Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.

Liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes. VIDEO ABSTRACT.

Using the Intrinsic Geometry of Binodal Curves to Simplify the Computation of Ternary Liquid-Liquid Phase Diagrams.

Phase diagrams are powerful tools to understand the multi-scale behaviour of complex systems. Yet, their determination requires in practice both experiments and computations, which quickly becomes a daunting task. Here, we propose a geometrical approach to simplify the numerical computation of liquid-liquid ternary phase diagrams. We show that using the intrinsic geometry of the binodal curve, it is possible to formulate the problem as a simple set of ordinary differential equations in an extended 4D space. Consequently, if the thermodynamic potential, such as Gibbs free energy, is known from an experimental data set, the whole phase diagram, including the spinodal curve, can be easily computed. We showcase this approach on four ternary liquid-liquid diagrams, with different topological properties, using a modified Flory-Huggins model. We demonstrate that our method leads to similar or better results comparing those obtained with other methods, but with a much simpler procedure. Acknowledging and using the intrinsic geometry of phase diagrams thus appears as a promising way to further develop the computation of multiphase diagrams.

Practical deployment of automation to expedite aqueous two-phase extraction.

The feasibility of bioprocess development relies heavily on the successful application of primary recovery and purification techniques. Aqueous two-phase extraction (ATPE) disrupts the definition of "unit operation" by serving as an integrative and intensive technique that combines different objectives such as the removal of biomass and integrated recovery and purification of the product of interest. The relative simplicity of processing large samples renders this technique an attractive alternative for industrial bioprocessing applications. However, process development is hindered by the lack of easily predictable partition behaviours, the elucidation of which necessitates a large number of experiments to be conducted. Liquid handling devices can assist to address this problem; however, they are configured to operate using low viscosity fluids such as water and water-based solutions as opposed to highly viscous polymeric solutions, which are typically required in ATPE. In this work, an automated high throughput ATPE process development framework is presented by constructing phase diagrams and identifying the binodal curves for PEG6000, PEG3000, and PEG2000. Models were built to determine viscosity- and volume-independent transfer parameters. The framework provided an appropriate strategy to develop a very precise and accurate operation by exploiting the relationship between different liquid transfer parameters and process error. Process accuracy, measured by mean absolute error, and device precision, evaluated by the coefficient of variation, were both shown to be affected by the mechanical properties, particularly viscosity, of the fluids employed. For PEG6000, the mean absolute error improved by six-fold (from 4.82% to 0.75%) and the coefficient of variation improved by three-fold (from 0.027 to 0.008) upon optimisation of the liquid transfer parameters accounting for the viscosity effect on the PEG-salt buffer utilising ATPE operations. As demonstrated here, automated liquid handling devices can serve to streamline process development for APTE enabling wide adoption of this technique in large scale bioprocess applications.

Poly[bis-[µ2-1,3-bis-(pyridin-4-yl)urea-κ2N4:N4']bis-(µ2-5-methyl-isophthalato-κ2O1:O3)dizinc(II)], a parallel inter-penetrated slab-like coordination polymer with {3.648}{326.728} 4,4-connected binodal topology.

In the title compound, [Zn2(C9H6O4)2(C11H10N4O)2]n, diperiodic coordination polymer slabs with {3.648}{326.728} 4,4-connected binodal topology are held into a parallel inter-penetrated triperiodic crystal structure by means of N-H⋯O hydrogen-bonding patterns.

SpiDec: Computing binodals and interfacial tension of biomolecular condensates from simulations of spinodal decomposition.

Phase separation of intrinsically disordered proteins (IDPs) is a phenomenon associated with many essential cellular processes, but a robust method to compute the binodal from molecular dynamics simulations of IDPs modeled at the all-atom level in explicit solvent is still elusive, due to the difficulty in preparing a suitable initial dense configuration and in achieving phase equilibration. Here we present SpiDec as such a method, based on spontaneous phase separation via spinodal decomposition that produces a dense slab when the system is initiated at a homogeneous, low density. After illustrating the method on four model systems, we apply SpiDec to a tetrapeptide modeled at the all-atom level and solvated in TIP3P water. The concentrations in the dense and dilute phases agree qualitatively with experimental results and point to binodals as a sensitive property for force-field parameterization. SpiDec may prove useful for the accurate determination of the phase equilibrium of IDPs.

