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One of the most accepted hypothesis to explain the anomalous behavior of water is the presence of a critical point between two liquids, the liquid–liquid critical point (LLCP), buried within the deep supercooled regime. Unfortunately, such hypothesis is hard to be experimentally confirmed due to fast freezing. Here, we show that the TIP4P/Ice water potential shifted by 400 bar can reproduce with unprecedented accuracy the experimental isothermal compressibility of water and its liquid equation of state for a wide pressure and temperature range. We find, both by extrapolation of response function maxima and by a Maxwell construction, that the location of the model LLCP is consistent with previous calculations. According to the pressure shift needed to recover the experimental behavior of supercooled water, we estimate the experimental LLCP to be located around 1250 bar and 195 K. We use the model to estimate the ice nucleation rate (J) in the vicinity of the hypothesized LLCP experimental location and obtain J = 1024 m−3 s−1. Thereby, experiments where the ratio between the cooling rate and the sample volume is equal or larger than the estimated nucleation rate could probe liquid–liquid equilibrium before freezing. Such conditions are not accessible in common experiments with microdroplets cooled at a few kelvin per second, but they could be, for instance, using nanodroplets of around 50 nm radius observed in a millisecond timescale.

Colloidal systems possess unique features to investigate the governing principles behind liquid-to-solid transitions. The phase diagram and crystallization landscape of colloidal particles can be finely tuned by the range, number and angular distribution of attractive interactions between the constituent particles. In this work, we present a computational study of colloidal patchy particles with high-symmetry bonding—six patches displaying octahedral symmetry—that can crystallize into distinct competing ordered phases: a cubic simple (CS) lattice, a body-centered cubic (BCC) phase, and two face-centered cubic (FCC) solids (orientationally ordered and disordered). We investigate the underlying mechanisms by which these competing crystals emerge from a disordered fluid at different pressures. Strikingly, we identify instances where the structure of the crystalline embryo corresponds to the stable solid, while in others it corresponds to a metastable crystal whose nucleation is enabled by its lower interfacial free energy with the liquid. Moreover, we find the exceptional phenomenon that, due to a subtle balance between volumetric enthalpy and interfacial free energy, the CS phase nucleates via crystalline cubic nuclei rather than through spherical clusters as the majority of crystal solids in nature. Finally, by examining growth beyond the nucleation stage, we uncover a series of alternating one-phase and two-phase crystallization mechanisms, depending on whether or not the same phase that nucleates keeps growing. Taken together, we show that an octahedral distribution of attractive sites in colloidal particles results in an extremely rich crystallization landscape where subtle differences in pressure crucially determine the crystallizing polymorph.

Computational studies of liquid water and its phase transition into vapor have traditionally been performed using classical water models. Here, we utilize the Deep Potential methodology—a machine learning approach—to study this ubiquitous phase transition, starting from the phase diagram in the liquid–vapor coexistence regime. The machine learning model is trained on ab initio energies and forces based on the SCAN density functional, which has been previously shown to reproduce solid phases and other properties of water. Here, we compute the surface tension, saturation pressure, and enthalpy of vaporization for a range of temperatures spanning from 300 to 600 K and evaluate the Deep Potential model performance against experimental results and the semiempirical TIP4P/2005 classical model. Moreover, by employing the seeding technique, we evaluate the free energy barrier and nucleation rate at negative pressures for the isotherm of 296.4 K. We find that the nucleation rates obtained from the Deep Potential model deviate from those computed for the TIP4P/2005 water model due to an underestimation in the surface tension from the Deep Potential model. From analysis of the seeding simulations, we also evaluate the Tolman length for the Deep Potential water model, which is (0.091 ± 0.008) nm at 296.4 K. Finally, we identify that water molecules display a preferential orientation in the liquid–vapor interface, in which H atoms tend to point toward the vapor phase to maximize the enthalpic gain of interfacial molecules. We find that this behavior is more pronounced for planar interfaces than for the curved interfaces in bubbles. This work represents the first application of Deep Potential models to the study of liquid–vapor coexistence and water cavitation.

Various physics- and data-driven sequence-dependent protein coarse-grained models have been developed to study biomolecular phase separation and elucidate the dominant physicochemical driving forces. Here we present Mpipi, a multiscale coarse-grained model that describes almost quantitatively the change in protein critical temperatures as a function of amino acid sequence. The model is parameterized from both atomistic simulations and bioinformatics data and accounts for the dominant role of π–π and hybrid cation–π/π–π interactions and the much stronger attractive contacts established by arginines than lysines. We provide a comprehensive set of benchmarks for Mpipi and seven other residue-level coarse-grained models against experimental radii of gyration and quantitative in vitro phase diagrams, demonstrating that Mpipi predictions agree well with experiments on both fronts. Moreover, Mpipi can account for protein–RNA interactions, correctly predicts the multiphase behavior of a charge-matched poly-arginine/poly-lysine/RNA system, and recapitulates experimental liquid–liquid phase separation trends for sequence mutations on FUS, DDX4 and LAF-1 proteins. Combining bioinformatics data and atomistic simulations, this study develops a sequence-dependent coarse-grained model for biomolecular phase separation. This model achieves a quantitative agreement with experimental observations. Extensive benchmarks exemplify its performance.

