Lipid Phase Research Articles

Exploring protein-protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching.

Pulmonary surfactant (PS) is a lipid-protein complex that forms films reducing surface tension at the alveolar air-liquid interface. Surfactant protein C (SP-C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N-terminal segment of SP-C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C-terminal region comprises a transmembrane α-helix that contains a ALLMG motif, supposedly analogous to a well-studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP-C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP-C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self-quenching and FRET assays to analyze dimerization of native palmitoylated SP-C and a non-palmitoylated recombinant version of SP-C (rSP-C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP-C remains primarily monomeric. In contrast, non-palmitoylated recombinant SP-C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α-helical conformation of SP-C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher-order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.

Custom Design of a Humidifier Chamber for InMeso Crystallization.

Membrane proteins are indispensable for every living organism, yet their structural organization remains underexplored. Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography, which have significantly increased the structural coverage of membrane proteins across various kingdoms, certain scientific methods, such as time-resolved crystallography, still mostly rely on crystallization techniques, such as lipidic cubic phase (LCP) or in meso crystallization. In this study, we present an open-access blueprint for a humidity control chamber designed for LCP/in meso crystallization experiments using a Gryphon crystallization robot. Using this chamber, we have obtained crystals of a transmembrane aspartate transporter GltTk from Thermococcus kodakarensis in a lipidic environment using in meso crystallization. The data collected from these crystals allowed us to perform an analysis of lipids bound to the transporter. With this publication of our open-access design of a humidity chamber, we aim to improve the accessibility of in meso protein crystallization for the scientific community.

Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. It is generally assumed that the composition of individual GUVs is the same as the nominal stock composition; however, there may be significant compositional variability between individual GUVs. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H-/13C-, which is highly sensitive to compositional differences between individual GUVs and circumvents the need for calibration by using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display standard deviations in composition (n = 30 GUVs) on the order of 1-4 mol %, consistent with variability seen in previous indirect measurements.

FRET-GP - A Local Measure of the Impact of Transmembrane Peptide on Lipids.

Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of proteins with lipids. In this work, we describe the FRET-GP method, which utilizes Förster resonance energy transfer (FRET) to specifically probe the nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe is quantified using generalized polarization (GP) to report on lipid mobility in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contributes to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination of GPFRET - the local analogue of classical GP parameter. The presence of WALP significantly increased both parameters but the local effects were considerably stronger (GPFRET ≫ GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby enhancing the depth of information obtained from the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions.

AN OVERVIEW ON CUBOSOMES AS REMARKABLE NANOCARRIER FOR DRUG DELIVERY

Lipids have been extensively used as main ingredients in various drug delivery systems, such as liposomes, solid lipid nanoparticles, nanostructured lipid carriers and lipid-based lyotropic liquid crystals. Over the past few years, lipid-based lyotropic, bicontinuous cubic phase liquid crystals have been investigated for their applicability to controlled delivery of active ingredients. Lipid-based lyotropic liquid crystals have highly ordered, thermodynamically stable internal nanostructure, thereby offering the potential as a sustained drug release matrix. The emulsification of cubic lipid phases in water results in the production of cubosomes that can be defined as nanoparticulate disperse systems characterised by high biocompatibility and bioadhesivity. The unique microstructure of cubosomes have the potentials to control the release of active ingredients, improve drug bioavailability and reduce toxicity, enhance the stability of drugs and to increase the penetrability of drug after topical application. This reflection will provide an overview of the lipids used to prepare cubic phase at physiological temperature, as well as the influencing factors on the phase transition of liquid crystals. In particular, the most current research progresses on cubic phase as drug delivery systems and its applications will be discussed. It might act as smart lipid nanoparticles for drug delivery.

Antimicrobial and antibiofilm effects of shikonin with tea tree oil nanoemulsion against Candida albicans and Staphylococcus aureus

The current work aims to develop a shikonin and tea tree oil loaded nanoemulsion system stabilized by a mixture of GRAS grade surfactants (Tween 20 and monoolein) and a cosurfactant (Transcutol P). This system was designed to address the poor aqueous solubility and photostability issues of shikonin. The authenticity of shikonin employed in this study was confirmed using nuclear magnetic resonance (NMR) spectroscopy. The optimized nanoemulsion exhibited highly favorable characteristics in terms of zeta potential (−23.8 mV), polydispersity index (0.216) and particle size (22.97 nm). These findings were corroborated by transmission electron microscopy (TEM) micrographs which confirmed the spherical and uniform nature of the nanoemulsion globules. Moreover, attenuated total reflectance (ATR) and X-ray diffraction analysis (XRD) analysis affirmed improved chemical stability and amorphization, respectively. Photodegradation studies were performed by exposing pure shikonin and the developed nanoemulsion to ultraviolet light for 1 h using a UV lamp, followed by high performance liquid chromatography (HPLC) analysis. The results confirmed that the developed nanoemulsion system imparts photoprotection to pure shikonin in the encapsulated system. Furthermore, the research investigated the effect of the nanoemulsion on biofilms formed by Candida albicans and methicillin resistant Staphylococcus aureus (MRSA). Scanning electron microscopy, florescence microscopy and phase contrast microscopy unveiled a remarkable reduction in biofilm area, accompanied by disruptions in the cell wall and abnormalities on the cell surface of the tested microorganisms. In conclusion, the nanoencapsulation of shikonin with tea tree oil as the lipid phase showcased significantly enhanced antimicrobial and antibiofilm potential compared to pure shikonin against resistant strains of Candida albicans and Staphylococcus aureus.

Self-emulsifying drug delivery systems (SEDDS) disrupt the gut microbiota and trigger an intestinal inflammatory response in rats

Self-emulsifying drug delivery systems (i.e. SEDDS, SMEDDS and SNEDDS) are widely employed as solubility and bioavailability enhancing formulation strategies for poorly water-soluble drugs. Despite the capacity for SEDDS to effectively facilitate oral drug absorption, tolerability concerns exist due to the capacity for high concentrations of surfactants (typically present within SEDDS) to induce gastrointestinal toxicity and mucosal irritation. With new knowledge surrounding the role of the gut microbiota in modulating intestinal inflammation and mucosal injury, there is a clear need to determine the impact of SEDDS on the gut microbiota. The current study is the first of its kind to demonstrate the detrimental impact of SEDDS on the gut microbiota of Sprague-Dawley rats, following daily oral administration (100 mg/kg) for 21 days. SEDDS comprising a lipid phase (i.e. Type I, II and III formulations according to the Lipid Formulation Classification Scheme) induced significant changes to the composition and diversity of the gut microbiota, evidenced through a reduction in operational taxonomic units (OTUs) and alpha diversity (Shannon’s index), along with statistically significant shifts in beta diversity (according to PERMANOVA of multi-dimensional Bray-Curtis plots). Key signatures of gut microbiota dysbiosis correlated with the increased expression of pro-inflammatory cytokines within the jejunum, while mucosal injury was characterised by significant reductions in plasma citrulline levels, a validated biomarker of enterocyte mass and mucosal barrier integrity. These findings have potential clinical ramifications for chronically administered drugs that are formulated with SEDDS and stresses the need for further studies that investigate dose-dependent effects of SEDDS on the gastrointestinal microenvironment in a clinical setting.

New Insights into the Loss of Antioxidant Effectiveness of Phenolic Compounds in Vegetable Oils in the Presence of Phosphatidylcholine.

It has been proposed that lipid oxidation reactions in edible oils primarily occur in reverse micelles (RM) of amphiphilic components. While the prooxidative effect of RM has been demonstrated, the mechanism involved is not fully understood. Both reductions and enhancements in the antioxidant efficacy (AE) of α-tocopherol and Trolox have been observed in different studies when phosphatidylcholine (PC) was added and PC RM were formed. However, most of these investigations employed lipid systems consisting of stripped vegetable oil diluted in saturated medium-chain triacylglycerols (MCT) and utilized antioxidant concentrations well below those found in edible oils. These two specific factors were investigated in the present study. The effect of RM of purified egg yolk PC on the AE of 1.16 mmol kg-1 α-tocopherol or Trolox in stripped sunflower oil (SSO) was studied by the Rancimat (100 °C) and oven (50 °C) tests. Increasing PC concentrations (50-1000 ppm) had no significant impact on α-tocopherol, but substantial reductions in AE were observed for Trolox. This phenomenon may be attributed to the partitioning of Trolox into the pre-existing PC micelles, suggesting that primary oxidation reactions occurred in the continuous lipid phase. In addition, the effectiveness of both antioxidants decreased significantly in the presence of PC when a low antioxidant concentration (0.06 mmol kg-1) was assayed in SSO:MCT (1:3, w/w).

Aspirin-Induced Ordering and Faster Dynamics of a Cationic Bilayer for Drug Encapsulation.

The lipid dynamics and phase play decisive roles in drug encapsulation and delivery to the intracellular target. Thus, understanding the dynamic and structural alterations of membranes induced by drugs is essential for targeted delivery. To this end, united-atom molecular dynamics simulations of a model bilayer, dioctadecyldimethylammonium bromide (DODAB), are performed in the absence and presence of the usual nonsteroidal anti-inflammatory drug (NSAID), aspirin, at 298, 310, and 345 K. At 298 and 310 K, the bilayers are in the interdigitated two-dimensional square phases, which become rugged in the presence of aspirin, as evident from height fluctuations. At 345 K, the bilayer is in the fluid phase in both the absence and presence of aspirin. Aspirin is preferentially located near the oppositely charged headgroup and creates void space, which leads to an increase in the interdigitation and order parameters. Although the center of mass of lipids experiences structural arrest, they reach the diffusive regime faster and have higher lateral diffusion constants in the presence of aspirin. Results are found to be consistent with recent quasi-elastic neutron scattering studies that reveal that aspirin acts as a plasticizer and enhances lateral diffusion of lipids in both ordered and fluid phases. Different relaxation time scales of the bonds along the alkyl tails of DODAB due to the multitude of lipid motions become faster upon the addition of aspirin. Our results show that aspirin insertion is most favorable at physiological temperature. Thus, the ordered, more stable, and faster DODAB bilayer can be a potential drug carrier for the protected encapsulation of aspirin, followed by targeted and controlled drug release with antibacterial activity in the future.

Optimization, cellular uptake, and in vivo evaluation of Eudragit S100-coated bile salt-containing liposomes for oral colonic delivery of 5-aminosalicylic acid

Eudragit S100-coated bile salt-containing liposomes were prepared and optimized by experimenting with different variables, including bile salt type and concentration, and the method of incorporation into liposomes using a model hydrophilic compound, 5-aminosalicylic acid (5-ASA). After optimizing the formulation, cellular uptake, and animal pharmacokinetic experiments were performed. The inclusion of sodium glycocholate (SG) into liposomes decreased liposome particle size and entrapment efficiency significantly but had no effect on zeta potential. The method of incorporating SG into the lipid or aqueous phase of the liposome did not notably impact the characteristics of the liposomes but the hydration media had a substantial effect on the entrapment efficiency of 5-ASA. In vitro drug release in different fluids simulating distinct gastrointestinal tract sections, indicated pH-dependent disintegration of the coating layer of coated SG-containing liposomes. The majority of the drug was retained when subjected to simulated gastric fluid (SGF) and fed-state simulated intestinal fluid (FeSSIF) (≈ 37% release after 2 h in SGF pH 1.2, followed by 3 h in FeSSIF pH 5). The remaining drug was subsequently released in phosphate-buffered saline pH 7.4 (≈ 85% release within 24 h). Increasing SG concentration in the liposomes decreased the amount of drug released in FeSSIF. Similar results were observed when SG was replaced with sodium taurocholate. Cellular uptake studies in Caco-2 cells demonstrated that all liposomal formulations (conventional liposomes, bile salt-containing liposomes, and coated bile salt-containing liposomes) have shown to be equally effective at increasing the cellular uptake compared to free fluorescein solution. In the pharmacokinetic study, coated bile salt-containing liposomes showed a lower Cmax and prolonged residence in the gastrointestinal tract in comparison to conventional liposomes. Taken together, these findings suggest that the polymer-coated bile salt-containing liposomes have the potential to serve as a drug delivery system targeted at the colon.

