Membrane Assembly Research Articles

Nanoelectrospray based synthesis of large, transportable membranes with integrated membrane proteins

Membrane proteins tend to be difficult to study since they need to be integrated into a lipid bilayer membrane to function properly. This study presents a method to synthesize a macroscopically large and freely transportable membrane with integrated membrane proteins which is useful for studying membrane proteins and protein complexes in isolation. The method could serve as a blueprint for the production of larger quantities of functionalised membranes for integration into technical devices similar to the MinION DNA sequencer. It is possible to self-assemble larger biological membranes on solid surfaces. However, they cannot be removed from their solid support without destroying them. In transportable form, self-assembled membranes are limited to sizes of about 17 nm in nanodiscs. Here we electrospray a series of molecular layers onto the liquid surface of a buffer solution which creates a flat, liquid environment on the surface that directs the self-assembly of the membrane. This method enables us to experimentally control the membrane composition and to succeed in producing large membranes with integrated OmpG, a transmembrane pore protein. The technique is compatible with the assembly of membrane based protein complexes. Listeriolysin O and pneumolysin efficiently assemble into non-covalent membrane pore complexes of approximately 30 units or more within the surface layer.

Proteomic Profiling of Endothelial Cell Secretomes After Exposure to Calciprotein Particles Reveals Downregulation of Basement Membrane Assembly and Increased Release of Soluble CD59

Calciprotein particles (CPPs) are essential circulating scavengers of excessive Ca2+ and PO43− ions, representing a vehicle that removes them from the human body and precludes extraskeletal calcification. Having been internalised by endothelial cells (ECs), CPPs induce their dysfunction, which is accompanied by a remarkable molecular reconfiguration, although little is known about this process’s extracellular signatures. Here, we applied ultra-high performance liquid chromatography-tandem mass spectrometry to perform a secretome-wide profiling of the cell culture supernatant from primary human coronary artery ECs (HCAECs) and internal thoracic artery ECs (HITAECs) treated with primary CPPs (CPP-P), secondary CPPs (CPP-S), magnesiprotein particles (MPPs), or Ca2+/Mg2+-free Dulbecco’s phosphate-buffered saline (DPBS) for 24 h. Incubation with CPP-P/CPP-S significantly altered the profiles of secreted proteins, delineating physiological and pathological endothelial secretomes. Neither pathway enrichment analysis nor the interrogation of protein–protein interactions detected extracellular matrix- and basement membrane-related molecular terms in the protein datasets from CPP-P/CPP-S-treated ECs. Both proteomic profiling and enzyme-linked immunosorbent assay identified an increased level of protectin (CD59) and reduced levels of osteonectin (SPARC), perlecan (HSPG2), and fibronectin (FN1) in the cell culture supernatant upon CPP-P/CPP-S treatment. Elevated soluble CD59 and decreased release of basement membrane components might be considered as potential signs of dysfunctional endothelium.

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from Mycobacterium tuberculosis.

Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis, an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro, inside macrophages, and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.

Hybrid Protocells Based on Coacervate-Templated Fatty Acid Vesicles Combine Improved Membrane Stability with Functional Interior Protocytoplasm.

Prebiotically-plausible compartmentalization mechanisms include membrane vesicles formed by amphiphile self-assembly and coacervate droplets formed by liquid-liquid phase separation. Both types of structures form spontaneously and can be related to cellular compartmentalization motifs in today's living cells. As prebiotic compartments, they have complementary capabilities, with coacervates offering excellent solute accumulation and membranes providing superior boundaries. Herein, protocell models constructed by spontaneous encapsulation of coacervate droplets by mixed fatty acid/phospholipid and by purely fatty acid membranes are described. Coacervate-supported membranes form over a range of coacervate and lipid compositions, with membrane properties impacted by charge-charge interactions between coacervates and membranes. Vesicles formed by coacervate-templated membrane assembly exhibit profoundly different permeability than traditional fatty acid or blended fatty acid/phospholipid membranes without a coacervate interior, particularly in the presence of magnesium ions (Mg2+). While fatty acid and blended membrane vesicles are disrupted by the addition of Mg2+, the corresponding coacervate-supported membranes remain intact and impermeable to externally-added solutes. With the more robust membrane, fluorescein diacetate (FDA) hydrolysis, which is commonly used for cell viability assays, can be performed inside the protocell model due to the simple diffusion of FDA and then following with the coacervate-mediated abiotic hydrolysis to fluorescein.

