Membrane Molecules Research Articles

Complementation of an Escherichia coli K-12 Mutant Strain Deficient in KDO Synthesis by Forming D-Arabinose 5-Phosphate from Glycolaldehyde with Fructose 6-Phosphate Aldolase (FSA)

KDO (2-keto-3-deoxy-D-manno-octulosonate) is a landmark molecule of the Gram-negative outer membrane. Mutants without KDO formation are known to be barely viable. Arabinose 5-phosphate (A5P) is a precursor of KDO biosynthesis and is normally derived from ribulose 5-phosphate by A5P isomerases, encoded by kdsD and gutQ genes in E. coli K-12. We created a kdsD gutQ-deficient double mutant of strain BW25113 and confirmed that these cells are A5P auxotrophs. Fructose 6-phosphate aldolase (FSA) is known to utilize (among other donors such as dihydroxyacetone or hydroxyacetone) glycolaldehyde (GoA) as a donor compound and to provide A5P in vitro when glyceraldehyde 3-phosphate is the acceptor. We show here that this FSA function in vivo fully reverses the growth defect and the A5P deficiency in kdsD gutQ double mutants. Expression of both plasmid-encoded fsaA, fsaAA129S, or fsaB genes as well as a chromosomally integrated form of fsaAA129S led to maximal OD600 values of >2.2 when GoA was added exogenously (together with glucose as a C source) at a concentration of 100 µM (Ks values in the range of 4–10 µM). Thus, a novel bio-orthogonal bypass to overcome an A5P deficiency was opened. Lower GoA concentrations led to lower growth yields. Interestingly, mutant strains with recombinant fsa genes showed considerable growth yields even without exogenous GoA addition, pointing to yet unknown endogenous GoA sources in E. coli metabolism. This is a further example of the usefulness of FSA in rewiring central metabolic pathways in E. coli.

A synthetic flavonoid derivate in the plasma membrane transforms the voltage-clamp fluorometry signal of CiHv1.

Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.

Iron Oxide Nanoparticles Engineered Macrophage-Derived Exosomes for Targeted Pathological Angiogenesis Therapy.

Engineering exosomes with nanomaterials usually leads to the damage of exosomal membrane and bioactive molecules. Here, pathological angiogenesis targeting exosomes with magnetic imaging, ferroptosis inducing, and immunotherapeutic properties is fabricated using a simple coincubation method with macrophages being the bioreactor. Extremely small iron oxide nanoparticle (ESIONPs) incorporated exosomes (ESIONPs@EXO) are acquired by sorting the secreted exosomes from M1-polarized macrophages induced by ESIONPs. ESIONPs@EXO suppress pathological angiogenesis in vitro and in vivo without toxicity. Furthermore, ESIONPs@EXO target pathological angiogenesis and exhibit an excellent T1-weighted contrast property for magnetic resonance imaging. Mechanistically, ESIONPs@EXO induce ferroptosis and exhibit immunotherapeutic ability toward pathological angiogenesis. These findings demonstrate that a pure biological method engineered ESIONPs@EXO using macrophages shows potential for targeted pathological angiogenesis therapy.

Redundancy and absurd names in immunology.

In this short review, examples of unnecessary multiple names of cell membrane molecules, for example, immune checkpoints and cytokines, are presented. Moreover, ridiculous or inaccurate names, such as 'Regulated on activation, normal T-cell expressed and secreted' and 'tissue factor', are discussed.

HLA-G Expression/Secretion and T-Cell Cytotoxicity in Missed Abortion in Comparison to Normal Pregnancy.

