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.     

X-ray and neutron surface scattering for studying lipid/polymer assemblies at the air–liquid and solid–liquid interfaces

Simple mono- and bilayers, built of amphiphilic molecules and prepared at air–liquid or solid–liquid interfaces, can be used as models to study such effects as water penetration, hydrocarbon chain packing, and structural changes due to head group modification. In the paper, we will discuss neutron and X-ray reflectometry and grazing incidence X-ray diffraction techniques used to explore structures of such ultra-thin organic films in different environments. We will illustrate the use of these methods to characterize the morphologies of the following systems: (i) polyethylene glycol-modified distearoylphosphatidylethanolamine monolayers at air–liquid and solid–liquid interfaces; and (ii) assemblies of branched polyethyleneimine polymer and dimyristoylphophatidylcholine lipid at solid–liquid interfaces.     

Liquid-surface immobilization system and liquid–liquid interface bioreactor: Application to fungal hydrolysis

Novel fungal cultivation and bioconversion systems are proposed. Spores and mycelia of a fungus suspended in a liquid medium were effectively floated on a liquid surface by the aid of a ballooned microsphere (MS). Many fungi such as Aspergillus and Penicillium formed a thick and physically strong fungus-MS mat on the liquid surface followed by stationary cultivation (LSI). The fungus-MS mat of Absidia coerulea IFO 4423 was overlaid by a solution of 2-ethylhexyl acetate (1) in n-decane (liquid–liquid interface bioreactor, L-L IBR). The strain could efficiently catalyze the hydrolysis of 1 to 2-ethyl-1-hexanol (2). The accumulation of 2 in the L-L IBR was significantly higher than those in emulsion and organic-aqueous two-liquid-phase systems and a formerly reported interface bioreactor (solid–liquid interface bioreactor, S-L IBR). Furthermore, lipase production in the LSI system was also higher than that in a submerged cultivation system.     

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.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

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.     

Comparison of simple perturbation-theory estimates for the liquid–solid and the liquid–vapor interfacial free energies of Lennard-Jones systems

The most naive perturbation method to estimate interfacial free energies is based on the assumption that the interface between coexisting phases is infinitely sharp. Although this approximation does not yield particularly accurate estimates for the liquid–vapor surface tension, we find that it works surprisingly well for the interface between a dense liquid and a solid. As an illustration we estimate the liquid–solid interfacial free energy of a Lennard-Jones system with truncated and shifted interactions and compare the results with numerical data that have been reported in the literature. We find that the agreement between theory and simulation is excellent. In contrast, if we apply the same procedure to estimate the variation of the liquid–vapor surface tension, for different variants of the Lennard-Jones potential (truncated/shifted/force-shifted), we find that the agreement with the available simulation data is, at best, fair. The present method makes it possible to obtain quick and easy estimate of the effect on the surface free energy of different potential-truncation schemes used in computer simulations.     

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.     

Trace quantification of 1-octacosanol and 1-triacontanol and their main metabolites in plasma by liquid–liquid extraction coupled with gas chromatography–mass spectrometry

A method for the simultaneous determination of 1-octacosanol and 1-triacontanol and their main metabolites in rat plasma was developed. The procedure involved ethanolic NaOH saponification of the sample, acidification, liquid–liquid extraction, and derivatization of the analytes to its trimethylsilylether/ester, followed analysis by gas chromatography–mass spectrometry (GC–MS) in selected ion monitoring (SIM) mode. Quantification was performed by the internal standard method using betulin. The method had a good linearity over the range 8.4–540 ng/ml (r ≥ 0.998) and showed an excellent intra-day (R.S.D. = 0.59–3.06%) and inter-day (R.S.D. = 2.99–5.22%) precision according to the acceptance criteria. The detection limits ranged between 1.32 and 3.47 ng/ml. The method was applied successfully to study the total plasmatic concentration of 1-octacosanol, octacosanoic acid, 1-triacontanol, and triacontanoic acid, after an oral dose of policosanols mixture, using plasma samples of 100 μl.     

Radial liquid distribution in gas–liquid concurrent downflow through packed beds

Radial liquid velocity profiles under concurrent air-water downflow through a packed bed containing cylinders were experimentally obtained at different flow rates of both the phases. The variation in liquid velocity with radial position of the column was estimated. A simple correlation for predicting the liquid phase velocity in terms of single phase velocities of gas and liquid, and dynamic liquid saturation was proposed.     

Characterization of immunoreactive acetyl–Ser–Asp–Lys–Pro in human plasma and urine by liquid chromatography–electrospray mass spectrometry

The tetrapeptide AcSDKP, a natural and specific substrate of angiotensin I-converting enzyme (ACE), is a negative regulator of hematopoiesis. AcSDKP has been measured in various biological media using an enzyme immunoassay (EIA), but its presence in human plasma and urine has not been formally established. By using immunoaffinity extraction and liquid chromatography–electrospray mass spectrometry, we demonstrate that AcSDKP-like immunoreactivity measured with EIA in plasma and urine samples from untreated, captopril- (an ACE inhibitor) and AcSDKP-treated subjects corresponds to AcSDKP. The present study confirms that AcSDKP is naturally present in human plasma and urine and that EIA is reliable for its measurement in such media.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

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     

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.     

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.     

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.