Mixed dimensional assembly of biodegradable and antifouling wood-based covalent organic framework composite membranes for rapid and efficient dye/salt separation

Manipulating materials of different dimensions into heterogeneous nanofiltration membranes with unique physicochemical properties and molecular sieving channels provides an effective way for accurate and fast molecular separation. Here we introduce a heterogeneous structure hybrid connection strategy to fabricate biodegradable wood-based covalent organic framework (COF) composite membranes. As a proof of concept, 3D Picea jezoensis (Siebold & Zucc.) Carrière was selected as the substrate of the membrane and in situ growth of 2D iCOF selective layers. Effective modulation of iCOF layers by 1D sulfonated polyaryletherketone (SPEEK-Na) using the “needle and thread” method. The rearrangement of the above multidimensional materials formed charge-regulated properties of laminar nano-channels and smooth hydrophilic contact area, thereby endowing specific molecular transport pathways and sieving capability for efficient dye/salt separation under ultra-low pressure of 0.5 bar. The wood-based heterostructured membranes exhibited high dye rejection (>97 %), low salt rejection (<10 %), and high permeance (172.34 L m−2 h−1 bar−1), which is superior to many reported dye/salt separation membrane materials. In addition, the system exhibited a certain degree of operational stability, good antifouling, and soil biodegradability. Overall, this work enables the design and fabrication of heterostructured separation membranes to be obtained from nature and used in nature, resulting in efficient and sustainable water purification applications.

The Rac1-PAK1-Arp2/3 signaling axis regulates CHIKV nsP1-induced filopodia and optimal viral genome replication.

Alphavirus infection induces dramatic remodeling of host cellular membranes, producing filopodia-like and intercellular extensions. The formation of filopodia-like extensions has been primarily assigned to the replication protein nsP1, which binds and reshapes the host plasma membrane when expressed alone. While reported decades ago, the molecular mechanisms behind nsP1 membrane deformation remain unknown. Using mammalian epithelial cells and Chikungunya virus (CHIKV) as models, we characterized nsP1-induced membrane deformations as highly dynamic actin-rich lamellipodia and filopodia-like extensions. Through pharmacological inhibition and genetic invalidation, we identified the critical contribution of the Rac1 GTPase and its downstream effectors PAK1 and the actin nucleator Arp2 in nsP1-induced membrane deformation. An intact Rac1-PAK1-Arp2 signaling axis was also required for optimal CHIKV genome replication. Therefore, our results designate the Rac1-PAK1-Arp2 pathway as an essential signaling node for CHIKV infection and establish a parallel requirement for host factors involved in nsP1-induced plasma membrane reshaping and assembly of a functional replication complex.IMPORTANCEThe alphavirus nsP1 protein dramatically remodels host cellular membranes, resulting in the formation of filopodia-like extensions. Although described decades ago, the molecular mechanisms controlling these membrane deformations and their functional importance remain elusive. Our study provides mechanistic insight, uncovering the critical role of the Rac1 GTPase, along with its downstream effectors PAK1 and the actin nucleator Arp2, in the nsP1-associated phenotype. Furthermore, we demonstrate that the Rac1-PAK1-Arp2 pathway is essential for optimal CHIKV genome replication. Our findings establish a parallel in the cellular mechanisms governing nsP1-induced plasma membrane reshaping and the production of a functional replication complex in infected cells.

Transcriptome Profiling of Phenylalanine-Treated Human Neuronal Model: Spotlight on Neurite Impairment and Synaptic Connectivity.