The main role of HLA-G is to protect the semi-allogeneic embryo from immune rejection by proper interaction with its cognate receptors on the maternal immune cells. Spontaneous abortion is the most common adverse pregnancy outcome, with an incidence rate between 10% and 15%, with immunologic dysregulation being thought to play a role in some of the cases. In this study, we aimed to detect the membrane and soluble HLA-G molecule at the maternal-fetal interface (MFI) and in the serum of women experiencing missed abortion (asymptomatic early pregnancy loss) in comparison to the women experiencing normal early pregnancy. In addition, the proportion of T cells and their cytotoxic profile was evaluated. We observed no difference in the spatial expression of HLA-G at the MFI and in its serum levels between the women with missed abortions and those with normal early pregnancy. In addition, comparable numbers of peripheral blood and decidual total T and γδT cells were found. In addition, as novel data we showed that missed abortion is not associated with altered extravilous invasion into uterine blood vessels and increased cytotoxicity of γδT cells. A strong signal for HLA-G on non-migrating extravilous trophoblast in the full-term normal placental bed was detected. In conclusion, HLA-G production at the MFI or in the blood of the women could not be used as a marker for normal pregnancy or missed abortions.

Superwetting membrane-based strategy for highly efficient separation of dimethyl carbonate and methanol

Dimethyl carbonate (DMC) produced through transesterification, which involves the reaction of methanol (MeOH) with a suitable ester, has been considered as an attractive route for industrial production. Although high-purity DMC can be obtained from DMC/MeOH mixture by energy-intensive extractive distillation or time-consuming pervaporation (PV), it is still challenging to produce pure DMC in an energy-saving and high-efficiency manner. Here we demonstrate an energy-economy yet efficient superwetting membrane (SWM)-based strategy to produce pure DMC by combining porous polytetrafluoroethylene (PTFE) as the solid membrane and eco-friendly water molecules as the liquid inductive agent. The relatively low polar DMC can rapidly and selectively permeate the nonpolar PTFE membrane by nonpolar interaction between PTFE and DMC. Meanwhile, the high polar water molecules generate strong hydration with MeOH, which makes the relatively high polar MeOH/H2O being blocked by the nonpolar PTFE membrane. The SWM system exhibits outstanding separation flux of 1090 L m−2 h−1, with DMC purity higher than 93 wt%. Furthermore, taking advantage of the easy assembly of this SWM with conventional PV, we can produce DMC with purity of 99.4 wt%, showing great potential in practical DMC production.

Membrane molecule bouncer regulates sperm binding activity in immature oocytes in the viviparous teleost species Poecilia reticulata (guppy).

Generally, in vertebrates, the first step toward fertilization is the ovulation of mature oocytes, followed by their binding to sperm cells outside of the ovary. Exceptionally, the oocytes of poeciliid fish are fertilized by sperm cells within the follicle, and the developmental embryo is subsequently released into the ovarian lumen before delivery. In the present study, we aimed to identify the factor(s) responsible for intrafollicular fertilization in a viviparous teleost species, Poecilia reticulata (guppy). Sperm tracking analysis in this regard indicated that in this species, sperm cells reached immature oocytes including the germinal vesicle, and the insemination assay indicated that the immature oocytes robustly adhered to the sperm cells; similar binding was not observed in Danio rerio (zebrafish) and Oryzias latipes (medaka). We also identified the Ly6/uPAR protein bouncer as the factor responsible for the observed sperm binding activity of the immature oocytes in this species. The recombinant bouncer peptide acted as an inhibitory decoy for the sperm-oocyte binding in guppy. On the other hand, ectopic expression of guppy bouncer in zebrafish oocytes resulted in interspecific sperm-oocyte binding. These results argue that bouncer is responsible for sperm-immature oocyte binding. Our findings highlight the unique reproductive strategies of guppy fish and enhance our understanding of the diverse reproductive mechanisms in vertebrates.

Coherent anti-Stokes Raman scattering spectroscopy system for observation of water molecules in anion exchange membrane

Anion exchange membrane fuel cells (AEMFCs) provide one of the most feasible remedies to fuel cells’ dependency on the dwindling Pt group catalysts. Nevertheless, AEMFCs still suffer reduced durability, which requires an in-depth understanding of their membranes. The low thermal endurance of the anion exchange membranes (AEMs) usually limits the direct application of powerful techniques, such as Raman spectroscopy. We sought to establish a system for coherent anti-Stokes Raman scattering (CARS) spectroscopy capable of taking measurements inside an AEM rapidly and accurately without photodamage. A 785 nm CARS system was newly developed to study the water species in an AEM (QPAF-4) located vertically in a fuel cell. From the results of water measurement in a QPAF-4 membrane, the OH-related region was deconvoluted into nine Gaussian peaks: Five H-bonded OH peaks, non-H-bonded OH, OH–, and two CH peaks. The H-bonded species increased with increasing relative humidity, but the other species remained constant. These results open unlimited possibilities for studying and comparing different AEMFCs, enabling more rapid technology optimization.