Dataset for the interfacial tension and phase properties of the ternary system water - 2-butoxyethanol - toluene.

Two-phase samples containing water, 2-butoxyethanol, and toluene in the different mass ratios were gravimetrically prepared in the jacketed cells at T=293.15 K and p=0.100 MPa and equilibrated for 24 h. The samples were volumetrically titrated until homogeneous. Then new samples were prepared in the two-phase region with compositions in the immediate proximity to the expected separation boundary and titrated until homogeneous. The critical point was located, keeping the phase ratio of 1:1 during the titration. The density of homogeneous samples obtained during titration was measured using the density meter. These data were used to construct an interpolation of the density along the separation boundary. New two-phase samples were prepared; the interfacial tension, density, and viscosity were measured. Thus, interfacial tension isotherm and viscosity isotherm were obtained using density interpolation to determine the composition of the equilibrated phases. The obtained data can be used to prepare the two-phase samples with desired properties, design the oil-water separation processes, and develop new oil spill dispersants containing 2-butoxyethanol. This article is a co-submission with a paper [1].

Thermodynamic Modeling and Validation of the Temperature Influence in Ternary Phase Polymer Systems.

The effect of the temperature, as a process variable in the fabrication of polymeric membranes by the non-solvent induced phase separation (NIPS) technique, has been scarcely studied. In the present work, we studied the influence of temperature, working at 293, 313 and 333 K, on the experimental binodal curves of four ternary systems composed of PVDF and PES as the polymers, DMAc and NMP as the solvents and water as the non-solvent. The increase of the temperature caused an increase on the solubility gap of the ternary system, as expected. The shift of the binodal curve with the temperature was more evident in PVDF systems than in PES systems indicating the influence of the rubbery or glassy state of the polymer on the thermodynamics of phase separation. As a novelty, the present work has introduced the temperature influence on the Flory-Huggins model to fit the experimental cloud points. Binary interaction parameters were calculated as a function of the temperature: (i) non-solvent/solvent (g12) expressions with UNIFAC-Dortmund methodology and (ii) non-solvent/polymer (χ13) and solvent/polymer (χ23) using Hansen solubility parameters. Additionally, the effect of the ternary interaction term was not negligible in the model. Estimated ternary interaction parameters (χ123) presented a linear relation with temperature and negative values, indicating that the solubility of the polymers in mixtures of solvent/non-solvent was higher than expected for single binary interaction. Finally, PES ternary systems exhibited higher influence of the ternary interaction parameter than PVDF systems.

Advances in precursor system for silica-based aerogel production toward improved mechanical properties, customized morphology, and multifunctionality: A review.

Conventional silica-based aerogels are among the most promising materials considering their special properties, such as extremely low thermal conductivity (~15 mW/mK) and low-density (∼0.003-0.5 g.cm-3) as well as high surface area (500-1200 m2. g-1). However, they have relatively low mechanical properties and entail extensive and energy-consuming processing steps. Silica-based aerogels are mostly fragile and possess minimal mechanical properties as well as a long processing procedure which hinders their application range. The key point in improving the mechanical properties of such a material is to increase the connectivity in the aerogel backbone. Several methods of mechanical improvement of silica-based aerogels have been explored by researchers such as (i) use of flexible silica precursors in silica gel backbone, (ii) surface-crosslinking of silica particles with a polymer, (iii) prolonged aging step in different solutions, (iv) distribution of flexible nanofillers into the silica solution prior to gelation, and, most recently, (v) polymerizing the silica precursor prior to gelation. The polymerized silica precursor, as in the most recent approach, can be gelled either by binodal decomposition (nucleation and growth), resulting in a particulate structure, or by spinodal decomposition, resulting in a non-particulate structure. By optimizing the material composition and processing conditions of materials, the aerogel can be tailored with different functional capabilities. This review paper presents a literature survey of precursor modification toward increased connectivity in the backbone, and the synthesis of inorganic and hybrid systems containing siloxane in the backbone of the silica-based aerogels and its composite version with carbon nanofillers. This review also explains the novel properties and applications of these material systems in a wide area. The relationship among the materials-processing-structure-properties in these kinds of aerogels is the most important factor in the development of aerogel products with given morphologies (particulate, fiber-like, or non-particulate) and their resultant properties. This approach to advancing precursor systems leads to the next-generation, multifunctional silica-based aerogel materials.

Walking Along a Protein Phase Diagram to Determine Coexistence Points by Static Light Scattering.

The physical process of liquid-liquid phase separation (LLPS), where the drive to minimize global free energy causes a solution to demix into dense and light phases, plays many important roles in biology. It is implicated in the formation of so-called "membraneless organelles" such as nucleoli, nuclear speckles, promyelocytic leukemia protein bodies, P bodies, and stress granules along with the formation of biomolecular condensates involved in transcription, signaling, and transport. Quantitative studies of LLPS in vivo are complicated by the out-of-equilibrium, multicomponent cellular environment. While in vitro experiments with purified biomolecules are inherently an oversimplification of the cellular milieu, they allow probing of the rich physical chemistry underlying phase separation. Critically, with the application of suitable models, the thermodynamics of equilibrium LLPS can inform on the nature of the intermolecular interactions that mediate it. These same interactions are likely to exist in out-of-equilibrium condensates within living cells. Phase diagrams map the coexistence points between dense and light phases and quantitatively describe LLPS by mapping the local minima of free energy versus biomolecule concentration. Here, we describe a light scattering method that allows one to measure coexistence points around a high-temperature critical region using sample volumes as low as 10 µl.

Phase Separation of Intrinsically Disordered Proteins.

There is growing interest in the topic of intracellular phase transitions that lead to the formation of biologically regulated biomolecular condensates. These condensates are membraneless bodies formed by phase separation of key protein and nucleic acid molecules from the cytoplasmic or nucleoplasmic milieus. The drivers of phase separation are referred to as scaffolds whereas molecules that preferentially partition into condensates formed by scaffolds are known as clients. Recent advances have shown that it is possible to generate physical and functional facsimiles of many biomolecular condensates in vitro. This is achieved by titrating the concentration of key scaffold proteins and solution parameters such as salt concentration, pH, or temperature. The ability to reproduce phase separation in vitro allows one to compare the relationships between information encoded in the sequences of scaffold proteins and the driving forces for phase separation. Many scaffold proteins include intrinsically disordered regions whereas others are entirely disordered. Our focus is on comparative assessments of phase separation for different scaffold proteins, specifically intrinsically disordered linear multivalent proteins. We highlight the importance of coexistence curves known as binodals for quantifying phase behavior and comparing driving forces for sequence-specific phase separation. We describe the information accessible from full binodals and highlight different methods for-and challenges associated with-mapping binodals. In essence, we provide a wish list for in vitro characterization of phase separation of intrinsically disordered proteins. Fulfillment of this wish list through key advances in experiment, computation, and theory should bring us closer to being able to predict in vitro phase behavior for scaffold proteins and connect this to the functions and features of biomolecular condensates.

Purification of bacterial inulinase in aqueous two-phase systems.

In this study, extracellular inulinase from Bacillus sp. 11/3 was partially purified and concentrated using aqueous two-phase system (ATPS). Two different phase forming salts and four types of polyethylene glycol (PEG) were used. Binodal curves and tie-length lines (TLLs) for eight ATPS were developed. For inulinase purification, concentrations of PEG and salt according to binodal curves (between 17 and 26%) were chosen. All ATPSs for inulinase purification were characterized. An ATPS consisted of 26% PEG1000 and 26% MgSO4 was found to be the most suitable for inulinase purification. This ATPS has 28.47% TLL, 1.03 of volume ratio, purification factor of 4.65 fold and recovery yield of 66.17%. On the SDS-PAGE electrophoresis two protein bands with molecular weight of around 24 and 56 kDa were observed. The partially purified enzymes had optimal activity at pH 8.0 and 6.5, optimal temperature at 30 and 70°C and kinetic parameters Km = 26.32 mmol and Vmax = 526 mmol/min.

Modified binodal model describes phase separation in aqueous two-phase systems in terms of the effects of phase-forming components on the solvent features of water.

The binodal model pioneered by Guan et al. [Y. Guan, T. H. Lilley, T. E. Treffry, J. Chem. Soc. Faraday Trans., 89 (1993) 4283-4298] remains the most successful in regard to the quantitative description of phase diagrams among various theoretical models proposed to describe phase separation in aqueous mixtures of polymers. This is a semi-empirical model based on the assumption that any point on the binodal line may be viewed as a saturated solution of the phase-forming compound-1 in the solution of the phase-forming compound-2. Although this model is originally based on the excluded volume concept, we suggest that the solubility of the compound-1 in solutions of compound-2 may depend on the solvent properties of water in solutions of compound-2. The binodal model described in these terms was very successfully applied to the phase diagrams of aqueous two-phase systems formed by different pairs of polymers (dextran, Ficoll, poly(ethylene glycol)-8000, and Ucon). Phase diagram of a new aqueous two-phase system formed by trimethylamine N-oxide (TMAO) and polypopylene glycol-400 and previously reported phase diagram for system formed by TMAO and poly(ethylene glycol)-600 were also described by this model quite well. It was found that the modified binodal model is also applicable to single polymer-salt and polymer-ionic liquid aqueous two-phase systems. The most important conclusion of our study is that the effects of different compounds (polymers, salts, ionic liquids) on the solvent features of water in their aqueous solutions cause changes in the water structure, resulting in phase separation in the mixtures of these compounds.

Effects of Phase Separation Behavior on Morphology and Performance of Polycarbonate Membranes.

The phase separation behavior of bisphenol-A-polycarbonate (PC), dissolved in N-methyl-2-pyrrolidone and dichloromethane solvents in coagulant water, was studied by the cloud point method. The respective cloud point data were determined by titration against water at room temperature and the characteristic binodal curves for the ternary systems were plotted. Further, the physical properties such as viscosity, refractive index, and density of the solution were measured. The critical polymer concentrations were determined from the viscosity measurements. PC/NMP and PC/DCM membranes were fabricated by the dry-wet phase inversion technique and characterized for their morphology, structure, and thermal stability using field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis, respectively. The membranes' performances were tested for their permeance to CO2, CH4, and N2 gases at 24 ± 0.5 °C with varying feed pressures from 2 to 10 bar. The PC/DCM membranes appeared to be asymmetric dense membrane types with appreciable thermal stability, whereas the PC/NMP membranes were observed to be asymmetric with porous structures exhibiting 4.18% and 9.17% decrease in the initial and maximum degradation temperatures, respectively. The ideal CO2/N2 and CO2/CH4 selectivities of the PC/NMP membrane decreased with the increase in feed pressures, while for the PC/DCM membrane, the average ideal CO2/N2 and CO2/CH4 selectivities were found to be 25.1 ± 0.8 and 21.1 ± 0.6, respectively. Therefore, the PC/DCM membranes with dense morphologies are appropriate for gas separation applications.

Bis(triethanolamine)bis(µ2-trimesato)dicobalt(II): a Co(II) dimer with an unreported two-dimensional supramolecular topology formed from triethanolamine and trimesic acid ligands.

