Air–liquid and liquid–liquid interfaces influence the formation of the urothelial permeability barrier in vitro

Optimizing culture conditions is known to be crucial for the differentiation of urothelial cell cultures and the formation of the permeability barrier. However, so far, no data exist to confirm if air–liquid (AL) and liquid–liquid (LL) interfaces are physiologically relevant during urothelial differentiation and barrier formation. To reveal the influence of interfaces on the proliferation, differentiation, and barrier formation of the urothelial cells (UCs) in vitro, we cultured UCs under four different conditions, i.e., at the AL or LL interfaces with physiological calcium concentration and without serum or without physiological calcium concentration and with serum. For each of the four models, the urothelial integrity was tested by measuring the transepithelial resistance (TER), and the differentiation stage was examined by immunolabeling of differentiation-related markers and ultrastructural analysis. We found that the UCs at a LL interface, regardless of the presence or absence of calcium or serum, form the urothelium with more cell layers and achieve a higher TER than UCs at an AL interface. However, UCs grown at an AL interface with physiological concentration of calcium in medium form only one- to two-layered urothelium of UCs, which are larger and express more differentiation-related proteins uroplakins than UCs in other models. These results demonstrate that the interface itself can play a major, although so-far neglected, role in urothelial physiology, particularly in the formation of the urothelial permeability barrier in vitro and the regulatory mechanisms related with urothelial differentiation. In the study, the culturing of UCs in three successive steps is proposed.     

Rare events out of equilibrium: mobility through the liquid–liquid interface

Transition state theory provides a well established means to compute the rate at which rare events occur; however, this is strictly an equilibrium approach. Here we consider a nonequilibrium problem of this nature in the form of transport through a liquid–liquid interface. When two immiscible liquids are coexisting in equilibrium, there will be a certain amount of mixing between the two phases, resulting in a finite linear mobility across the liquid–liquid interface. We derive an exact relationship between the mobility and the local diffusion in the direction perpendicular to the interface. We compute the mobility using both nonequilibrium molecular dynamics and a variety of linear response type approaches, with accurate agreement being obtained for the best of these. Our analysis makes it clear how the local diffusion is influenced by the inhomogeneities of the interface, even when at a distance from it. This nonlocal character to the mobility has not been appreciated before and results in a strong variation in the local diffusion, which is formally coupled to the variation in the potential of mean force. The nonlocal aspect of the diffusion requires the velocity autocorrelation function to be integrated out to far longer times than is the case for homogeneous liquids, and requires special care with regard to the choice of numerical approach.     

Investigations on the biodegradation of alkylpolyglucosides by means of liquid chromatography–electrospray mass spectrometry

The biodegradation of alkylpolyglucosides (APGs) was studied under the conditions of the OECD Screening Test with activated sludge as an inoculum. An influence of alkyl and sugar chain length on the biodegradation rate and a central scission pathway of the biodegradation were investigated. The liquid chromatography-electrospray mass spectrometry technique was used for alkylpolyglucoside analysis and for identification and semiquantitative determination of metabolites. It was found that APGs with a longer alkyl chain were biodegraded faster than those with a shorter one. However, a longer sugar chain caused slower biodegradation of APGs. The central scission pathway of biodegradation was also confirmed.     

Effect of nutrients on the accumulation of glutamyl endopeptidase in the culture liquid ofBacillus intermedius 3–19

The effect of nutrients and growth conditions on the accumulation of glutamyl endopeptidase in the culture liquid ofBacillus intermedius 3–19 was studied. Glucose and other readily metabolizable carbon sources were found to suppress the production of the enzyme, whereas inorganic phosphate and ammonium cations enhanced it. Protein substrates, such as casein, gelatin, and hemoglobin, did not affect enzyme production. Some bivalent cations (Ca²⁺, Mg²⁺, Co²⁺) increased the production of glutamyl endopeptidase, but others (Zn²⁺, Fe²⁺, Cu²⁺) acted in the opposite way. The rate of enzyme accumulation in the culture liquid increased as the growth rate of the bacterium decreased, so that the maximum enzyme activity was observed in the stationary growth phase. Based on the results of this investigation, an optimal medium for the maximum production of glutamyl endopeptidase byB. intermedius 3–19 was elaborated.     

A model for the rate of enzymatic hydrolysis of cellulose in heterogeneous solid–liquid systems

Hydrolysis of cellulose by cellulase enzymes has been studied in a stirred batch reactor at 50°C. A kinetic model has been devised by which the behaviour of such a reaction could be described. The model has been developed on the basis of shrinking particle theory and Langmuir isotherm concept. The applicability of the model has been tested by comparing the experimental results for diverse reaction systems, obtained in the present study or taken from the literature, and those predicted from the model. The degree of agreement was within ±2–11%.     

Voltammetric studies of glutathione transfer across arrays of liquid–liquid microinterfaces for sensing applications

Amino Acids - The simple and facilitated transfer of tripeptide glutathione across the water/2-nitrophenyl octhyl ether interface was studied via cyclic voltammetry at interface between two...     

Vapor–liquid equilibria and thermophysical behavior of the SPC-HW model for heavy water

The vapor–liquid coexistence curve of the simple point charge heavy-water model (SPC-HW), [J. Chem. Phys., 114, 8064–8067 (2001)] is determined by Gibbs Ensemble Monte-Carlo (GEMC) simulation. The estimated critical conditions of the model based on the Wegner-type expansion for the order parameters and the rectilinear diameter are ρ_c = 0.300 g/cc, T _c = 661 K and P _c = 156 bars. The dielectric constant determined by isothermal–isochoric molecular dynamics is underpredicted along the coexistence curve by 29–44% in comparison with the experimental values. The analysis of the orthobaric temperature dependence of the system microstructure, in terms of the three site–site radial distribution functions, indicates that the first coordination numbers for the oxygen–oxygen and the oxygen–deuterium interactions are ～4.3 ± 0.1 and ～1.9 ± 0.1 at T = 300 K, and decrease by 15 and 55%, respectively, at criticality. The dipole–dipole correlation functions show that the orientational order in heavy water is quickly lost beyond the first oxygen–oxygen coordination shell. The model's second virial coefficient is determined by Monte-Carlo integration and used to aid the interpretation of the predicted phase equilibrium results.     

In situ recovery of 2,3-butanediol from fermentation by liquid–liquid extraction

End-product conversion, low product concentration and large volumes of fermentation broth, the requirements for large bioreactors, in addition to the high cost involved in generating the steam required to distil fermentation products from the broth largely contributed to the decline in fermentative products. These considerations have motivated the study of organic extractants as a means to remove the product during fermentation and minimize downstream recovery. The aim of this study is to assess the practical applicability of liquid–liquid extraction in 2,3-butanediol fermentations. Eighteen organic solvents were screened to determine their biocompatibility, and bioavailability for their effects on Klebsiella pneumoniae growth. Candidate solvents at first were screened in shake flasks for toxicity to K. pneumoniae. Cell density and substrate consumption were used as measures of cell toxicity. The possibility of employing oleyl alcohol as an extraction solvent to enhance end product in 2,3-butanediol fermentation was evaluated. Fermentation was carried out at an initial glucose concentration of 80 g/l. Oleyl alcohol did not inhibit the growth of the fermentative organism. 2,3-Butanediol production increased from 17.9 g/l (in conventional fermentation) to 23.01 g/l (in extractive fermentation). Applying oleyl alcohol as the extraction solvent, about 68% of the total 2,3-butanediol produced was extracted. An erratum to this article can be found at     

Clean-up and re-use of kieselguhr (Extrelut) for liquid–liquid extraction of urinary cortisol

Cortisol was isolated from human urine using kieselguhr (Extrelut)-filled columns. After use, Extrelut was cleaned-up once with distilled water and twice with ethanol. Before re-use, the cleaned-up kieselguhr was dried for 24 h by a warm air stream. The comparison of cortisol recovery from human urine and HPLC chromatograms of urinary extracts show that Extrelut can be repeatedly used for liquid–liquid extraction of urinary cortisol.     

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid–liquid phase separation of tau

Liquid–liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.     

PEG–salt aqueous two-phase systems: an attractive and versatile liquid–liquid extraction technology for the downstream processing of proteins and enzymes

Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)–salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid–liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG–salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid–liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.     