Microglial Modulation of Synaptic Maturation, Activity, and Plasticity.

Microglia, which are the resident immune cells of the CNS, also have important functions in physiological conditions. In this chapter, we review the experimental evidence that microglia modulate neuronal and synaptic activity during normal development and in adults. We show that microglia can regulate the maturation and function of both inhibitory and excitatory synapses that can be stimulated or repressed. We further review the fact that these regulations occur in various brain regions, through soluble and membrane molecules, directly or through other cell partners. This review emphasizes the fact that microglia are genuine and highly context-dependent and thus adaptable regulators of neuronal activity.

Experimental and numerical investigation of microdialysis probes for ethanol metabolism studies.

Microdialysis is an important technique for in vivo sampling of tissue's biochemical composition. Understanding the factors that affect the performance of the microdialysis probes and developing methods for sample analysis are crucial for obtaining reliable results. In this work, we used experimental and numerical procedures to study the performance of microdialysis probes having different configurations, membrane materials and dimensions. For alcohol research, it is important to understand the dynamics of ethanol metabolism, particularly in the brain and in other organs, and to simultaneously measure the concentrations of ethanol and its metabolites - acetaldehyde and acetate. Our work provides a comprehensive characterization of three microdialysis probes, in terms of recovery rates and backpressure, allowing for interpretation and optimization of experimental procedures. In vivo experiments were performed to measure the time course concentration of ethanol, acetaldehyde, and acetate in the rat brain dialysate. Additionally, the combination of in vitro experimental results with numerical simulations enabled us to calculate diffusion coefficients of molecules in the microdialysis membranes and study the extent of the depletion effect caused by continuous microdialysis sampling, thus providing additional insights for probe selection and data interpretation.

Finely tuned water structure and transport in functionalized carbon nanotube membranes during desalination.

Molecular dynamics simulations were performed to tune the transport of water molecules in nanostructured membrane in a desalination process. Four armchair-type (7,7), (8,8), (9,9) and (10,10) carbon nanotubes (CNTs) with pore diameters around 1 nm were chosen, their interior surfaces were modified with -OH, -CH3 and -F groups. Simulation results show that water transport in nanochannel depends on confined water structures which could be regulated by precisely controlled channel diameter and chemical functionalization. Increasing CNT diameter changes water structures from single-file-like to be square and hexagonal-like, then into a disordered pattern, resulting in a concave-shaped trend of water permeance. The -OH functional groups promote structural ordering of water molecules in (7,7) CNT, but disrupt water structures in (8,8) and (9,9) CNTs, and reduce the order degree of water molecules in (10,10) CNT, moreover, exert an attraction to enhance surface friction inside channel. The -CH3 groups induce more strictly single-file movement of water molecules in (7,7) CNT, turning water structures in (8,8) and (9,9) CNTs into two and triangular column arrangements, improving water transport, however, causing again square-like water structure in (10,10) CNT. Fluorinations of CNT make water structure more disordered in (7,7), (9,9) and (10,10) CNTs, while enhance the square water structure in (8,8) CNT with a lower water permeance. Through changing channel diameter and functionalization, the low tetrahedral order corresponds to a more single-file-like water structure, associated with rapid water diffusion and high permeability; an increase in tetrahedrality results in more ice-like water structures, lower water diffusion coefficients, and permeability. The results of this study demonstrate that water transport could be finely regulated via a functionalized CNT membrane.

Cholesterol inhibits oxygen permeation through biological membranes: mechanism against double-bond peroxidation.