Supramolecular networks are an important subset in the field of coordination polymer (CP) frameworks and are widely encountered in crystal engineering research. The search for novel topologies continues to be a significant goal in CP chemistry. The dimeric compound bis(µ-5-carboxybenzene-1,3-dicarboxylato-κ(2)O(1):O(3))bis[(triethanolamine-κ(4)N,O,O',O'')cobalt(II)], [Co2(C9H4O6)2(C6H15NO3)2], formed from the coligands 5-carboxybenzene-1,3-dicarboxylate (tmaH(2-)) and triethanolamine (teaH3), namely [Co(µ2-tmaH)(teaH3)]2, was synthesized and characterized by single-crystal and powder X-ray diffraction analyses, IR spectroscopy, thermogravimetric analysis (TGA) and magnetic measurements. The crystal structure features a zero-dimensional molecular structure consisting of centrosymmetric macrocyclic dinuclear complexes. Four classical hydrogen bonds between carboxylate groups and hydroxyethyl arms stabilize and extend the molecules into a two-dimensional supramolecular network. The topological analysis indicates that an unreported (3,5)-binodal supramolecular topology with a short Schläfli symbol of (4.5.6)(4.5(5).6(3).7) can be achieved by means of intermolecular hydrogen bonds. The crystal structure accounts for the potential to obtain unique topological types from two excellent hydrogen-bonding candidates, i.e. tmaH3 and teaH3. A variable-temperature magnetic study shows the existence of antiferromagnetic behaviour in the complex.

Determination of aqueous two phase system binodal curves using a microfluidic device.

Aqueous two phase systems (ATPS) offer great potential for selective separation of a wide range of biomolecules by exploring differences in solubility in each of the two phases. However, their use has been greatly hindered due to poor theoretical understanding of the principles behind ATPS formation and the empirical and time-consuming techniques used for the determination of optimal extraction parameters including the binodal curves. In this work, characteristic ATPS binodal curves were determined by a novel technique in which the formation of an ATPS system is measured in a microfluidic device. Two solutions containing separate ATPS solution precursors were loaded into the side inlets of a three inlet microfluidic channel while milli-Q water was loaded into the middle inlet. By varying the flow rates of the three solutions, a wide range of concentrations inside the microchannel could be rapidly tested using limited volumes. Using optical microscopy, depending on the concentrations inside the microchannel, three different states could be observed at the end of the microchannel (i) the presence of an interface; (ii) no presence of an interface; or (iii) the presence of an unstable interface. The binodal curve was calculated using the points corresponding to unstable interfaces and compared to binodal curves obtained through the standard turbidometric titration method for both PEG/salt and PEG/dextran systems.

Quantification of feather structure, wettability and resistance to liquid penetration.

Birds in the cormorant (Phalacrocoracidae) family dive tens of metres into water to prey on fish while entraining a thin layer of air (a plastron film) within the microstructures of their feathers. In addition, many species within the family spread their wings for long periods of time upon emerging from water. To investigate whether wetting and wing-spreading are related to feather structure, microscopy and photographic studies have previously been used to extract structural parameters for barbs and barbules. In this work, we describe a systematic methodology to characterize the quasi-hierarchical topography of bird feathers that is based on contact angle measurements using a set of polar and non-polar probing liquids. Contact angle measurements on dip-coated feathers of six aquatic bird species (including three from the Phalacrocoracidae family) are used to extract two distinguishing structural parameters, a dimensionless spacing ratio of the barbule (D*) and a characteristic length scale corresponding to the spacing of defect sites. The dimensionless spacing parameter can be used in conjunction with a model for the surface topography to enable us to predict a priori the apparent contact angles of water droplets on feathers as well as the water breakthrough pressure required for the disruption of the plastron on the feather barbules. The predicted values of breakthrough depths in water (1-4 m) are towards the lower end of typical diving depths for the aquatic bird species examined here, and therefore a representative feather is expected to be fully wetted in a typical deep dive. However, thermodynamic surface energy analysis based on a simple one-dimensional cylindrical model of the feathers using parameters extracted from the goniometric analysis reveals that for water droplets on feathers of all six species under consideration, the non-wetting 'Cassie-Baxter' composite state represents the global energy minimum of the system. By contrast, for other wetting liquids, such as alkanes and common oils, the global energy minimum corresponds to a fully wetted or Wenzel state. For diving birds, individual feathers therefore spontaneously dewet once the bird emerges out of water, and the 'wing-spreading' posture might assist in overcoming kinetic barriers associated with pinning of liquid droplets that retard the rate of drying of the wet plumage of diving birds.