phase separation in cGAS-STING signaling

Biomolecular condensates formed by phase separation are widespread and play critical roles in many physiological and pathological processes.cGAS-STING signaling functions to detect aberrant DNA signals to initiate anti-infection defense and antitumor immunity.At the same time,cGAS-STING signaling must be carefully regulated to maintain immune homeostasis.Interestingly,exciting recent studies have reported that biomolecular phase separation exists and plays important roles in different steps of cGAS-STING signaling,including cGAS condensates,STING condensates,and IRF3 condensates.In addition,several intracellular and extracellular factors have been proposed to modulate the condensates in cGAS-STING signaling.These studies reveal novel activation and regulation mechanisms of cGAS-STING signaling and provide new opportunities for drug discovery.Here,we summarize recent advances in the phase separation of cGAS-STING signaling and the development of potential drugs targeting these innate immune condensates.

Phase separation-induced nanoprecipitation for making polymer nanoparticles with high drug loading Special Collection:Distinguished Australian Researchers

Increasing drug loading remains a critical challenge in the development and translation of nanomedicine.High drug-loading nanoparticles have demonstrated unique advantages such as less carrier material used,better-controlled drug release,and improved efficacy and safety.Herein,we report a simple and efficient salt concentration screening method for making polymer nanoparticles with exceptionally high drug loading(up to 66.5 wt%)based on phase separation-induced nanoprecipitation.Upon addition of salt,phase separation occurs in a miscible solvent-water solution delaying the precipitation time of drugs and polymers to different extents,facilitating their co-precipitation thus the formation of high drug-loading nanoparticles with high encapsulation efficiency(>90%)and excellent stability(>1 month).This technology is versatile and easy to be adapted to various hydrophobic drugs,different polymers,and solvents.This salt-induced nanoprecipitation strategy offers a novel approach to fabricating polymer nanoparticles with tunable drug loading,and opens great potentials for future nanomedicines.

Regulating FUS Liquid-Liquid Phase Separation via Specific Metal Recognition

Liquid-liquid phase separation(LLPS)or biomolecular condensation that leads to formation of membraneless organelles plays a critical role in many biochemical processes.Mechanism study of regulating LLPS is therefore central to the understanding of protein aggregation and disease-relevant process.We report a fused in sarcoma protein(FUS)-derived low complexity(LC)sequence that undergoes LLPS in the presence of metal ions.The LC protein was constructed by fusing a hexhistidine-tag to the N-terminal low complexity domain(the residues 1–165 in QGSY-rich segment)of FUS.Spontaneous condensation of the intrinsic disordered protein into coacervate droplets was observed in the presence of metal ions that chelate oligohistidine moieties to form protein matrix.We demonstrate the key role of metal ion-histidine coordination in governing LLPS behaviours and the fluidity of biomolecular condensates.By taking advantage of competitive binding using chelators,we show the possibility of regulating dynamic behaviors of disease-relevant protein droplets,and developing a potential approach towards controllable biological encapsulation/release.

Self-assembly of Amphiphilic Diblock Copolymers Induced by Liquid-Liquid Phase Separation

The liquid-liquid phase separation(LLPS)widely exists in biology,synthetic chemistry,crystallization kinetics and other fields,and it is very important to realize the related functions.The research on the competition between LLPS and micellization/vesiculation has made considerable progress.However,the way to effectively control the formation paths from homogeneous state to aggregates has not been completely solved,which is vital to determine its structure and properties and even its future functions.Here we describe the phenomenon of LLPS and its effect on the dynamic process of self-assembly of amphiphilic diblock copolymers(BCPs).Starting from the establishment of phase diagram,we explore the existence conditions of LLPS state,the internal morphology and external size of large droplets,and its significant implications to the dynamic path of vesicle formation.Vesicles formed via LLPS have larger sized outer dimensions and inner cavities,and contain more solvents during certain stages.The detailed research of LLPS and its self-assembly simulation has contributed to completing its theoretical basis and practical applications in the future in various fields.