The presence of oxygen molecules (O2) in biological membranes promotes lipid peroxidation of phospholipids with unsaturated acyl chains. On the other hand, cholesterol is considered to be an antioxidant molecule as it has a significant barrier effect on the permeation of O2 across membranes. However, a comprehensive explanation of how cholesterol affects the distribution and diffusion of O2 within lipid bilayers is yet to be established. In this study, we investigated the interaction of oxygen molecules with polyunsaturated lipid bilayers using molecular dynamics (MD) simulations. The degree of lipid unsaturation and the concentration of cholesterol were varied to study the permeation of O2. The free energy profile of O2 diffusing from the water phase to the lipid bilayer was calculated using biased umbrella MD simulations. The results show that O2 passively translocates into the membrane without changing the physical properties of the bilayer. Interestingly, in the unsaturated lipid bilayers the presence of cholesterol led to a significantly decreased permeation of O2 and an increase in the lipid chain order. Our results indicate that the hydroxyl groups of cholesterol strongly interact with the O2 molecules effectively inhibiting interactions between the oxygens and the double bonds in unsaturated lipid tails. In addition, a linear relationship between permeation and the ratio of membrane thickness and area per lipid was found. These insights can help our understanding of how the degree of unsaturation in a lipid tail and cholesterol affect lipid peroxidation at the molecular level.

Characterization of outer membrane vesicles released by clinical isolates of Neisseria gonorrhoeae.

The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo-oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as IL-1αandIL-1β. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

Development of Chemical Tools Based on GSK-7975A to Study Store-Operated Calcium Entry in Cells

Many physiological functions, such as cell differentiation, proliferation, muscle contraction, neurotransmission and fertilisation, are regulated by changes of Ca2+ levels. The major Ca2+ store in cells is the endoplasmic reticulum (ER). Certain cellular processes induce ER store depletion, e.g. by activating IP3 receptors, that in turn induces a store refilling process known as store-operated calcium entry (SOCE). This refilling process entails protein-protein interactions between Ca2+ sensing stromal interaction molecules (STIM) in the ER membrane and Orai proteins in the plasma membrane. Fully assembled STIM/Orai complexes then form highly selective Ca2+ channels called Ca2+ release activated Ca2+ Channels (CRAC) through which Ca2+ ions flow into the cytosol and subsequently are pumped into the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Abnormal SOCE has been associated with numerous human diseases and cancers, and therefore key players STIM and Orai have attracted significant therapeutic interest. Several potent experimental and clinical candidate compounds have been developed and have helped to study SOCE in various cell types. We have synthesized multiple novel small-molecule probes based on the known SOCE inhibitor GSK-7975A. Here we present GSK-7975A derivatives, which feature photo-caging, photo-crosslinking, biotin and clickable moieties, and also contain deuterium labels. Evaluation of these GSK-7975A probes using a fluorometric imaging plate reader (FLIPR)-Tetra-based Ca2+ imaging assay showed that most synthetic modifications did not have a detrimental impact on the SOCE inhibitory activity. The photo-caged GSK-7975A was also used in patch-clamp electrophysiology experiments. In summary, we have developed a number of active, GSK-7975A-based molecular probes that have interesting properties and therefore are useful experimental tools to study SOCE in various cells and settings.

Fabrication of 1D/2D ATP/GO-COOH membrane with low-resistance transport pathways for highly efficient separation of oil-in-water emulsion

Attapulgite/carbonylated graphene oxide (ATP/GO-COOH) composite membranes have been successfully prepared by vacuum-assisted filtration of an aqueous ATP/GO-COOH dispersion on a microfiltration substrate. Due to the interaction between the OH groups of ATPs and COOH groups of GO-COOH, ATP rods showed excellent compatibility with GO-COOH nanosheets, and the ATP mass fraction was significantly improved. The large amount of ATP can establish a fast transport channel for the diffusion of water molecules in the composite membrane. As a result, the optimum membrane gave a permeation flux with 13582 L/(m2 h), which is significantly higher than that of the pure GO-COOH membrane (2281 L/(m2 h)). In addition, the underwater superoleophobic properties of the ATP/GO-COOH composite membrane enable it to have a low oil-adhesive water/membrane interfaces. The ATP/GO-COOH composite membrane showed good separation and separation stability for oil-in-water emulsion systems with different concentrations, pH, and oil types.

