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.     

Mucus liquid crystallinity: is function related to microstructural domain size?

The size of liquid crystalline domains formed in partially dried giraffe saliva is found to be an order of magnitude greater than that previously documented for slug pedal mucus. A correlation between (a) intrinsic liquid crystalline domain size and (b) the scale of surface topography over which the mucus is required to provide lubrication is postulated. The scale of mucus microstructure can be related, via a simple model, to two significant material constants: the elastic constant K for distortion of molecular alignment in the liquid crystalline state, and the anchoring energy I at the liquid crystal/substrate interface. In turn, the quantity K is expected to depend on fundamental molecular characteristics of the constituent mucin, such as molecular weight and degree of glycosylation. The possibility that observations of mucus microstructure might serve as a diagnostic tool for mucus defects at the molecular level is suggested.     

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.     

A new kinetic Monte Carlo scheme with Gibbs ensemble to determine vapour–liquid equilibria

We present a new kinetic Monte Carlo scheme, as an alternative to the Gibbs ensemble Monte Carlo (GEMC) method, to determine vapour–liquid equilibria using a canonical ensemble in a system composed of two boxes. To illustrate the method, we have tested it with two systems: (1) argon over a range of temperatures from below the triple point to close to the critical point; (2) methane and ethane mixtures of various compositions at 180 K. The advantage of the new scheme is that chemical potentials of all components are accurately determined in both boxes. In particular, the chemical potential in the liquid box is determined much more accurately than with the Widom method employed in conventional GEMC simulations.     

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.     

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.     

Correction to: Improving micropropagation of Mentha × piperita L. using a liquid culture system

In Vitro Cellular & Developmental Biology - Plant - This correction reflects Sadanand A. Dhekney’s updated e-mail address and affiliation.     

A liquid chromatography - mass spectrometry method to measure ¹³C-isotope enrichment for DNA stable-isotope probing

DNA stable-isotope probing (DNA-SIP) is a cultivation-independent technique that makes it possible to associate metabolic function and taxonomic identity in a wide range of terrestrial and aquatic environments. In DNA-SIP, DNA is labeled via the assimilation of a labeled growth substrate that is subsequently used to identify microorganisms involved in assimilation of the substrate. However, the labeling time has to be sufficient to obtain labeled DNA but not so long such that cross-feeding of 13C-labeled metabolites from the primary consumers to nontarget species can occur. Confirmation that the DNA is isotopically labeled in DNA-SIP assays can be achieved using an isotope ratio mass spectrometer. In this study, we describe the development of a method using liquid chromatography (HPLC) coupled to a quadrupole mass spectrometer (QMS) to measure the 13C enrichment of thymine incorporated into DNA in Escherichia coli cultures fed with [13C]acetate. The method involved the hydrolysis of DNA extracted from the cultures that released the nucleotides, followed by the separation of the thymine by HPLC on a reverse-phase C? column in isocratic elution mode and the detection and quantification of 13C-labeled thymine by QMS. To mimic a DNA-SIP assay, a DNA mixture was made using 13C-labeled E. coli DNA with DNA extracted from five bacterial species. The HPLC-MS method was able to measure the correct proportion of 13C-DNA in the mix. This method can then be used as an alternative to the use of isotope ratio mass spectrometry in DNA-SIP assays.     

Basis of coffee seed sensitivity to liquid nitrogen exposure: oxidative stress or imbibitional damage?

Two hypotheses, namely the occurrence of post‐thaw oxidative stress or imbibitional damage, were tested to explain the high sensitivity of coffee seeds to liquid nitrogen (LN) exposure. Oxidative stress was studied by measuring primary and secondary products of lipid peroxidation in seeds during the desiccation and rehydration periods. The 4‐hydroxynonenal (4‐HNE) content of seeds remained constant throughout the desiccation step. No significant difference was observed between desiccated seeds and seeds desiccated and exposed to LN for the evolution of their 4‐HNE and hydroperoxide contents during rehydration. In both cases, an increase in 4‐HNE and hydroperoxide contents of seeds was observed during the first hours of culture under germination conditions, followed by a progressive decrease down to values comparable to those observed in desiccated seeds. The hydroperoxide composition of frozen seeds was not significantly different from that of control seeds. The (S)/(R) enantiomeric ratios of 9‐ and 13‐hydroxy‐octadecadienoic acid extracted from rehydrating seeds were chiral, suggesting that they originated from lipoxygenase activity. These results suggest that the high sensitivity of coffee seeds to LN exposure is not directly associated with the occurrence of an oxidative stress during post‐thaw rehydration. The effect on seed viability of different rehydration procedures previously identified to reduce membrane imbibitional injury was studied after desiccation and LN exposure. Desiccation tolerance increased with, by increasing order, seed osmoconditioning, pre‐heating and pre‐humidifying prior to their culture under germination conditions. Among the four combinations of pre‐humidification durations (24 or 48 h) and temperatures (25 or 37°C) tested, pre‐humidification for 24 h at 37°C gave the highest level of desiccation tolerance. This rehydration procedure also dramatically increased seed viability after LN exposure. Seed desiccation sensitivity modelling in combination with the calculation of the decrease in seed water activity during cooling facilitated the explanation of the beneficial effect of controlled rehydration after desiccation and LN exposure. These results support the hypothesis that imbibitional membrane damage is involved in the sensitivity of coffee seeds to LN exposure.     

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.     

Phosphorylation assay in liquid scintillation counter using C̆erenkov radiation of P: Application to photophosphorylation

A simple procedure is described for the assay of phosphorylation using C?erenkov radiation to detect ³²P in a liquid scintillation counter. Unreacted ³²P_i is first removed from the reaction mixture as the phosphomolybdate complex by butanol/benzene extraction. Addition of ammonium hydroxide to the remaining aqueous fraction avoids color quenching, phase separation, and instability in the counting rate during measurement of ³²P. Application of this procedure to several photophosphorylation systems is included.     

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.     

Using liquid chromatography–tandem mass spectrometry to quantify monohydroxylated metabolites of polycyclic aromatic hydrocarbons in urine

We present an assay which employs enzyme digestion and solid phase extraction followed by liquid chromatography–tandem mass spectrometry to simultaneously quantify 16 hydroxylated polycyclic aromatic hydrocarbons (OHPAHs) in 3-ml samples of urine. The analytes consisted of 2-, 3-, and 4-ring OHPAHs, namely, 1- and 2-hydroxynaphthalene (1- and 2-OHNAP), 2-hydroxyfluorine (2-OHFLU), 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene (1-, 2-, 3-, 4-, and 9-OHPHE), 1-hydroxypyrene (1-OHPYR), 1- and 2-hydroxybenzo(a)anthracene (1- and 2-OHBAA), 3- and 6-hydroxychrysene (3- and 6-OHCHR) and 3-, 7-, and 9-hydroxybenzo(a)pyrene (3-, 7-, and 9-OHBAP). The method was validated using urine samples from steel workers and control subjects. The coefficients of variation of the method for the particular analytes were between 7% and 27% and the limits of quantitation were between 0.002 and 0.010 μg/l urine. The 2- and 3-ring OHPAHs were easily quantified in all subjects. However, 1-OHPYR was the only representative of the 4- and 5-ring metabolites that could be quantified. Pairwise correlations showed that all OHPAHs were highly correlated with each other (0.553 ≤ r ≤ 0.910) and with 1-OHPYR (0.614 ≤ r ≤ 0.910), the metabolite most widely accepted as a short-term biomarker of exposure to PAHs. The analyte, 2-OHNAP exhibited the lowest pairwise correlations with the other OHPAHs (0.542 ≤ r ≤ 0.628), presumably due to confounding by smoking. Metabolites of phenanthrene, an abundant PAH and the smallest to possess a bay region, are promising OHPAHs for characterizing both exposures to PAHs and the various metabolic pathways.     

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.     

Optimized 25-hydroxyvitamin D analysis using liquid–liquid extraction with 2D separation with LC/MS/MS detection, provides superior precision compared to conventional assays

The analysis of 25-hydroxyvitamin D3 (25(OH)D3) and related metabolites represents a considerable challenge for both clinical and research laboratories worldwide. There is currently debate about the best methodology employed to assess vitamin D status and whether the 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3) should be separated and quantitated when measuring 25(OH)D3. Mass spectrometry techniques are generally regarded as the gold standard due to high specificity for vitamin D metabolites. However, many methods require high sample volumes for analysis. We have developed a new 2 dimensional (2D) ultra performance liquid chromatography (UPLC) separation coupled tandem mass spectrometry (MS/MS) detection to accurately quantitate 25(OH)D3, epi-25(OH)D3, and 25(OH)D2 in adults and children, requiring only 50 μL of human serum. The assay gives excellent separation of epi-25(OH)D3, and 25(OH)D2 from 25(OH)D3, has excellent precision with an intra-assay CV of 0.5 % at 74 nmol/L and can report down to 2 nmol/L for 25(OH)D3. Furthermore, the method shows excellent agreement with the vitamin D external quality assessment scheme (DEQAS) quality control program for vitamin D analysis. We present this approach as a candidate reference method for 25(OH)D3, epi-25(OH)D3, and 25(OH)D2 analysis in humans.     

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.