Despite the importance of ice nucleation, this process has been barely explored at negative pressures. Here, we study homogeneous ice nucleation in stretched water by means of molecular dynamics seeding simulations using the TIP4P/Ice model. We observe that the critical nucleus size, interfacial free energy, free energy barrier, and nucleation rate barely change between isobars from −2600 to 500 bars when they are represented as a function of supercooling. This allows us to identify universal empirical expressions for homogeneous ice nucleation in the pressure range from −2600 to 500 bars. We show that this universal behavior arises from the pressure dependence of the interfacial free energy, which we compute by means of the mold integration technique, finding a shallow minimum around −2000 bars. Likewise, we show that the change in the interfacial free energy with pressure is proportional to the excess entropy and the slope of the melting line, exhibiting in the latter a reentrant behavior also at the same negative pressure. Finally, we estimate the excess internal energy and the excess entropy of the ice Ih–water interface.

SUMMARYPRC1 (Polycomb repressive complex 1) plays a significant role in cellular differentiation and development by repressing lineage-inappropriate genes. PRC1 proteins phase separate to form Polycomb condensates (bodies) that are multi-component hubs for silencing Polycomb target genes; however, the molecular principles that underpin the condensate assembly and biophysical properties remain unknown. Here, by using biochemical reconstitution, cellular imaging, and multiscale molecular simulations, we show that PRC1 condensates are assembled via a scaffold-client liquid–liquid phase separation (LLPS) model by which Chromobox 2 (CBX2) is the scaffold and other subunits of the CBX2-PRC1 complex act as clients. The clients induce a reentrant phase transition of CBX2 condensates in a concentration-dependent manner. The composition of the multi-component, heterotypic LLPS systems directs the assembly and biophysical properties of CBX2-PRC1 condensates and selectively promotes the formation of CBX4-PRC1 condensates, but specifically dissolves condensates of CBX6-, CBX7-, and CBX8-PRC1. Additionally, the composition of CBX2-PRC1 condensates controls the enrichment of CBX4-, CBX7-, and CBX8-PRC1 into condensates but the exclusion of CBX6-PRC1 from condensates. Our results show the composition- and stoichiometry-dependent scaffold-client assembly of multi-component PRC1 condensates and supply a conceptual framework underlying the molecular basis and dynamics of Polycomb condensate assembly.

Maturation of functional liquid-like biomolecular condensates into solid-like aggregates has been linked to the onset of several neurodegenerative disorders. Low-complexity aromatic-rich kinked segments (LARKS) contained in numerous RNA-binding proteins can promote aggregation by forming inter-proteinβ-sheet fibrils that accumulate over time and ultimately drive the liquid-to-solid transition of the condensates. Here, we combine atomistic molecular dynamics simulations with sequence-dependent coarse-grained models of various resolutions to investigate the role of LARKS abundance and position within the amino acid sequence in the maturation of condensates. Remarkably, proteins with tail-located LARKS display much higher viscosity over time than those in which the LARKS are placed towards the center. Yet, at very long timescales, proteins with a single LARKS—independently of its location—can still relax and behave as high viscous liquids. However, phase-separated condensates of proteins containing two or more LARKS become kinetically trapped due to the formation of percolatedβ-sheet networks that display gel-like behaviour. Furthermore, as a work case example, we demonstrate how shifting the location of the LARKS-containing low-complexity domain of FUS protein towards its center effectively precludes the accumulation ofβ-sheet fibrils in FUS-RNA condensates, maintaining functional liquid-like behaviour without ageing.

Biomolecular condensates are important contributors to the internal organization of the cell material. While initially described as liquid-like droplets, the term biomolecular condensates is now used to describe a diversity of condensed phase assemblies with material properties extending from low to high viscous liquids, gels, and even glasses. Because the material properties of condensates are determined by the intrinsic behaviour of their molecules, characterising such properties is integral to rationalising the molecular mechanisms that dictate their functions and roles in health and disease. Here, we apply and compare three distinct computational methods to measure the viscoelasticity of biomolecular condensates in molecular simulations. These methods are the shear stress relaxation modulus integration (SSRMI), the oscillatory shear (OS) technique, and the bead tracking (BT) method. We find that, although all of these methods provide consistent results for the viscosity of the condensates, the SSRMI and OS techniques outperform the BT method in terms of computational efficiency and statistical uncertainty. We, thus, apply the SSRMI and OS techniques for a set of 12 different protein/RNA systems using a sequence-dependent high-resolution coarse-grained model. Our results reveal a strong correlation between condensate viscosity and density, as well as with protein/RNA length and the number of stickersvs.spacers in the amino-acid protein sequence. Moreover, we couple the SSRMI and the OS technique to nonequilibrium molecular dynamics simulations that mimic the progressive liquid-to-gel transition of protein condensates due to the accumulation of inter-proteinβ-sheets. We compare the behaviour of three different protein condensates—i.e., those formed by either hnRNPA1, FUS, or TDP-43 proteins—whose liquid-to-gel transitions are associated with the onset of amyotrophic lateral sclerosis and frontotemporal dementia. We find that both SSRMI and OS techniques successfully predict the transition from functional liquid-like behaviour to kinetically arrested states once the network of inter-proteinβ-sheets has percolated through the condensates. Overall, our work provides a comparison of different modelling rheological techniques to assess the viscosity of biomolecular condensates, a critical magnitude that provides information on the behaviour of biomolecules inside condensates.

Funder: European Research Council (ERC), The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is promoted by protein-DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through electrostatic interactions via AT hooks 2 and 3. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure.