Influence of silicon nanocone on cell membrane self-sealing capabilities for targeted drug delivery—Computer simulation study

Efficient and non-invasive techniques of cargo delivery to biological cells are the focus of biomedical research because of their great potential importance for targeted drug therapy. Therefore, much effort is being made to study the characteristics of using nano-based biocompatible materials as systems that can facilitate this task while ensuring appropriate self-sealing of the cell membrane. Here, we study the effects of indentation and withdrawal of nanocone on phospholipid membrane by applying steered molecular dynamics (SMD) technique. Our results show that the withdrawal process directly depends on the initial position of the nanocone. The average force and work are considerably more significant in case of the withdrawal starting from a larger depth. This result is attributed to stronger hydrophobic interactions between the nanocone and lipid tails of the membrane molecules. Furthermore, when the indenter was started from the lower initial depth, the number of lipids removed from the membrane was several times smaller than the deeper indentation. The choice of the least invasive method for nanostructure-assisted drug delivery is crucial for possible applications in medicine. Therefore, the results presented in this work might be helpful in efficient and safe drug delivery with nanomaterials.

Probing Conformational Transition of TRPV5 Induced by Mechanical Force Using Coarse-Grained Molecular Dynamics.

Transient receptor potential vanilloid 5 (TRPV5) is a calcium-selective TRP channel that plays a crucial role in calcium homeostasis regulation. However, there are still many issues that need to be addressed, such as the specific conformational transition of TRPV5 and the specific functions of each structure in cation gating. Here, we build a model of the calcium ion transport protein from Xenopus oocytes in the presence of the lipid membrane and water molecules. Due to the activation process of ion channels are global and collective, coarse-grained molecular dynamics (CG-MD) simulations of the potential of mean force along the conformational transition pathway are performed. The CG-MD simulations show that the S6 helix plays a vital role in the TRPV5 conformational transition. Most importantly, these simulated trajectories indicate that the activation of ion channels happens before the extension and rotation of S6 helices, revealing that TRPV5 has a unique gating mechanism different from TRPV6. The present work demonstrates how the mechanical force acting on the S6 helix opens the TRPV5 channel gates. These results deepen our understanding of the TRPV5 gating mechanism.

The role of peroxidasin in solid cancer progression.

Peroxidasin is a heme-containing peroxidase enzyme that plays a vital role in the cross-linking of collagen IV molecules in basement membranes. Collagen IV cross-links are essential for providing structure and mechanical stability throughout tissue development, homeostasis, and wound healing. During cancer progression, the basement membrane is degraded, and proteins typically found in the basement membrane, including peroxidasin and collagen IV, can be found spread throughout the tumour microenvironment where they interact with cancer cells and alter cell behaviour. Whilst peroxidasin is reported to be up-regulated in a number of different cancers, the role that it plays in disease progression and metastasis has only recently begun to be studied. This review highlights the current literature exploring the known roles of peroxidasin in normal tissues and cancer progression, regulators of peroxidasin expression, and the reported relationships between peroxidasin expression and patient outcome in cancer.

Microbial Primer: Multidrug efflux pumps.

Multidrug efflux pumps are molecular machines that sit in the bacterial cell membrane and pump molecules out from either the periplasm or cytoplasm to outside the cell. While involved in a variety of biological roles, they are primarily known for their contribution to antibiotic resistance by limiting the intracellular accumulation of antimicrobial compounds within bacteria. These transporters are often overexpressed in clinical isolates, leading to multidrug-resistant phenotypes. Efflux pumps are classified into several families based on their structure and understanding the characteristics of each family is important for the development of novel therapies to restore antibiotic potency.

Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis.

Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB) has been observed. However, sample preparation remains a significant challenge in the field. Here, we evaluated the MPs solved using cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å. The most critical parameters for sample preparation are as follows: (i) the surfactant used for protein extraction from the membrane, (ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, (iii) the vitrification method employed, and (iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures.