The Transport of Charged Molecules across Three Lipid Membranes Investigated with Second Harmonic Generation.

Subtle variations in the structure and composition of lipid membranes can have a profound impact on their transport of functional molecules and relevant cell functions. Here, we present a comparison of the permeability of bilayers composed of three lipids: cardiolipin, DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)). The adsorption and cross-membrane transport of a charged molecule, D289 (4-(4-diethylaminostyry)-1-methyl-pyridinium iodide), on vesicles composed of the three lipids were monitored by second harmonic generation (SHG) scattering from the vesicle surface. It is revealed that structural mismatching between the saturated and unsaturated alkane chains in POPG leads to relatively loose packing structure in the lipid bilayers, thus providing better permeability compared to unsaturated lipid bilayers (DOPG). This mismatching also weakens the efficiency of cholesterol in rigidifying the lipid bilayers. It is also revealed that the bilayer structure is somewhat disturbed by the surface curvature in small unilamellar vesicles (SUVs) composed of POPG and the conical structured cardiolipin. Such subtle information on the relationship between the lipid structure and the molecular transport capability of the bilayers may provide clues for drug development and other medical and biological studies.

Understanding the different cross-membrane transport kinetics of two charged molecules on the DOPG lipid surface with second harmonic generation and MD simulation.

A clear physical picture of the dynamic behavior of molecules on the surface of the lipid membrane is highly desired and has attracted great attention from researchers. In this study, a step forward in this direction based on previous studies was presented with second harmonic generation (SHG) and molecular dynamic (MD) simulation. Specifically, details on the orientation flipping and cross-membrane transport of two charged molecules, 4-(4-diethylaminostyry)-1-methyl-pyridinium iodide (D289) and malachite green (MG), on the surface of 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) lipids were presented. Firstly, the orientation flipping of the two molecules on the surface of lipids before their cross-membrane transport was confirmed by the MD simulation. Then, the concentration dependent rate of the cross membrane transport for MG/D289 was analyzed. It was found that a simplified model could satisfactorily interpret the faster cross-membrane transport of MG under higher bulk concentrations. A different concentration dependent dynamics was observed with D289 and the reason behind it was also discussed. With this investigation, the surface structures and dynamics of D289 and MG on the DOPG lipid surface were clearly presented.

Combined thermodynamic and kinetic analysis of GroEL interacting with CXCR4 transmembrane peptides.

GroEL along with ATP and its co-chaperonin GroES has been demonstrated to significantly enhance the folding of newly translated G-protein-coupled receptors (GPCRs). This work extends the previous studies to explore the guest capture and release processes in GroEL-assisted folding of GPCRs, by the reduced approach of employing CXCR4 transmembrane peptides as model substrates. Each of the CXCR4-derived peptides exhibited high affinity for GroEL with a binding stoichiometry near seven. It is found that the peptides interact with the paired α helices in the apical domain of the chaperonin which are similar with the binding sites of SBP (strongly binding peptide: SWMTTPWGFLHP). Complementary binding study with a single-ring version of GroEL indicates that each of the two chaperonin rings is competent for accommodating all the seven CXCR4 peptides bound to GroEL under saturation condition. Meanwhile, the binding kinetics of CXCR4 peptides with GroEL was also examined; ATP alone, or in combination of GroES evidently promoted the release of the peptide substrates from the chaperonin. The results obtained would be beneficial to understand the thermodynamic and kinetic nature of GroEL-GPCRs interaction which is the central molecular event in the assisted folding process.

Klotho-mediated activation of the anti-oxidant Nrf2/ARE signal pathway affects cell apoptosis, senescence and mobility in hypoxic human trophoblasts: involvement of Klotho in the pathogenesis of preeclampsia.

The anti-aging gene Klotho is implicated in the pathogenesis of preeclampsia (PE), which is a pregnancy disease characterized by hypertension and proteinuria. Oxidative stress is closely associated with the worse outcomes in PE, and Klotho can eliminate Reactive Oxygen Species (ROS), but it is still unclear whether Klotho regulates PE pathogenesis through modulating oxidative damages. Here, by analyzing the clinical data, we found that Klotho was aberrantly downregulated in PE umbilical cord serum and placental tissues, compared to their normal counterparts. In in vitro experiments, the human trophoblasts were subjected to hypoxic pressure to establish the PE models, and we confirmed that hypoxia also decreased the expression levels of Klotho in those trophoblasts. In addition, through performing functional experiments, we confirmed that hypoxia promoted oxidative damages, cell apoptosis and senescence, whereas suppressed cell invasion in human trophoblasts, which were all reversed overexpressing Klotho. The following mechanical experiments verified that Klotho increased the levels of nuclear Nrf2, total Nrf2, SOD2 and NQO1 to activate the anti-oxidant Nrf2/ARE signal pathway, and silencing of Nrf2 abrogated the protective effects of Klotho overexpression on hypoxic human trophoblasts. Consistently, in in vivo experiments, Klotho overexpression restrained oxidative damages and facilitated cell mitosis in PE rats' placental tissues. In conclusion, this study validated that Klotho activated the Nrf2/ARE signal pathway to eliminate hypoxia-induced oxidative damages, cell apoptosis and senescence to recover normal cellular functions in human trophoblasts, and our data supported that Klotho could be used as novel biomarker for PE diagnosis and treatment.

Temperature-Modulated Evolution of Surface Structures Induces Significant Enhancement of Two-Photon Fluorescent Emission from a Dye Molecule.

Fluorescence is an essential property of molecules and materials that plays a pivotal role across various areas such as lighting, sensing, imaging, and other applications. For instance, temperature-sensitive fluorescence emission is widely utilized for chemo-/biosensing but usually decreases the intensity upon the increase in temperature. In this study, we observed a temperature-induced enhancement of up to ∼150 times in two-photon fluorescence (TPF) emission from a dye molecule, 4-(4-diethylaminostyry)-1-methylpyridinium iodide (D289), as it interacted with binary complex vesicles composed of two commonly applied surfactants: sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). By employing second harmonic generation (SHG) and TPF techniques, we clearly revealed the temperature-dependent kinetic behavior of D289 on the surface of the vesicles and utilized it to interpret the origin of the significant TPF enhancement. Additionally, we also demonstrated a similar heating-induced enhancement of the TPF emission from D289 on the membrane of phospholipid vesicles, indicating the potential application of TPF in temperature sensing in the biology systems. The embedding of D289 in the tightly packed alkane chains was identified as the key factor in enhancing the TPF emission from D289. This finding may provide valuable information for synthesizing fluorescence materials with a high optical yield.

Evaluating the cross-membrane dynamics of a charged molecule on lipid films with different surface curvature.

Does the curvature of a phospholipid membrane influence the permeability of the lipid bilayers? This is a question of great importance yet hard to answer. In this work the permeability of a positively charged rod like probing molecule (D289 dye) on the bilayers of DOPG lipid vesicles was investigated using angle resolved second harmonic generation method. It was revealed that the permeability of D289 on the surface of small vesicles with âˆ¼ 100 nm diameter was notably lower than that on giant vesicles with âˆ¼ 1000 nm diameter. With the increasing of temperature or the introducing of dimethyl sulfoxide (DMSO) in the solutions, the D289 permeability of the lipid bilayers was notably enhanced as expected, on both the small and the giant vesicles. Still, the D289 permeability of the lipid film with more curvature is lower than the relatively flat film in all these cases. This work demonstrated a general protocol for the investigating of surface permeability of lipid films with various curvature.

Oil-in-water Pickering emulsions using a protein nano-ring as high-grade emulsifiers.

Pickering emulsion-based delivery of liposoluble bioactive ingredients employing protein nanoparticles as biocompatible emulsifiers is a promising choice for food, cosmetic, and medical industries. This paper reports a novel design of a protein nano-ring (termed SR') derived from chaperonin GroEL as an emulsifying agent, which has a naturally evolved hydrophobic binding rim in addition to its well-defined shape. It is shown that SR' adsorbed at rosemary oil/water interface and formed stable oil-in-water Pickering emulsions, with dispersed droplet size being dependent on the SR' concentration and oil/water ratio as well. The optimal formulation yielding stable nano-emulsions was determined to be at a SR' concentration between 0.30 wt.% and 0.45 wt.%, and an oil/water ratio of 0.05-0.20 (v/v). Meanwhile, we demonstrate that nano-sized Pickering droplets could be easily prepared irrespective of the examined external factors including pH, temperature and ionic strength, with the lowest droplet sizes being produced at pH = 7.0, temperature ≤ 40 °C, and ionic strength (NaCl concentration) ≤ 50 mM. Besides, rheological analysis revealed the gelation propensity of SR'-stabilized emulsions with high oil/water ratios, an advantageous property that would further enhance the emulsion stability. Finally, it is shown that the SR' emulsified system is able to protect ß-carotene, which was used as a model of bioactive but labile compound. This work, in the context of the current drive for biocompatibility and sustainability, is believed to provide opportunities for emulsion-based applications to switch towards greener solutions.

Highly Surface-Active Chaperonin Nanobarrels for Oil-in-Water Pickering Emulsions and Delivery of Lipophilic Compounds.

Stabilization of Pickering emulsions via particles of biological origin exhibits a great potential to be widely applied in food, cosmetic, or biomedicine formulation because of their excellent biocompatibility, biodegradability, and functional properties. This paper describes the successful development of a bioderived GroEL protein nanobarrel as a Pickering stabilizer and its protective properties on ß-carotene in dispersed oil phase, as a model of labile bioactive compounds. It is shown that the GroEL nanobarrel is highly surface-active and allows the formation of Pickering emulsion by physical adsorption at the oil/water interface. The optimized formulation for generating a stable submicron oil droplet by ultrasonication includes a GroEL concentration of 0.05-0.45 wt % with an oil/water volume ratio of 0.05-0.35. The as-prepared Pickering emulsion shows pH-responsive emulsification/demulsification transition and excellent stability at temperatures less than 65 °C and ionic strength (with NaCl addition) up to 500 mM. Meanwhile, the emulsion tends to form a gel-like network structure with the oil/water ratio increasing. Finally, we demonstrate that possible factors of oxidant, reducing agent, UV radiation, and sucrose have sequentially decreasing to no effect on the stability of ß-carotene encapsulated in GroEL-stabilized Pickering emulsion and that higher GroEL concentration can significantly reduce ß-carotene degradation rate, thus ensuring more efficient long-term storage. We believe that the emulsion system supported by the GroEL nanobarrel could be developed to a viable tool for delivering lipophilic bioactive compounds.

MicroRNA-505 predicts prognosis and acts as tumor inhibitor in cervical carcinoma with inverse association with FZD4.

PURPOSE: We investigated the expression and mechanisms of microRNA-505 (miR-505) and its downstream target gene Frizzled-4 (FZD4) in cervical cancer. METHODS: miR-505 expression was evaluated by qRT-PCR in cervical cancer cell lines and human carcinomas. Cancer patients' clinicopathological factors and survival were analyzed based on their tumorous miR-505 levels. Ca-Ski and HeLa cells were transduced with lentivirus to upregulate or downregulate miR-505. Their impacts on cervical cancer were evaluated by in vitro proliferation, invasion and in vivo tumorigenicity assays, respectively. Target gene of miR-505, FZD4, was evaluated by dual-luciferase reporter assay. Its expression in cervical cancer cell was evaluated by qRT-PCR. FZD4 was either upregulated or downregulated in cervical cancer cells to further assess its impact on modulating cervical cancer development in vitro. RESULTS: MiR-505 is lowly expressed in cervical cancer cell lines and human carcinomas. Low tumorous miR-505 expression was associated with patients' advanced tumor stage and short survival. In Ca-Ski and HeLa cells, lentivirus-mediated miR-505 upregulation suppressed cancer proliferation and invasion in vitro, and tumorigenicity in vivo, whereas miR-505 downregulation had no functional effects. FZD4 was confirmed to be a downstream target of miR-505, and found to be upregulated in cervical cancer. Genetic modification of FZD4 in cervical cancer cells yielded a significant change in cancer growth, as FZD4 upregulation suppressed whereas FZD4 downregulation promoted cervical cancer proliferation and invasion In vitro. CONCLUSION: MiR-505 may act as a cancer inhibitor and prognostic factor in cervical cancer. FDZ4 is reversely expressed as miR-505, and has dramatic regulatory function in cervical cancer.