Phenylketonuria (PKU) is the most common inherited disorder of amino acid metabolism, characterized by high levels of phenylalanine (Phe) in the blood and brain, leading to cognitive impairment without treatment. Nevertheless, Phe-mediated brain dysfunction is not fully understood. The objective of this study was to address gene expression alterations due to excessive Phe exposure in the human neuronal model and provide molecular advances in PKU pathophysiology. Hence, we performed NT2/D1 differentiation in culture, and, for the first time, we used Phe-treated NT2-derived neurons (NT2/N) as a novel model for Phe-mediated neuronal impairment. NT2/N were treated with 1.25 mM, 2.5 mM, 5 mM, 10 mM, and 30 mM Phe and subjected to whole-mRNA short-read sequencing. Differentially expressed genes (DEGs) were analyzed and enrichment analysis was performed. Under three different Phe concentrations (2.5 mM, 5 mM, and 10 mM), DEGs pointed to the PREX1, LRP4, CDC42BPG, GPR50, PRMT8, RASGRF2, and CDH6 genes, placing them in the context of PKU for the first time. Enriched processes included dendrite and axon impairment, synaptic transmission, and membrane assembly. In contrast to these groups, the 30 mM Phe treatment group clearly represented the neurotoxicity of Phe, exhibiting enrichment in apoptotic pathways. In conclusion, we established NT2/N as a novel model for Phe-mediated neuronal dysfunction and outlined the Phe-induced gene expression changes resulting in neurite impairment and altered synaptic connectivity.

An effective vaccine against influenza A virus based on the matrix protein 2 (M2)

The ever-increasing antigenic diversity of the hemagglutinin (HA) of influenza A virus (IAV) poses a significant challenge for effective vaccine development. Notably, the matrix protein 2 (M2) is a highly conserved 97 amino acid long transmembrane tetrameric protein present in the envelope of IAV. More than 99 % of IAV strains circulating in American swine herds share the identical pandemic (pdm) isoform of M2, making it an ideal target antigen for a vaccine that could elicit broadly protective immunity. Here, using soluble nanoscale membrane assemblies termed nanodiscs (NDs), we designed this membrane mimetic nanostructures displaying full-length M2 in its natural transmembrane configuration (M2ND). Intramuscular (IM) immunization of swine with M2ND mixed with conventional emulsion adjuvant elicited M2-specific IgG antibodies in the serum that recognized influenza virions and M2-specific interferon-γ secreting cells present in the blood. Intranasal (IN) immunization with M2ND adjuvanted with a mycobacterial extract elicited M2-specific IgA in mucosal secretions that also recognized IAV. Immunization with an influenza whole inactivated virus (WIV) vaccine supplemented with a concurrent IM injection of M2ND mixed with an emulsion adjuvant increased the level of protective immunity afforded by the former against a challenge with an antigenically distinct H3N2 IAV, as exhibited by an enhanced elimination of virus from the lung. The lone IM administration of the M2ND vaccine mixed with an emulsion adjuvant provided measurable protection as evidenced by a >10-fold reduction or complete elimination of the challenge virus from the lung, but it did not diminish the viral load in nasal secretions nor the extent of pneumonia that ensued after the virus challenge. In contrast, an improved formulation of the M2ND vaccine that incorporated synthetic CpG oligodeoxynucleotides (CpG-ODN) in the nanostructures administered alone, via the IN and IM routes combined, provided a significant level of protective immunity against IAV as evidenced by a decreased viral load in both the upper and lower respiratory tracts and fully eliminated the occurrence of pneumonia in 89 % of the pigs immunized with this biologic. Notably, to be effective, the M2 protein must be displayed in the ND assemblies, as shown by the observation that simply mixing M2 with empty NDs incorporating CpG-ODN (eND-CpG-ODN) did not provide protective immunity. This novel M2-based vaccine offers great promise to help increase the breadth of protection afforded by conventional WIV vaccines against the diversity of IAV in circulation and, plausibly, as a broadly protective stand-alone biologic.

Capillary-Assisted Assembly of Polymer Gel-Supported Lipid Bilayers.

The polymer-supported lipid bilayer represents an attractive supramolecular assembly in numerous biophysical and bioanalytical applications. The assembly of polymer-supported membranes with a polymer layer thickness of just a few nanometers is now well-established, but bilayer properties in such a membrane architecture are still influenced by the nearby solid substrate. Polymer-supported lipid bilayer systems with a several micrometers thick polymer layer will overcome this shortcoming. However, formation of a fluid lipid bilayer on a fully hydrated, micrometer thick polymer film using traditional methods (e.g., vesicle fusion and lipid monolayer deposition techniques) remains a challenging task due to the rather unfavorable interfacial conditions for bilayer formation in such a system. Here, we report for the first time on the facile capillary-assisted formation of a lipid bilayer on the surface of a fully hydrated, several micrometers thick polyacrylamide (PAA) gel, in which forced molecular crowding of lipids at the air-water interface of the capillary results in monolayer instability and collapse, thereby forming a lipid bilayer on the top of the polymer gel inside the capillary. Stable bilayer attachment on the surface of the polymer gel can be achieved via physisorption or specific chemical linkages (tethering) on both cross-linked and non-cross-linked PAA films. Unlike the traditional solid-supported lipid bilayer (SLB), the lipid lateral diffusion in the polymer gel-supported lipid bilayer is not anymore perturbed by a solid substrate. Instead, more like a plasma membrane, it is mainly influenced by the properties of the underlying polymer and the nature/distribution of polymer-bilayer attachments. Polymer gel-supported lipid bilayers built using the capillary-assisted assembly approach show attractive self-healing properties, resulting in superior long-term stability relative to the SLB. We hypothesize that the described capillary-assisted assembly method can be applied to a wide range of polymeric materials and lipid compositions, opening exciting opportunities as an advanced model membrane system.

RpoS sigma factor mediates adaptation and virulence in Vibrio mimicus

The alternative sigma factor RpoS functions as a regulator of stress and virulence response in numerous bacterial species. Vibrio mimicus is a critical opportunistic pathogen causing huge losses to aquaculture. However, the exact role of RpoS in V. mimicus remains unclear. In this study, rpoS deletion mutant of V. mimicus was constructed through allelic exchange and the phenotypic and transcriptional changes were investigated to determine the function of RpoS. The abilities of growth, motility, biofilm production, hemolytic activity and pathogenicity were significantly impaired in ΔrpoS strain. Stationary-phase cells of ΔrpoS strain showed lower tolerance to H2O2, heat, ethanol, and starvation stress than the wild-type strain. Transcriptome analyses revealed the involvement of rpoS in various cellular processes, notably bacterial-type flagellum synthesis and assembly, membrane synthesis and assembly and response to various stimuli. Phenotypic and RNA-seq analysis revealed that RpoS is required for biofilm formation, stress resistance, and pathogenicity in V. mimicus. Furthermore, β-galactosidase activity showed that rpoS is essential for optimal transcription of the flgK, fliA, cheA, mcpH mRNA. These results offer significant insight into the function and regulatory network of rpoS/RpoS, thereby improving our understanding and facilitating selection of molecular targets for future prevention strategies against V. mimicus.

Outer membrane protein assembly mediated by BAM-SurA complexes

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis.

Native MS-guided lipidomics to define endogenous lipid microenvironments of eukaryotic receptors and transporters.

The mammalian membrane is composed of various eukaryotic lipids interacting with extensively post-translationally modified proteins. Probing interactions between these mammalian membrane proteins and their diverse and heterogeneous lipid cohort remains challenging. Recently, native mass spectrometry (MS) combined with bottom-up 'omics' approaches has provided valuable information to relate structural and functional lipids to membrane protein assemblies in eukaryotic membranes. Here we provide a step-by-step protocol to identify and provide relative quantification for endogenous lipids bound to mammalian membrane proteins and their complexes. Using native MS to guide our lipidomics strategies, we describe the necessary sample preparation steps, followed by native MS data acquisition, tailored lipidomics and data interpretation. We also highlight considerations for the integration of different levels of information from native MS and lipidomics and how to deal with the various challenges that arise during the experiments. This protocol begins with the preparation of membrane proteins from mammalian cells and tissues for native MS. The results enable not only direct assessment of copurified endogenous lipids but also determination of the apparent affinities of specific lipids. Detailed sample preparation for lipidomics analysis is also covered, along with comprehensive settings for liquid chromatography-MS analysis. This protocol is suitable for the identification and quantification of endogenous lipids, including fatty acids, sterols, glycerolipids, phospholipids and glycolipids and can be used to interrogate proteins from recombinant sources to native membranes.

Gasdermin D cysteine residues synergistically control its palmitoylation-mediated membrane targeting and assembly.

Gasdermin D (GSDMD) executes the cell death program of pyroptosis by assembling into oligomers that permeabilize the plasma membrane. Here, by single-molecule imaging, we elucidate the yet unclear mechanism of Gasdermin D pore assembly and the role of cysteine residues in GSDMD oligomerization. We show that GSDMD preassembles at the membrane into dimeric and trimeric building blocks that can either be inserted into the membrane, or further assemble into higher-order oligomers prior to insertion into the membrane. The GSDMD residues Cys39, Cys57, and Cys192 are the only relevant cysteines involved in GSDMD oligomerization. S-palmitoylation of Cys192, combined with the presence of negatively-charged lipids, controls GSDMD membrane targeting. Simultaneous Cys39/57/192-to-alanine (Ala) mutations, but not Ala mutations of Cys192 or the Cys39/57 pair individually, completely abolish GSDMD insertion into artificial membranes as well as into the plasma membrane. Finally, either Cys192 or the Cys39/Cys57 pair are sufficient to enable formation of GSDMD dimers/trimers, but they are all required for functional higher-order oligomer formation. Overall, our study unveils a cooperative role of Cys192 palmitoylation-mediated membrane binding and Cys39/57/192-mediated oligomerization in GSDMD pore assembly. This study supports a model in which Gasdermin D oligomerization relies on a two-step mechanism mediated by specific cysteine residues.

Structural basis for coupling of the WASH subunit FAM21 with the endosomal SNX27–Retromer complex

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains.

The intrinsic macrolide resistome of Escherichia coli.

Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and molecular mechanisms remain to be fully elucidated. Here, we used transposon directed insertion-site sequencing (TraDIS) to identify chromosomal non-essential genes involved in Escherichia coli intrinsic resistance to a macrolide antibiotic, tilmicosin. We constructed two highly saturated transposon mutant libraries of >290,000 and >390,000 unique Tn5 insertions in a clinical enterotoxigenic strain (ETEC5621) and in a laboratory strain (K-12 MG1655), respectively. TraDIS analysis identified genes required for growth of ETEC5621 and MG1655 under 1/8 MIC (n = 15 and 16, respectively) and 1/4 MIC (n = 38 and 32, respectively) of tilmicosin. For both strains, 23 genes related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump, and peptidoglycan metabolism were enriched in the presence of the antibiotic. Individual deletion of genes (n = 10) in the wild-type strains led to a 64- to 2-fold reduction in MICs of tilmicosin, erythromycin, and azithromycin, validating the results of the TraDIS analysis. Notably, deletion of surA or waaG, which impairs the outer membrane, led to the most significant decreases in MICs of all three macrolides in ETEC5621. Our findings contribute to a genome-wide understanding of intrinsic macrolide resistance in E. coli, shedding new light on the potential role of the peptidoglycan layer. They also provide an in vitro proof of concept that E. coli can be sensitized to macrolides by targeting proteins maintaining the outer membrane such as SurA and WaaG.

Unlocking Secrets: Lipid Metabolism and Lipid Droplet Crucial Roles in SARS-CoV-2 Infection and the Immune Response.

Lipid droplets (LD) are crucial for maintaining lipid and energy homeostasis within cells. LDs are highly dynamic organelles that present a phospholipid monolayer rich in neutral lipids. Additionally, LDs are associated with structural and non-structural proteins, rapidly mobilizing lipids for various biological processes. Lipids play a pivotal role during viral infection, participating during viral membrane fusion, viral replication, and assembly, endocytosis, and exocytosis. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection often induces LD accumulation, which is used as a source of energy for the replicative process. These findings suggest that LDs are a hallmark of viral infection, including SARS-CoV-2 infection. Moreover, LD participates in the inflammatory process and cell signaling, activating pathways related to innate immunity and cell death. Accumulating evidence demonstrates that LD induction by SARS-CoV-2 is a highly coordinated process, aiding replication and evading the immune system, and may contribute to the different cell death process observed in various studies. Nevertheless, recent research in the field of LDs suggests these organelles according to the pathogen and infection conditions may also play roles in immune and inflammatory responses, protecting the host against viral infection. Understanding how SARS-CoV-2 influences LD biogenesis is crucial for developing novel drugs or repurposing existing ones. By targeting host lipid metabolic pathways exploited by the virus, it is possible to impact viral replication and inflammatory responses. This review seeks to discuss and analyze the role of LDs during SARS-CoV-2 infection, specifically emphasizing their involvement in viral replication and the inflammatory response.

Validation Experiments for In Situ Ne Isotope Analysis on Mars: Gas Separation Flange Assembly Using Polyimide Membrane and Metal Seal

The Martian atmospheric Ne may reflect recent gas supply from its mantle via volcanic degassing, due to its short (∼100 Myr) escape timescale. The isotopic ratio of the Martian atmospheric Ne would therefore provide insights into that of the Martian mantle, further suggesting the origin of Mars volatiles during planetary formation. Mass spectrometric analysis of the Martian atmospheric Ne, however, has faced challenges from interference between 20Ne+ and 40Ar++. Previous studies using a polyimide membrane for 20Ne/40Ar separation were limited by the drawbacks of elastomeric O-rings to support the membrane, such as low-temperature intolerance, outgassing, and the need to endure environmental conditions during the launch and before/after landing on Mars. This study proposes a new method employing a metal C-ring to secure a 100 μm polyimide sheet within vacuum flanges. Environmental tests, including vibration, shock, extreme temperatures, and radiation exposure, were conducted on the gas separation flanges. Pre- and post-test analyses for He, Ne, and Ar demonstrated the membrane-flange system’s resilience. Gas permeation measurements using terrestrial air effectively permeated 4He and 20Ne, while reducing 40Ar by more than six orders of magnitude. This study achieved a <3% accuracy in determining the 20Ne/22Ne ratio, sufficient for assessing the origins of Ne in the Martian mantle. Furthermore, experiments with a 590 Pa gas mixture simulating the Martian atmosphere achieved a 10% accuracy for the 20Ne/22Ne isotope ratio, with gas abundances consistent with numerical predictions based on individual partial pressures. These results validate the suitability of the developed polyimide membrane assembly for in situ Martian Ne analyses.

The Impairment of Endothelial Autophagy Accelerates Renal Senescence by Ferroptosis and NLRP3 Inflammasome Signaling Pathways with the Disruption of Endothelial Barrier.

Autophagy is a cellular process that degrades damaged cytoplasmic components and regulates cell death. The homeostasis of endothelial cells (ECs) is crucial for the preservation of glomerular structure and function in aging. Here, we investigated the precise mechanisms of endothelial autophagy in renal aging. The genetic deletion of Atg7 in the ECs of Atg7flox/flox;Tie2-Cre mice accelerated aging-related glomerulopathy and tubulointerstitial fibrosis. The EC-specific Atg7 deletion in aging mice induced the detachment of EC with the disruption of glomerular basement membrane (GBM) assembly and increased podocyte loss resulting in microalbuminuria. A Transwell co-culture system of ECs and kidney organoids showed that the iron and oxidative stress induce the disruption of the endothelial barrier and increase vascular permeability, which was accelerated by the inhibition of autophagy. This resulted in the leakage of iron through the endothelial barrier into kidney organoids and increased oxidative stress, which led to ferroptotic cell death. The ferritin accumulation was increased in the kidneys of the EC-specific Atg7-deficient aging mice and upregulated the NLRP3 inflammasome signaling pathway. The pharmacologic inhibition of ferroptosis with liproxstatin-1 recovered the disrupted endothelial barrier and reversed the decreased expression of GPX4, as well as NLRP3 and IL-1β, in endothelial autophagy-deficient aged mice, which attenuated aging-related renal injury including the apoptosis of renal cells, abnormal structures of GBM, and tubulointerstitial fibrosis. Our data showed that endothelial autophagy is essential for the maintenance of the endothelial barrier during renal aging and the impairment of endothelial autophagy accelerates renal senescence by ferroptosis and NLRP3 inflammasome signaling pathways. These processes may be attractive therapeutic targets to reduce cellular injury from renal aging.

Unveiling the Correlation Between the Membrane Assembly of P-gp and Drug Resistance in Multiple Myeloma Using Super-Resolution Fluorescence Imaging.

Drug resistance in multiple myeloma (MM) poses a significant challenge to treatment efficacy, primarily attributed to P-glycoprotein (P-gp) dysfunction. This study delves into the elusive spatial organization of P-gp, aiming to enhance our understanding of its role in MM drug resistance by exploring the intricate relationship between molecular function and spatial arrangement. Employing super-resolution imaging of P-gp with the inhibitor probe Tariquidar-TAMR labeling on MM cell membranes, the research uncovered a more pronounced clustering distribution of P-gp in drug-resistant cells (MM1R) compared to drug-sensitive counterparts (MM1S). Further exploration revealed the clustering distribution of P-gp was heightened as cellular drug resistance increased in hypoxic condition, directly emphasizing the strong correlation between P-gp cluster morphology and drug resistance. Additionally, stable P-gp cluster formation was influenced by cross-linking of membrane carbohydrates, and disrupting these glycoprotein clusters could reduce cellular drug resistance, suggesting that altering distribution patterns of P-gp can modulate drug responsiveness. Finally, dexamethasone (Dex) treatment was revealed to enhance P-gp clustering distribution, particularly in MM1S cells, indicating that change degree in P-gp distribution correlate with the modifiable space of cellular drug responsiveness. This study provides insights into the correlation between P-gp assembly and cellular drug responsiveness, deepening our understanding of functional changes in MM drug resistance and offering valuable perspectives for overcoming this challenge.

A novel Janus composite membrane and its enhanced antifouling strategy for emulsion purification

The development of advanced membranes for the efficient separation of oil-water emulsions and the degradation of soluble pollutants is crucial for environmental protection. This study presents a Janus composite membrane with Co(CO3)0.5OH·0.11H2O nanoneedle arrays, fabricated through electrospinning, membrane assembly, surface hydrophobization, and chemical bath deposition (CBD). By controlling the amount of carbon nanofiber (CNF) and CBD time, the membrane's morphology can be optimized for improved performance. In dead-end filtration driven by gravity, the membrane effectively separates stabilized oil-in-water emulsions and demonstrates strong anti-fouling capabilities. Furthermore, the membrane exhibits significant catalytic activation for peroxymonosulfate (PMS), enabling complete restoration of water flux of polluted membranes and purifying emulsions with soluble organic pollutants. To further enhance anti-fouling performance, a novel separation strategy integrating pulse current is proposed. Under the influence of a pulsed electric field, the membrane achieves a permeate flux nearly twice as high as that observed without this external stimulus, while maintaining high separation efficiency. This research advances Janus nanofiber membrane development via electrospinning techniques and supports sustainable treatment of oily wastewater.