Biophysics of endocytic vesicle formation: A focus on liquid–liquid phase separation

Endocytosis is a fine-tuned mechanism of cellular communication through which cells internalize molecules on the plasma membrane, such as receptors and their bound ligands. Through receptor clustering in endocytic pits, recruitment of active receptors to different endocytic routes and their trafficking towards different fates, endocytosis modulates cell signaling and ultimately leads to a variety of biological responses. Many studies have focused their attention on specialized endocytic mechanisms depending on the nature of the internalizing cargo and cellular context, distinct sets of coat proteins, endocytic adaptors and membrane lipids. Here, we review recent advances in our understanding of the principles underlying endocytic vesicle formation, integrating both biochemical and biophysical factors, with a particular focus on intrinsically disordered regions (IDRs) creating weakly interconnected protein networks assembled through liquid–liquid phase separation (LLPS) and driving membrane bending especially in clathrin-mediated endocytosis (CME). We finally discuss how these properties impinge on receptor fate and signaling.     

Equilibrium studies on enantioselective liquid–liquid extraction of homophenylalanine enantiomers with metal-BINAP complexes

Enantioselective liquid–liquid extraction of homophenylalanine (Hph) enantiomers was investigated with metal-BINAP complexes as enantioselective extractants. The metal complexes were synthesized by the complexation of (s)-2,2′-Bis(diphenylphosphino)-1,1′-binaphthalene (BINAP) with different central ions, among which, copper(I) complex allowed the separation of the Hph enantiomers with the highest operational selectivity. Efficiency of the extraction depends, often strongly, on a number of process variables, including types of organic solvents, pH of the aqueous phase, concentration of host and substrate, and temperature. In order to better understand the extraction process, equilibrium of the system were modeled by a homogeneous reaction model and an interfacial reaction model, respectively. Important parameters required by the modeling, such as complexation equilibrium constant and physical distribution coefficients were determined experimentally. When coupled with the parameters, extraction performance can be predicted by the models. Comparison between the experimental values and the model predictions indicates that the homogeneous reaction model can predict more accurately. By modeling and experiment, an optimal extraction condition concerning pH of 8 and host concentration of 2 mmol/L was obtained with high enantioselective (α) of 1.837 and performance factor (pf) of 0.086.     

Atomistic simulations of liquid–liquid coexistence in confinement: comparison of thermodynamics and kinetics with bulk

We review a few simulation methods and results related to the structure and non-equilibrium dynamics in the coexistence region of immiscible symmetric binary fluids, in bulk as well as under confinement, with special emphasis on the latter. Monte Carlo methods to estimate interfacial tensions for flat and curved interfaces have been discussed. The latter, combined with a thermodynamic integration technique, provides contact angles for coexisting fluids attached to the wall. For such three-phase coexistence, results for the line tension are also presented. For the kinetics of phase separation, various mechanisms and corresponding theoretical expectations have been discussed. A comparative picture between the domain growth in bulk and confinement (including thin-film and semi-infinite geometry) has been presented from molecular dynamics simulations. Applications of finite-size scaling technique have been discussed in both equilibrium and non-equilibrium contexts.     

Application of kinetic Monte Carlo method to the vapour–liquid equilibria of associating fluids and their mixtures

Canonical kinetic Monte Carlo (C-kMC) simulations have been carried out to assess their feasibility and potential for calculating the vapour–liquid equilibria of various pure components with increasingly strong electrostatic interactions (carbon dioxide, methanol, ammonia and water) over a wide range of temperatures and for methanol/water mixtures at 298 K. The simulation results show that C-kMC is successful as a method for studying phase equilibria and thermodynamic properties. For all the examples investigated, the performance of the C-kMC method is at least as good as that of the conventional Monte Carlo (MC) methods and is efficient at low temperature where these fail. It also provides a route that is superior to the Widom method for the calculation of chemical potential. We recommend this method for this purpose and as an alternative to conventional MC for simulations of strongly associating fluids and at low temperatures.     

Gibbs ensemble Monte Carlo simulations of binary vapour–liquid–liquid equilibrium: application to n-hexane–water and ethane–ethanol systems

Gibbs ensemble Monte Carlo (GEMC) simulations in the isochoric–isothermal (NVT) ensemble were used to simulate vapour–liquid–liquid equilibrium (VLLE) for binary n-hexane–water and ethane–ethanol mixtures. The GEMC simulation of binary VLLE data proved to be extremely difficult and that is probably the reason why the open literature is so sparse with simulations for these types of systems. The results presented in this paper are to our knowledge the first successful binary three-phase GEMC simulations of non-idealised fluid systems. This paper also shows that the isobaric–isothermal (NPT) ensemble is unsuitable for the simulation of phase equilibria of binary three-phase systems.