Spatial heterogeneity in liquid–liquid phase transition

Molecular dynamics simulations are performed to investigate the liquid-liquid phase transition （LLPT） and the spatial heterogeneity in A1-Pb monotectic alloys. The results reveal that homogeneous liquid AI-Pb alloy undergoes an LLPT, separating into Al-rich and Pb-rich domains, which is quite different from the isocompositional liquid water with a transition between low-density liquid （LDL） and high-density liquid （HDL）. With spatial heterogeneity becoming large, LLPT takes place correspondingly. The relationship between the cooling rate, relaxation temperature and percentage of A1 and the spatial heterogeneity is also reported. This study may throw light on the relationship between the structure heterogeneity and LLPT, which provides novel strategies to control the microstructures in the fabrication of the material with high performance.

Liquid-liquid phase separation of Arabidopsis transcriptional repressor VRN1

With the support by the National Natural Science Foundation of China and the Ministry of Science and Technology and China,the research team led by Prof.Lai LuHua(来鲁华)at BNLMS,College of Chemistry and Molecular Engineering,Peking-Tsinghua Center for Life Sciences,and Center for Quantitative Biology,Peking University recently reported that Arabidopsis transcriptional repressor VRN1undergoes liquid-liquid phase separation with DNA in Angew Chem Int Ed(2019,58:4858—4862).This research uncovers the mechanism of DNA induced VRN1phase separation and provides novel insight of phase separation mediated transcriptional repression.Zhou HuaBin,agraduate student from Lai's group,is the first author of this paper.

相分离调控植物胁迫感知和应答的研究进展

生物大分子凝聚体(biomolecular condensates)是由相分离(phase separation)驱动形成的具有特定功能的无膜细胞器(MLOs),为特定的生化反应提供微环境,对细胞生命活动进行精细的时空调控。相分离是一种高度动态的过程,对温度、pH、盐浓度等理化因素的变化非常敏感。因此,相分离可以快速响应外界刺激,作为生物感受器感知压力信号,参与胁迫应答。近年来,相分离在植物中的应用受到越来越多的关注,特别是在胁迫感知和逆境响应方面。本文基于近期关于相分离响应胁迫的研究,探讨了相分离在胁迫信号感知和应答中的机制,综述了相分离在植物响应胁迫的研究成果,旨在为进一步研究植物胁迫感知和逆境响应中相分离的作用提供参考依据。

Phase-separated bienzyme compartmentalization as artificial intracellular membraneless organelles for cell repair

Implanting artificial organelles in living cells is capable of correcting cellular dysfunctionalities for cell repair and biomedical applications. In this work, phase-separated bienzyme-loaded coacervate microdroplets are established as a model of artificial membraneless organelles in endothelial dysfunctional cells for the cascade enzymatic production of nitric oxide(NO) with a purpose of correcting cellular NO deficiency. We prepared the coacervate microdroplets via liquid-liquid phase separation of oppositely charged polyelectrolytes, in which glucose oxidase/horseradish peroxidase-mediated cascade reaction was compartmented. After the coacervate microdroplets were implanted in NO-deficient dysfunctional cells, the compartments maintained a phase-separated liquid droplet structure, which facilitated a significant enhancement of NO production in the dysfunctional cells. The recovery of NO production was further exploited to inhibit clot formation in blood plasma located in the cell suspension. This demonstrated a proof-of-concept design of artificial organelles in dysfunctional cells for cell repair and anticoagulation-related medical applications. Our results demonstrate an approach for the construction of coacervate droplets through phase separation for the generation of artificial membraneless organelles, which can be designed to provide an array of functionalities in living organisms that have the potential to be used in the field of cell engineering and medical therapy.

液⁃液相分离与神经系统退行性疾病

细胞区室化(compartmentation)有助于分隔不同的生化反应,使其相互不产生干扰。有膜细胞器是通过生物膜把细胞内不同的空间分隔,进而实现细胞区室化。研究发现,细胞区室化也会通过细胞中无膜细胞器实现,但核仁等无膜区室的形成机制一直未得到明确。“液-液相分离”(liquid-liquid phase separation,LLPS)机制的发现,为解开无膜区室的谜题提供了全新的思路。现在认为,多种蛋白质和RNA也是通过LLPS机制形成局部的高浓度冷凝物,发挥更强或独特的基因表达调控、细胞信号转导等功能。LLPS的形成主要依赖于蛋白质和/或核酸之间的多价态非共价键相互作用,主要包括由低复杂序列区域介导的相分离及由多个重复结构域间特异性作用介导的相分离。组分浓度、pH和翻译后修饰等条件均能改变分子多价相互作用的强度,从而调节相分离和相变过程。蛋白质相分离的失调与肌萎缩性侧索硬化症、阿尔茨海默病、亨廷顿舞蹈症和帕金森病等多种神经退行性疾病的发生有关。在这些退行性疾病中,已发现TDP-43、FUS、Ataxin-2、Tau蛋白等致病基因的突变,以及修饰会导致LLPS异常而形成病理特征性的聚集体。但这些聚集体是否是神经退行性致病的直接致病因素尚未明确。