Biomolecular condensates, thought to form via liquid–liquid phase separation of intracellular mixtures, are multicomponent systems that can include diverse types of proteins and RNAs. RNA is a critical modulator of RNA-protein condensate stability, as it induces an RNA-concentration dependent reentrant phase transition—increasing stability at low RNA concentrations and decreasing it at high concentrations. Beyond concentration, RNAs inside condensates can be heterogeneous in length, sequence, and structure. Here, we use multiscale simulations to understanding how different RNA parameters interact with one another to modulate the properties of RNA-protein condensates. To do so, we perform residue/nucleotide-resolution coarse-grained Molecular Dynamics simulations of multicomponent RNA-protein condensates containing RNAs of different lengths and concentrations, and either FUS or PR25proteins. Our simulations reveal that RNA length regulates the reentrant phase behaviour of RNA-protein condensates: increasing RNA length sensitively rises the maximum value that the critical temperature of the mixture reaches, and the maximum concentration of RNA that the condensate can incorporate before beginning to become unstable. Strikingly, RNA of different lengths are organised heterogeneously inside condensates, which allows them to enhance condensate stability via two distinct mechanisms: shorter RNA chains accumulate at the condensate’s surface acting as natural biomolecular surfactants, whilst longer RNA chains concentrate inside the core to saturate their bonds and enhance the density of molecular connections in the condensate. Using a patchy particle model, we demonstrate that the combined impact of RNA length and concentration on condensate properties is dictated by the valency, binding affinity, and polymer length of the various biomolecules involved. Our results postulate that diversity on RNA parameters within condensates allows RNAs to increase condensate stability by fulfilling two different criteria: maximizing enthalpic gain and minimizing interfacial free energy; hence, RNA diversity should be considered when assessing the impact of RNA on biomolecular condensates regulation.

Costimulation blockade (CoB)–based immunosuppression offers the promise of improved transplantation outcomes with reduced drug toxicity. However, it is hampered by early acute rejections, mediated at least in part by differentiated, CoB-resistant T cells, such as CD57+PD1− CD4 T cells. In this study, we characterize these cells pretransplant, determine their fate posttransplant, and examine their proliferative capacity in vitro in humans. Our studies show that CD57+PD1− CD4 T cells are correlated with increasing age and CMV infection pretransplant, and persist for up to 1 y posttransplant. These cells are replication incompetent alone but proliferated in the presence of unsorted PBMCs in a contact-independent manner. When stimulated, cells sorted by CD57/PD1 status upregulate markers of activation with proliferation. Up to 85% of CD57+PD1− cells change expression of CD57/PD1 with stimulation, typically, upregulating PD1 and downregulating CD57. PD1 upregulation is accentuated in the presence of rapamycin but prevented by tacrolimus. These data support a general theory of CoB-resistant cells as Ag-experienced, costimulation-independent cells and suggest a mechanism for the synergy of belatacept and rapamycin, with increased expression of the activation marker PD1 potentiating exhaustion of CoB-resistant cells.

Funder: Ernest Oppenheimer Memorial Trust, Biomolecular condensates, some of which are liquid-like during health, can age over time becoming gel-like pathological systems. One potential source of loss of liquid-like properties during ageing of RNA-binding protein condensates is the progressive formation of inter-protein β-sheets. To bridge microscopic understanding between accumulation of inter-protein β-sheets over time and the modulation of FUS and hnRNPA1 condensate viscoelasticity, we develop a multiscale simulation approach. Our method integrates atomistic simulations with sequence-dependent coarse-grained modelling of condensates that exhibit accumulation of inter-protein β-sheets over time. We reveal that inter-protein β-sheets notably increase condensate viscosity but does not transform the phase diagrams. Strikingly, the network of molecular connections within condensates is drastically altered, culminating in gelation when the network of strong β-sheets fully percolates. However, high concentrations of RNA decelerate the emergence of inter-protein β-sheets. Our study uncovers molecular and kinetic factors explaining how the accumulation of inter-protein β-sheets can trigger liquid-to-solid transitions in condensates, and suggests a potential mechanism to slow such transitions down.

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A wide range of macromolecules undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed in a reversible manner, and carry out important functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics and mechanics in time and space, that FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases co-exist simultaneously within the same condensate, resulting in highly inhomogeneous structures. We introduce two new optical techniques, dynamic spatial mapping and reflective confocal dynamic speckle microscopy, and use these to further show that the liquid-to-solid transition is initiated at the interface between the dense phase within condensates and the dilute phase. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a key element in driving pathological protein aggregation.

Freezing of water is the most common liquid-to-crystal phase transition on Earth; however, despite its critical implications on climate change and cryopreservation among other disciplines, its characterization through experimental and computational techniques remains elusive. In this work, we make use of computer simulations to measure the nucleation rate ( J) of water at normal pressure under different supercooling conditions, ranging from 215 to 240 K. We employ two different water models: mW, a coarse-grained potential for water, and TIP4P/ICE, an atomistic nonpolarizable water model that provides one of the most accurate representations of the different ice phases. To evaluate J, we apply the Lattice Mold technique, a computational method based on the use of molds to induce the nucleus formation from the metastable liquid under conditions at which observing spontaneous nucleation would be unfeasible. With this method, we obtain estimates of the nucleation rate for ice Ih and Ic and a stacking mixture of ice Ih/Ic, reaching consensus with most of the previously reported rates, although differing with some others. Furthermore, we confirm that the predicted nucleation rates obtained by the TIP4P/ICE model are in better agreement with experimental data than those obtained through the mW potential. Taken together, our study provides a reliable methodology to measure nucleation rates in a simple and computationally efficient manner that contributes to benchmarking the freezing behavior of two popular water models.

Hard-sphere crystallization has been widely investigated over the last six decades by means of colloidal suspensions and numerical methods. However, some aspects of its nucleation behaviour are still under debate. Here, we provide a detailed computational characterisation of the polymorphic nucleation competition between the face-centered cubic (fcc) and the hexagonal-close packed (hcp) hard-sphere crystal phases. By means of several state-of-the-art simulation techniques, we evaluate the melting pressure, chemical potential difference, interfacial free energy and nucleation rate of these two polymorphs, as well as of a random stacking mixture of both crystals. Our results highlight that, despite the fact that both polymorphs have very similar stability, the interfacial free energy of the hcp phase could be marginally higher than that of the fcc solid, which in consequence, mildly decreases its propensity to nucleate from the liquid compared to the fcc phase. Moreover, we analyse the abundance of each polymorph in grown crystals from different types of inserted nuclei: fcc, hcp and stacking disordered fcc/hcp seeds, as well as from those spontaneously emerged from brute force simulations. We find that post-critical crystals fundamentally grow maintaining the polymorphic structure of the critical nucleus, at least until moderately large sizes, since the only crystallographic orientation that allows stacking close-packed disorder is the fcc (111) plane, or equivalently the hcp (0001) one. Taken together, our results contribute with one more piece to the intricate puzzle of colloidal hard-sphere crystallization.

10 pags., 7 figs., 2 tabs., The seeding method is an approximate approach to investigate nucleation that combines molecular dynamics simulations with classical nucleation theory. Recently, this technique has been successfully implemented in a broad range of nucleation studies. However, its accuracy is subject to the arbitrary choice of the order parameter threshold used to distinguish liquid-like from solid-like molecules. We revisit here the crystallization of NaCl from a supersaturated brine solution and show that consistency between seeding and rigorous methods, like Forward Flux Sampling (from previous work) or spontaneous crystallization (from this work), is achieved by following a mislabelling criterion to select such threshold (i.e. equaling the fraction of the mislabelled particles in the bulk parent and nucleating phases). This work supports the use of seeding to obtain fast and reasonably accurate nucleation rate estimates and the mislabelling criterion as one giving the relevant cluster size for classical nucleation theory in crystallization studies., This project has been funded by grants FIS2016-78117-P and PID2019-105898GB-C21 of MEC. E. G. N. thanks Agencia Estatal de Investigacion and Fondo Europeo de Desarrollo Regional (FEDER), Grant No FIS2017-89361-C3-2-P. C. P. L. thanks Ministerio de Educacion y Formacio´n Profesional for a predoctoral Formacion Profesorado Universitario Grant No. FPU18/03326 and also Ayuntamiento de Madrid for a Residencia de Estudiantes grant. The authors acknowledge the computer resources and technical assistance provided by RES. PMdH acknowledges financial support from the FPI grant no. BES6712017-080074. J. R. E. acknowledges funding from the Oppenheimer Research fellowship and the Roger Ekins Research Fellowship of Emmanuel College. M. M. C. thanks financial support from PID2019-105898GA-C22 of the MICINN and CAM and UPM through the CavitieS project No. APOYOJOVENES-01HQ1S-129-B5E4MM from ‘‘Accion financiada por la Comunidad de Madrid en el marco del Convenio Plurianual con la Universidad Politecnica de Madrid en la linea de actuacion estimulo a la investigacion de jovenes doctores’’. The authors gratefully acknowledge Universidad Politecnica de Madrid (www.upm.es) for providing computing resources on Magerit Supercomputer.

A biosolid was produced from the composted digestate of the organic fraction of urban solid wastes (OFUSW). During a first hydrolytic-acidogenic stage, the milled OFUSW mass is transformed into volatile fatty acids and other soluble or suspended substances in the leachate while the residual solids constitute the digestate. A biosolid was produced from the composted digestate of the organic fraction of urban solid wastes (OFUSW). During a first hydrolytic-acidogenic stage, the milled OFUSW mass is transformed into volatile fatty acids and other soluble or suspended substances in the leachate while the residual solids constitute the digestate. To obtain a composting mass (CM) with a C/N between 25 and 35 and a matrix to allow aeration, mixtures of digestate (fD), OFUSW (fF) and sawdust (fS) were prepared using a simplex centroid design. Results were adjusted to a multiple regression model with volatile solids degradation efficiency (ηVS) as a response variable. It was found that the operation zone with the highest ηVS, were CM with the following compositions: fD: 0.425-0.625, fF: 0.275-0.450; and fS: 0.1-0.15. Three assays were carried out in a bench scale reactor, the final biosolids had a pH = 7.5, a C/N = 15 and a germination index (GI) = 84%. A linear correlation between ηvs and CO2 production was proposed for process control. A rapid CM stabilization was reached, up to ηVS = 35% in 12 days and the biosolids with high GI, can be used to improve soils.

Crystallographic data comes from a space-time average over all the unit cells within the crystal, so dynamic phenomena do not contribute significantly to the diffraction data. Many efforts have been made to reconstitute the movement of the macromolecules and explore the microstates that the confined proteins can adopt in the crystalline network. We explored different strategies to simulate a heart fatty acid binding protein (H-FABP) crystal by means of Molecular Dynamics (MD) simulations. We evaluate the effect of introducing restraints according to experimental isotropic B-factors and we analyzed the H-FABP motions in the crystal using Principal Component Analysis (PCA), isotropic and anisotropic B-factors. We compared the behavior of the protein simulated in the crystal confinement versus in solution, and we observed the effect of that confinement in the mobility of the protein residues. Restraining one-third of Cα atoms based on experimental B-factors produce lower B-factors than simulations without restraints, showing that the position restraint of the atoms with the lowest experimental B-factor is a good strategy to maintain the geometry of the crystal with an obvious decrease in the degrees of motion of the protein. PCA shows that, as position restraint reduces the conformational space explored by the system, the motion of the crystal is better recovered, for an essential subspace of the same size, in the simulations without restraints. Restraining only one Cα seems to be a good balance between giving flexibility to the system and preserving its structure. Fil: Espinosa Silva, Yanis Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Universidad Industrial Santander; Colombia Fil: Alvarez, Hugo Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Universidad Nacional Arturo Jauretche; Argentina Fil: Howard, Eduardo Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Universidad Tecnológica Nacional; Argentina Fil: Carlevaro, Carlos Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Universidad Tecnológica Nacional; Argentina

The synthesis of triarylmethanes via Pd-catalyzed Suzuki-Miyaura reactions between diarylmethyl 2,3,4,5,6-pentafluorobenzoates and aryl boronic acids is described. The system operates at mild conditions and has a broad substrate scope, including the coupling of diphenylmethanol derivatives that do not contain extended aromatic substituents. This is significant as these substrates, which result in the types of triarylmethane products that are prevalent in pharmaceuticals, have not previously been compatible with systems for diarylmethyl ester coupling. Further, the reaction can be performed stereospecifically to generate stereo-inverted products. On the basis of DFT calculations, it is proposed that the oxidative addition of the diarylmethyl 2,3,4,5,6-pentafluorobenzoate substrate occurs via an S(N)2 pathway, which results in the inverted products. Mechanistic studies indicate that oxidative addition of the diarylmethyl 2,3,4,5,6-pentafluorobenzoate substrates to (IPr)Pd(0) results in the selective cleavage of the O–C(benzyl) bond in part because of a stabilizing η(3)-interaction between the benzyl ligand and Pd. This is in contrast to previously described Pd-catalyzed Suzuki-Miyaura reactions involving phenyl esters, which involve selective cleavage of the C(acyl)–O bond, because there is no stabilizing η(3)-interaction. It is anticipated that this fundamental knowledge will aid the development of new catalytic systems, which use esters as electrophiles in cross-coupling reactions.

Biomolecular condensates formed by the process of liquid–liquid phase separation (LLPS) play diverse roles inside cells, from spatiotemporal compartmentalisation to speeding up chemical reactions. Upon maturation, the liquid-like properties of condensates, which underpin their functions, are gradually lost, eventually giving rise to solid-like states with potential pathological implications. Enhancement of inter-protein interactions is one of the main mechanisms suggested to trigger the formation of solid-like condensates. To gain a molecular-level understanding of how the accumulation of stronger interactions among proteins inside condensates affect the kinetic and thermodynamic properties of biomolecular condensates, and their shapes over time, we develop a tailored coarse-grained model of proteins that transition from establishing weak to stronger inter-protein interactions inside condensates. Our simulations reveal that the fast accumulation of strongly binding proteins during the nucleation and growth stages of condensate formation results in aspherical solid-like condensates. In contrast, when strong inter-protein interactions appear only after the equilibrium condensate has been formed, or when they accumulate slowly over time, with respect to the time needed for droplets to fuse and grow, spherical solid-like droplets emerge. By conducting atomistic potential-of-mean-force simulations of NUP-98 peptides—prone to forming inter-protein β -sheets—we observe that formation of inter-peptide β -sheets increases the strength of the interactions consistently with the loss of liquid-like condensate properties we observe at the coarse-grained level. Overall, our work aids in elucidating fundamental molecular, kinetic, and thermodynamic mechanisms linking the rate of change in protein interaction strength to condensate shape and maturation during ageing., Adiran Garaizar is funded by the EPRSC Doctoral Programme Training number EP/N509620/.

The insertion of carbon dioxide into metal element σ-bonds is an important elementary step in many catalytic reactions for carbon dioxide valorization. Here, the insertion of carbon dioxide into a family of group 10 alkyl complexes of the type (RPBP)M(CH3) (RPBP = B(NCH2PR2)2C6H4−; R = Cy or tBu; M = Ni or Pd) to generate κ1-acetate complexes of the form (RPBP)M{OC(O)CH3} is investigated. This involved the preparation and characterization of a number of new complexes supported by the unusual RPBP ligand, which features a central boryl donor that exerts a strong trans-influence, and the identification of a new decomposition pathway that results in C–B bond formation. In contrast to other group 10 methyl complexes supported by pincer ligands, carbon dioxide insertion into (RPBP)M(CH3) is facile and occurs at room temperature because of the high trans-influence of the boryl donor. Given the mild conditions for carbon dioxide insertion, we perform a rare kinetic study on carbon dioxide insertion into a late-transition metal alkyl species using (tBuPBP)Pd(CH3). These studies demonstrate that the Dimroth–Reichardt parameter for a solvent correlates with the rate of carbon dioxide insertion and that Lewis acids do not promote insertion. DFT calculations indicate that insertion into (tBuPBP)M(CH3) (M = Ni or Pd) proceeds via an SE2 mechanism and we compare the reaction pathway for carbon dioxide insertion into group 10 methyl complexes with insertion into group 10 hydrides. Overall, this work provides fundamental insight that will be valuable for the development of improved and new catalysts for carbon dioxide utilization.

Funder: Oppenheimer Fellowship, Funder: Roger Ekins Fellowship, Funder: Derek Brewer Emmanuel College scholarship, Biomolecular condensates formed via liquid-liquid phase separation (LLPS) play a crucial role in the spatiotemporal organization of the cell material. Nucleic acids can act as critical modulators in the stability of these protein condensates. To unveil the role of RNA length in regulating the stability of RNA binding protein (RBP) condensates, we present a multiscale computational strategy that exploits the advantages of a sequence-dependent coarse-grained representation of proteins and a minimal coarse-grained model wherein proteins are described as patchy colloids. We find that for a constant nucleotide/protein ratio, the protein fused in sarcoma (FUS), which can phase separate on its own-i.e., via homotypic interactions-only exhibits a mild dependency on the RNA strand length. In contrast, the 25-repeat proline-arginine peptide (PR25), which does not undergo LLPS on its own at physiological conditions but instead exhibits complex coacervation with RNA-i.e., via heterotypic interactions-shows a strong dependence on the length of the RNA strands. Our minimal patchy particle simulations suggest that the strikingly different effect of RNA length on homotypic LLPS versus RBP-RNA complex coacervation is general. Phase separation is RNA-length dependent whenever the relative contribution of heterotypic interactions sustaining LLPS is comparable or higher than those stemming from protein homotypic interactions. Taken together, our results contribute to illuminate the intricate physicochemical mechanisms that influence the stability of RBP condensates through RNA inclusion.

Liquid–liquid phase separation underlies the formation of biological condensates. Physically, such systems are microemulsions which have a general propensity to fuse and coalesce; however, many condensates persist as independent droplets inside cells. This stability is crucial for their functioning, but the physicochemical mechanisms that control the emulsion stability of condensates remain poorly understood. Here, by combining single-condensate zeta potential measurements, optical microscopy, tweezer experiments, and multiscale molecular modelling, we investigate how the forces that sustain condensates impact their stability against fusion. By comparing PR25:PolyU and FUS condensates, we show that a higher condensate surface charge correlates with a lower fusion propensity, and that this behavior can be inferred from their zeta potentials. We reveal that overall stabilization against fusion stems from a combination of repulsive forces between condensates and the effects that surface electrostatics have on lowering surface tension, thus shedding light on the molecular determinants of condensate coalescence.

The molecular structure of membrane lipids is formed by mono- or polyunsaturations on their aliphatic tails that make them susceptible to oxidation, facilitating the incorporation of hydroperoxide (R-OOH) functional groups. Such groups promote changes in both composition and complexity of the membrane significantly modifying its physicochemical properties. Human Langerhans islets amyloid polypeptide (hIAPP) is the main component of amyloid deposits found in the pancreas of patients with type-2 diabetes (T2D). hIAPP in the presence of membranes with oxidized lipid species accelerates the formation of amyloid fibrils or the formation of intermediate oligomeric structures. However, the molecular bases at the initial stage of the anchoring and stabilization of the hIAPP in a hydroperoxidized membrane are not yet well understood. To shed some light on this matter, in this contribution, three bilayer models were modeled: neutral (POPC), anionic (POPS), and oxidized (POPC

The insertion of carbon dioxide into metal element σ-bonds is an important elementary step in many catalytic reactions for carbon dioxide valorization. Here, the insertion of carbon dioxide into a family of group 10 alkyl complexes of the type (

There are 14 forms of lipofuscinosis, among them type 6 in its late childhood form is found, it starts between three and eight years with epilepsy, motor disorders, myoclonus, dysarthria, ataxia and neurological regression associated with vision loss and motor skills, and early death. It occurs from mutations in the CLN6 gene, most patients have homozygote variants associated with consanguinity, and rarely, with compound heterozygote variants.Siblings, started at 4 and 5 years each, with unstable gear, frequent falls and difficult running. Subsequently, loss of gait, myoclonus, dysphagia, and hallucinations. On physical examination, present optic nerve atrophy, Babinski and trunk ataxia. Electroencephalogram with widespread slow wave bursts during non-REM sleep, non photoparoxystic response, MRI with periventricular white substance hyperintensity, cerebellar atrophy and cortical. Panel of lipofuscinosis report two mutations, c.552del and c.244GC, not described previously, in both patients. The mother was the carrier of the 552 deletion and the father and paternal grandmother of the GC substitution (Gly82Arg).Differential diagnosis in neuroregression disorders is difficult because clinical signs are nonspecific, like many other neurodegenerative disorders with progressive myoclonic epilepsy. We report the clinical findings in two Mexican siblings with the late childhood variant of CLN6 with two new heterozygote mutations that contribute to the knowledge of mutations in the Mexican population and point out the relevance of performing next-generation genetic sequencing studies which will allow a better genetic counseling practice.Lipofuscinosis ceroidea neuronal. Variante infantil tardía de tipo 6 en dos hermanos heterocigotos compuestos con mutaciones nuevas.Introducción. Existen 14 formas de lipofuscinosis. La de tipo 6, en su forma infantil tardía, comienza entre los 3 y 8 años con alteraciones motoras, mioclonos, disartria, ataxia, pérdida de la visión y las habilidades motoras, y muerte temprana. Ocurre por mutaciones en el gen CLN6. La mayoría de los pacientes presenta variantes en estado homocigoto, asociadas a consanguinidad o endogamia, y son poco frecuentes las variantes en estado heterocigoto compuesto. Casos clínicos. Hermanos con síntomas desde los 4 y 5 años, con marcha inestable, caídas frecuentes, posteriormente pérdida de la marcha, mioclonías, disfagia y alucinaciones visuales. En el examen físico presentaban atrofia del nervio óptico, Babinski y ataxia del tronco. El electroencefalograma mostraba brotes de ondas lentas generalizadas, sin respuesta fotoparoxística, y la resonancia magnética de cráneo, hiperintensidad de la sustancia blanca periventricular, y atrofia cerebelosa y cortical. El panel de lipofuscinosis reveló dos mutaciones nuevas en el gen CLN6, c.552del y c.244GC (p.Gly82Arg), no descritas previamente. La madre resultó portadora de la deleción 552, y el padre y la abuela paterna, de la sustitución GC (Gly82Arg). Conclusiones. El diagnóstico diferencial en los trastornos con neurorregresión se dificulta debido a que los signos clínicos son inespecíficos, similares a otras epilepsias mioclónicas progresivas. Presentamos los hallazgos clínicos en dos hermanos mexicanos con la variante infantil tardía de CLN6 por dos mutaciones heterocigotas nuevas que contribuyen al conocimiento de las mutaciones en la población mexicana y señalan la relevancia de realizar estudios genéticos aplicando la secuenciación de nueva generación para permitir un adecuado asesoramiento genético.