A new look at Hsp70 activity in phosphatidylserine-enriched membranes: chaperone-induced quasi-interdigitated lipid phase

70 kDa heat shock protein Hsp70 (also termed HSP70A1A) is the major stress-inducible member of the HSP70 chaperone family, which is present on the plasma membranes of various tumor cells, but not on the membranes of the corresponding normal cells. The exact mechanisms of Hsp70 anchoring in the membrane and its membrane-related functions are still under debate, since the protein does not contain consensus signal sequence responsible for translocation from the cytosol to the lipid bilayer. The present study was focused on the analysis of the interaction of recombinant human Hsp70 with the model phospholipid membranes. We have confirmed that Hsp70 has strong specificity toward membranes composed of negatively charged phosphatidylserine (PS), compared to neutral phosphatidylcholine membranes. Using differential scanning calorimetry, we have shown for the first time that Hsp70 affects the thermotropic behavior of saturated PS and leads to the interdigitation that controls membrane thickness and rigidity. Hsp70-PS interaction depended on the lipid phase state; the protein stabilized ordered domains enriched with high-melting PS, increasing their area, probably due to formation of quasi-interdigitated phase. Moreover, the ability of Hsp70 to form ion-permeable pores in PS membranes may also be determined by the bilayer thickness. These observations contribute to a better understanding of Hsp70-PS interaction and biological functions of membrane-bound Hsp70 in cancer cells.

Synthesis and Characterization of Paclitaxel-Loaded PEGylated Liposomes by the Microfluidics Method.

For cancer therapy, paclitaxel (PX) possesses several limitations, including limited solubility and untargeted effects. Loading PX into nanoliposomes to enhance PX solubility and target their delivery as a drug delivery system has the potential to overcome these limitations. Over the other conventional method to prepare liposomes, a microfluidic system is used to formulate PX-loaded PEGylated liposomes. The impact of changing the flow rate ratio (FRR) between the aqueous and lipid phases on the particle size and polydispersity index (PDI) is investigated. Moreover, the effect of changing the polyethylene glycol (PEG) lipid ratio on the particle size, PDI, stability, encapsulation efficiency % (EE %), and release profile is studied. The physicochemical characteristics of the obtained formulation were analyzed by dynamic light scattering, FTIR spectroscopy, and AFM. This work aims to use microfluidic technology to produce PEGylated PX-loaded liposomes with a diameter of <200 nm, low PDI < 0.25 high homogeneity, and viable 28 day stability. The results show a significant impact of FRR and PEG lipid ratio on the empty liposomes' physicochemical characteristics. Among the prepared formulations, two formulations produce size-controlled, low PDI, and stable liposomes, which make them preferable for PX encapsulation. The average EE % was >90% for both formulations, and the variation in the PEG lipid ratio affected the EE % slightly; a high packing for PX was reported at different drug concentrations. A variation in the release profiles was notified for the different PEG lipid ratios.

Encapsulation of vitamin D3 using rhamnolipids-based nanostructured lipid carriers.

This work had as main objective to encapsulate vitamin D3 (VD3) into nanostructured lipid carriers (NLCs) using rhamnolipids as surfactant. Glycerol monostearate and medium chain triglycerides with 2.625 % of VD3 were used as lipid materials. The three formulations of NLCs with VD3 (NLCs+VD3) were composed by 99 % of aqueous phase, 1 % of lipid phase and 0.05 % of surfactant. The difference between them was the ratio of solid:liquid in lipid phase. The NLCs+VD3 sizes ranged between 92.1 and 108.1nm. The most stable formulation maintaining their caracteristics for 60days at 4°C. The NLCs+VD3 cytotoxicity demonstrated that concentrations of 0.25mg/mL or lower up had a good biocompatibility in vitro. During the in vitro digestion, formulations with lower sizes and higher content on solid lipid had higher lipolysis rate and consequently higher VD3 bioaccessibility. The rhamnolipids-based NLCs are a good option for the encapsulation of VD3.

Influence of Auricularia cornea Polysaccharide Coating on the Stability and Antioxidant Activity of Liposomes Ginsenoside Rh2.

Liposomes (Lip) are microstructures containing lipid and aqueous phases for encapsulation and delivery of bioactivators. In this study, Ginsenoside Rh2 liposomes (Rh2-Lip) were prepared by a thin-film hydrated ultrasonic binding method. But they are not stable during storage. In addition, Rh2-Lip was wrapped with Auricultural cornea polysaccharide (ACP) and Chitosan (CS) as coating materials to improve stability. CS coating was used as a positive control. The particle sizes determined by dynamic light scattering (DLS) showed 183 ± 5.52 nm for liposomes, 197 ± 6.7 nm for Auricultural cornea polysaccharide coated liposomes (ACP-Rh2-Lip), and 198 ± 3.5 nm for Chitosan coated liposomes (CS-Rh2-Lip). The polydispersity index (PDI) of all liposomes was less than 0.3. Transmission electron microscopy (TEM) showed that ACP and CS were successfully encapsulated on the liposome surface. In vitro simulations of digestive stability in the gastrointestinal tract showed that ACP-Rh2-Lip and CS-Rh2-Lip were more stable in gastrointestinal fluids compared to Lip. The antioxidant experiment revealed that ACP-Rh2-Lip has greater antioxidant activity than Lip. The purpose of this study was to look into the effects of ACP-Rh2-Lip and to offer a reference for Ginsenoside Rh2 (Rh2) delivery.

Natural microbial surfactant containing self-nanoemulsifying formulation with improved performance of paclitaxel therapy: A newer avenue in breast cancer treatment

Due to the presence of a high concentration of surfactants (e.g., Cremophor EL, Tween 80), paclitaxel (PTX) drug delivery systems administered orally and intravenously demonstrate severe toxicity. In the current research work, a unique self-nanoemulsifying drug delivery system (SNEDDS) comprising surfactin, a microbial biosurfactant was used for reducing the formulation toxicity and improving the biopharmaceutical performance. The preformulation study including solubility analysis indicated suitability of corn oil as the natural lipophile. Nanoemulsion boundaries were delineated by aqueous titration of the lipid phase (ethyl oleate: plurol oleique), surfactants (Tween 80 and Surfactin) and cosolvent (Transcutol HP) to identify the optimum compositions on the basis of formulation quality attributes with globule size <250 nm, emulsification time <3 min and in vitro drug release >85% in 60 min. Biocompatibility evaluation of PTX-Surfactin SNEDDS on rat hepatocytes by H2DCF assay to determine the ROS level indicated an insignificant increase in fluorescence intensity, while PTX Tween 80-SNEDDS showed nearly 20% increase in ROS levels compared to the control cells. Furthermore, histopathology examination showed the absence of any signs of inflammation from intestinal segments treated with PTX Surfactin-SNEDDS, while PTX Tween 80-SNEDDS revealed infiltration of cells and the presence of inflammation on the intestinal villous microstructures. The anticancer activity evaluation by in vitro cytotoxicity on MCF-7 cells showed PTX Surfactin-SNEDDS and PTX Tween 80-SNEDDS with IC50 of 2.65 μM and 11.52 μM, respectively, while PTX suspension showed IC50 of 18.5 μM. Moreover, blank Surfactin-SNEDDS showed IC50 of 15.23 μM, while no cytotoxicity was observed for blank Tween 80-SNEDDS. Overall, the study interpreted high suitability of biosurfactant for developing the self-nanoemulsifying formulation of PTX with enhanced bioavailability, biocompatibility and synergistic improvement in the anticancer activity over the formulations containing synthetic surfactant.

A 3D Quantification Technique for Liver Fat Fraction Distribution Analysis Using Dixon Magnetic Resonance Imaging.

This study presents a 3D quantification methodology for the distribution of liver fat fraction (LFF) through the utilization of Dixon MRI image analysis. The central aim is to offer a highly accurate and non-invasive means of evaluating liver fat content. The process involves the acquisition of In-phase and Water-phase images from a Dixon sequence. LFF maps are then meticulously computed voxel by voxel by dividing the Lipid Phase images by the In-phase images. Simultaneously, 3D liver contours are extracted from the In-phase images. These crucial components are seamlessly integrated to construct a comprehensive 3D-LFF distribution model. This technique is not limited to healthy livers but extends to those afflicted by hepatic steatosis. The results obtained demonstrate the remarkable effectiveness of this approach in both visualizing and quantifying liver fat content. It distinctly discerns patterns that differentiate between normal and steatotic livers. By harnessing Dixon MRI to extract the 3D structure of the liver, this method offers precise LFF assessments spanning the entirety of the organ, thereby holding great promise for the diagnosis of hepatic steatosis with remarkable effectiveness.

Phase behavior of phospholipid-based myelin figures influenced by pH

The study of the effects of external stimuli on lipidic mesophases may enable a better understanding of alterations in biomembranes and improve the design of multilayered lipid structures. Here we demonstrate the influence of pH on the formation of elongated multilamellar assemblies, known as myelin figures (MFs). Polarized light microscopy allowed us to follow the growth process and thermal stability of phosphatidylcholine-based MFs. Our results reveal that the dimensions of these cylindrical microstructures change depending on the initial pH of the aqueous phase required for the self-assembly of lipids into MFs. Furthermore, we have confirmed the significant strength of the association between pH and characteristic parameters of MFs using Spearman’s rank correlation tests. Also, we have shown that one- and two-photon fluorescence microscopy can reveal the morphological changes that occur in MFs resulting from the lipid phase transition. These findings indicate the feasibility of using non-destructive methods to gain insight into liquid crystalline properties of biologically relevant multilamellar lipid structures in a wide pH range.

Metabolic Profiling Analysis Uncovers the Role of Carbon Nanoparticles in Enhancing the Biological Activities of Amaranth in Optimal Salinity Conditions

Enhancing the productivity and bioactivity of high-functional foods holds great significance. Carbon nanoparticles (CNPs) have a recognized capacity for boosting both plant growth and the efficacy of primary and secondary metabolites. Furthermore, while salinity diminishes plant growth, it concurrently amplifies the production of phytomolecules. To ensure the robust and sustainable production of nutritious food, it becomes essential to elevate biomolecule yield without compromising plant growth. Here, we assessed the CNPs priming on plant performance and metabolites of the glycophyte amaranth (Amaranthus hypochondriacus) sprouts at the threshold salinity (25 mM NaCl; i.e., salinity that does not reduce growth but enhances the metabolites of that plant). We measured growth parameters, pigment levels, and primary (carbohydrates, amino acids, organic acids, fatty acids) and secondary metabolites (phenolics, flavonoids, tocopherols). CNP priming significantly improved biomass accumulation (fresh and dry weight) and primary and secondary metabolites of amaranth sprouts. Increased photosynthetic pigments can explain these increases in photosynthesis. Enhanced photosynthesis induced carbohydrate production, providing a C source for producing bioactive primary and secondary metabolites. The priming effect of CNPs further enhanced the accumulation of essential amino acids, organic acids, unsaturated fatty acids, tocopherols, and phenolics at threshold salinity. The increase in bioactive metabolites under threshold salinity can explain the CNP priming impact on boosting the antioxidant activities (FRAP, DPPH, anti-lipid peroxidation, superoxide-anion-scavenger, hydroxyl-radical-scavenger, Fe-chelating and chain-breaking activity in aqueous and lipid phases) and antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi. Overall, this study suggested that threshold salinity and CNP priming could be useful for enhancing amaranth sprouts’ growth and nutritional quality.

Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

Analysis of Phase Heterogeneity in Lipid Membranes Using Single-Molecule Tracking in Live Cells.

In live cells, the plasma membrane is composed of lipid domains separated by hundreds of nanometers in dynamic equilibrium. Lipid phase separation regulates the trafficking and spatiotemporal organization of membrane molecules that promote signal transduction. However, visualizing domains with adequate spatiotemporal accuracy remains challenging because of their subdiffraction limit size and highly dynamic properties. Here, we present a single lipid-molecular motion analysis pipeline (lipid-MAP) for analyzing the phase heterogeneity of lipid membranes by detecting the instantaneous velocity change of a single lipid molecule using the excellent optical properties of nanoparticles, high spatial localization accuracy of single-molecule localization microscopy, and separation capability of the diffusion state of the hidden Markov model algorithm. Using lipid-MAP, individual lipid molecules were found to be in dynamic equilibrium between two statistically distinguishable phases, leading to the formation of small (∼170 nm), viscous (2.5× more viscous than surrounding areas), and transient domains in live cells. Moreover, our findings provide an understanding of how membrane compositional changes, i.e., cholesterol and phospholipids, affect domain formation. This imaging method can contribute to an improved understanding of spatiotemporal-controlled membrane dynamics at the molecular level.