PABP-driven secondary condensed phase within RSV inclusion bodies activates viral mRNAs for ribosomal recruitment

Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and viral mRNAs are concentrated.However,the mechanism of IBAG formation and the physiological function of IBAGs are unclear.Here,we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein(mRNP)condensates formed by secondary LLPS.Mechanistically,the RSV nucleoprotein(N)and M2-1 interact with and recruit PABP to IBs,promoting PABP to bind viral mRNAs transcribed in IBs by RNArecognition motif and drive secondary phase separation.Furthermore,PABP-eIF4G1 interaction regulates viral mRNP condensate composition,thereby recruiting specific translation initiation factors(eIF4G1,eIF4E,eIF4A,eIF4B and eIF4H)into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment.Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase.

生物分子液-液相分离的物理化学机制

近年来,有关生物分子通过液-液相分离机制进行组织定位、功能调控的研究发展迅速。相分离产生的聚集体在众多细胞活动事件中发挥了关键作用。这些聚集体的生物功能是以相分离的物理化学性质为基础的。本文将从相分离聚集体的基本性质、相图、微观结构,相分离的统计热力学、实验和分子模拟研究等方面阐释相分离物理化学机制研究相关进展。对于生物分子相分离的重要功能体系进行了列举和归纳,收集了相分离研究的模式体系,探讨了生物分子相分离的生物功能同物理化学机制之间的关系,总结了生物分子相分离的调控机制和调控分子的设计方法,并对生物分子相分离物理化学机制研究的未来发展方向进行了展望。

Liquid-liquid phase separation as a major mechanism of plant abiotic stress sensing and responses

Identification of environmental stress sensors is one of the most important research topics in plant abiotic stress research.Traditional strategies to identify stress sensors or early signaling components based on the cell membrane as a primary site of sensing and calcium signal as a second messenger have had only limited successes.Therefore,the current theoretical framework underlying stress sensing in plants should be reconsidered and additional mechanisms need to be introduced.Recently,accumulating evidence has emerged to suggest that liquid-liquid phase separation(LLPS)is a major mechanism for environmental stress sensing and response in plants.In this review,we briefly introduce LLPS regarding its concept,compositions,and dynamics,and then summarize recent progress of LLPS research in plants,emphasizing the contribution of LLPS to the sensing of various environmental stresses,such as dehydration,osmotic stress,and low and high temperatures.Finally,we propose strategies to identify key proteins that sense and respond to environmental stimuli on the basis of LLPS,and discuss the research directions of LLPS in plant abiotic stress responses and its potential application in enhancing stress tolerance in crops.

Solid-state NMR studies of proteins in condensed phases

Some proteins perform their biological functions by changing their material states through liquid-liquid phase separation.Upon phase separation,the protein condenses into a concentrated liquid phase and sometimes into a gel phase,changing its dynamic properties and intermolecular interactions,thereby regulating cellular functions.Although the biological significance of this phenomenon has been widely recognized by researchers,there is still a lack of a comprehensive understanding of the structural and dynamic properties of the protein in the condensed phase.In this phase,molecules usually contain domains with varied dynamic properties and undergo intermediate exchanges.Magic angle spinning(MAS)solid-state NMR(SSNMR)experiments are very powerful in studying rigid protein polymers such as amyloid.The incorporation of solution-like experiments into SSNMR and the development of J-coupling based MAS SSNMR techniques extend its ability to study partially mobile segments of proteins in a condensed liquid or gel phase which are not visible by solution NMR or dipolar-coupling based SSNMR.Therefore,it has been applied in studying protein condensation and has provided very important information that is hard to obtain by other techniques.