The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is critically promoted by protein–DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through cation–π and electrostatic interactions. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure.

Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multi-component mixtures of biomolecules (e.g. proteins and nucleic acids) when the different components present sufficient physicochemical diversity (e.g. in inter-molecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such diversity is highly coupled to the solution conditions (e.g. temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli, or progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of ageing. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically ageing condensates with non-conservative inter-molecular forces. Our nonequilibrium simulations of condensate ageing predict that single-component condensates that are initially homogeneous and liquid-like can transform into gel-core/liquid-shell or liquid-core/gel-shell multiphase condensates as they age, due to gradual and irreversible enhancement of inter-protein interactions. The type of multiphase architecture is determined by the ageing mechanism, the molecular organization of the gel and liquid phases, and the chemical make up of the protein. Notably, we predict that inter-protein disorder-to-order transitions within the prion-like domains of intracellular proteins could lead to the required non-conservative enhancement of inter-molecular interactions. Our study, therefore, predicts a potential mechanismSignificance StatementBiomolecular condensates are highly diverse systems spanning not only homogeneous liquid droplets, but also gels, glasses, and even multiphase architectures that contain various coexisting liquid-like and/or gel-like inner phases. Multiphase architectures form when the different biomolecular components in a multi-component condensate establish sufficiently imbalanced inter-molecular forces to sustain different coexisting phases. While such a requirement seems, at first glance, impossible to fulfil for a condensate formed exclusively of chemically-identical proteins (i.e., single-component), our simulations predict conditions under which this may be possible. During condensate ageing, a sufficiently large imbalance in inter-molecular interactions can emerge intrinsically from the accumulation of protein structural transitions—driving even single-component condensates into nonequilibrium liquid-core/gel-shell or gel-core/liquid-shell multiphase architectures.

Multivalent proteins and nucleic acids can self-assemble into biomolecular condensates that contribute to compartmentalize the cell interior. Computer simulations offer a unique view to elucidate the mechanisms and key intermolecular interactions behind the dynamic formation and dissolution of these condensates. In this work, we present a novel approach to include explicit water and salt in sequence-dependent coarse-grained (CG) models for proteins and RNA, enabling the study of biomolecular condensate formation in a salt-dependent manner. Our framework combines a reparameterized version of the HPS protein force field with the monoatomic mW water model and the mW-ion potential for NaCl. We show how our CG model qualitatively captures the experimental radius of the gyration trend of a subset of intrinsically disordered proteins and reproduces the experimental protein concentration and water percentage of the human fused in sarcoma (FUS) low-complexity-domain droplets at physiological salt concentration. Moreover, we perform seeding simulations as a function of salt concentration for two antagonist systems: the engineered peptide PR25 and poly-uridine/poly-arginine mixtures, finding good agreement with their reported in vitro phase behavior with salt concentration in both cases. Taken together, our work represents a step forward towards extending sequence-dependent CG models to include water and salt, and to consider their key role in biomolecular condensate self-assembly.

Hard-sphere crystallization has been widely investigated over the last six decades by means of colloidal suspensions and numerical methods. However, some aspects of its nucleation behaviour are still under debate. Here, we provide a detailed computational characterisation of the polymorphic nucleation competition between the face-centered cubic (fcc) and the hexagonal-close packed (hcp) hard-sphere crystal phases. By means of several state-of-the-art simulation techniques, we evaluate the melting pressure, chemical potential difference, interfacial free energy and nucleation rate of these two polymorphs, as well as of a random stacking mixture of both crystals. Our results highlight that, despite the fact that both polymorphs have very similar stability, the interfacial free energy of the hcp phase could be marginally higher than that of the fcc solid, which in consequence, mildly decreases its propensity to nucleate from the liquid compared to the fcc phase. Moreover, we analyse the abundance of each polymorph in grown crystals from different types of inserted nuclei: fcc, hcp and stacking disordered fcc/hcp seeds, as well as from those spontaneously emerged from brute force simulations. We find that post-critical crystals fundamentally grow maintaining the polymorphic structure of the critical nucleus, at least until moderately large sizes, since the only crystallographic orientation that allows stacking close-packed disorder is the fcc (111) plane, or equivalently the hcp (0001) one. Taken together, our results contribute with one more piece to the intricate puzzle of colloidal hard-sphere crystallization.

One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a chemically-accurate sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1, and TDP-43 proteins along with that of their corresponding prion-like domains and RNA recognition motifs from absence to moderately high RNA concentration. By characterizing the phase diagram, key molecular interactions, surface tension, and transport properties of the condensates, we report a dual RNA-induced behavior: on the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whereas at high concentration, it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity even at high concentration, but barely modify protein self-diffusion which mainly depends on RNA concentration and on the effect RNA has on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.

In this work, a low-cost and rapid electrochemical resistive DNA biosensor based on the current relaxation method is described. A DNA probe, complementary to the specific human papillomavirus type 16 (HPV-16) sequence, was immobilized onto a screen-printed gold electrode. DNA hybridization was detected by applying a potential step of 30 mV to the system, composed of an external capacitor and the modified electrode DNA/gold, for 750 µs and then relaxed back to the OCP, at which point the voltage and current discharging curves are registered for 25 ms. From the discharging curves, the potential and current relaxation were evaluated, and by using Ohm’s law, the charge transfer resistance through the DNA-modified electrode was calculated. The presence of a complementary sequence was detected by the change in resistance when the ssDNA is transformed in dsDNA due to the hybridization event. The target DNA concentration was detected in the range of 5 to 20 nM. The results showed a good fit to the regression equation ΔRtotal(Ω)=2.99&nbsp, ×&nbsp, [DNA]+81.55, and a detection limit of 2.39 nM was obtained. As the sensing approach uses a direct current, the electronic architecture of the biosensor is simple and allows for the separation of faradic and nonfaradaic contributions. The simple electrochemical resistive biosensor reported here is a good candidate for the point-of-care diagnosis of HPV at a low cost and in a short detection time.

To study gastroesophageal reflux (GER) in children undergoing gastrostomy in a single pediatric institution.A retrospective study of patients undergoing gastrostomy from 2000 to 2017 was carried out. Demographic data, clinical data, progression, and complications were recorded. GER was considered positive in patients with clinical signs requiring antisecretory treatment, prokinetic treatment, or anti-reflux surgery to control symptoms.207 patients with a median age of 2 years [R: 0.25-18] were included. Neurological impairment was the most frequent underlying condition (74%). Swallowing difficulty and undernourishment were the main surgical indications for gastrostomy. Prior to gastrostomy, 96 out of 207 patients (46%) showed GER symptoms. Combined fundoplication and gastrostomy was performed in 41 (43%) patients with preexisting GER, 6 of whom showed GER worsening (4 required redo fundoplication). 5 complications following fundoplication were noted - gastric perforation, sustained Dumping syndrome, and gastroesophageal stenosis. 55 out of 96 (57%) patients with preexisting GER underwent gastrostomy alone. Clinical signs disappeared in 16 of them (29%) and improved or stabilized in 19 (35%). GER worsening occurred in 20 patients (36%), with subsequent fundoplication being required in 10 cases. In patients with no previous clinical signs (111 out of 207), GER symptoms occurred following gastrostomy in just 18 cases (16%), and only 2 patients required fundoplication.In our experience, routine anti-reflux surgery combined with gastrostomy is not justified. Individualized fundoplication should be considered in case of medical treatment failure. Further studies with an adequate design are required to establish which patients could really benefit from this procedure.Estudio del reflujo gastroesofágico (RGE) en los pacientes en los que se ha realizado una gastrostomía en nuestro centro.Revisión de los pacientes intervenidos de gastrostomía en el periodo 2000-2017. Registro de datos demográficos, clínicos, evolución y complicaciones. Definimos RGE como la presencia de clínica compatible en pacientes que requirieron tratamiento médico o quirúrgico antirreflujo.Incluimos 207 pacientes con una mediana de edad de 2 años [r:0,25-18]. La patología subyacente más frecuente fue déficit neurológico (74%). Las indicaciones quirúrgicas fueron trastornos deglutorios y/o desnutrición. Previamente a la gastrostomía, 96/207 pacientes (46%) presentaban clínica de RGE. Se realizó funduplicatura asociada a gastrostomía en 41/96 (43%) de los pacientes con RGE previo. En 6/41 pacientes (15%) el RGE empeoró, requiriendo 4 de ellos una segunda funduplicatura. Se registraron 5 complicaciones tras funduplicatura (perforaciones gástricas, síndromes de Dumping prolongados y estenosis esofagogástrica). En 55/96 pacientes con RGE previo a la gastrostomía no se asoció funduplicatura. La clínica desapareció en 16/55 (29%), y mejoró o se estabilizó en 19/55 pacientes (35%). En 20/55 (36%) la sintomatología empeoró, y 10 de ellos precisaron una funduplicatura posterior. De los pacientes sin clínica previa de RGE (111/207), presentaron síntomas de RGE tras la gastrostomía 18/111 (16%), y solo 2 pacientes requirieron funduplicatura.Según nuestra experiencia, la funduplicatura de rutina asociada a la gastrostomía no está justificada. En caso de fracaso del tratamiento médico del RGE, una técnica antirreflujo debe plantearse de forma individualizada. Son necesarios estudios adecuadamente diseñados para definir qué pacientes realmente se beneficiarían de este procedimiento.

One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a novel sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1 and TDP-43 proteins along with that of their corresponding prion-like domains and RNA-recognition motifs, from absence to moderately high RNA concentration. By characterising the phase diagram, key molecular interactions, surface tension and transport properties of the condensates, we report a dual RNA-induced behavior: On the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whilst at high concentration it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity, even at high concentration, but barely modify protein self-diffusion, which mainly depends on RNA concentration and on its own effect on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.

Intracellular liquid-liquid phase separation enables the formation of biomolecular condensates, such as ribonucleoprotein granules, which play a crucial role in the spatiotemporal organization of biomolecules (e.g., proteins and RNAs). Here, we introduce a patchy-particle polymer model to investigate liquid-liquid phase separation of protein-RNA mixtures. We demonstrate that at low to moderate concentrations, RNA enhances the stability of RNA-binding protein condensates because it increases the molecular connectivity of the condensed-liquid phase. Importantly, we find that RNA can also accelerate the nucleation stage of phase separation. Additionally, we assess how the capacity of RNA to increase the stability of condensates is modulated by the relative protein-protein/protein-RNA binding strengths. We find that phase separation and multiphase organization of multicomponent condensates is favored when the RNA binds with higher affinity to the lower-valency proteins in the mixture than to the cognate higher-valency proteins. Collectively, our results shed light on the roles of RNA in ribonucleoprotein granule formation and the internal structuring of stress granules.

Liquid–liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates., Elucidating the molecular driving forces underlying liquid–liquid phase separation is a key objective for understanding biological function and malfunction. Here the authors show that a wide range of cellular proteins, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, which form condensates at low salt concentrations, can reenter a phase-separated regime at high salt concentrations.