Lipid loss and compositional change during preparation of simple two-component liposomes.

Liposomes are used as model membranes in many scientific fields. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures can produce liposomes at specific concentrations and lipid compositions, and researchers often assume that the concentration and composition of their liposomes are similar or identical to what would be expected if no lipid loss occurred.

Lipid loss and compositional change during preparation of liposomes by common biophysical methods.

Liposomes are widely used as model lipid membrane platforms in many fields, ranging from basic biophysical studies to drug delivery and biotechnology applications. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures have the potential to produce liposomes at specific concentrations and membrane compositions, and researchers often assume that the concentration and composition of their liposomes are similar to, if not identical, to what would be expected if no lipid loss occurred during preparation.

mechanotransduction: Effect of acute exercise on the metabolomic profiles of mouse synovial fluid.

Objective: Exercise is known to induce beneficial effects in synovial joints. However, the mechanisms underlying these are unclear. Synovial joints experience repeated mechanical loading during exercise.

Choice of buffer in mobile phase can substantially alter peak areas in quantification of lipids by HPLC-ELSD.

Evaporative light scattering detectors (ELSD) are commonly used with high-performance liquid chromatography (HPLC) to separate and quantify lipids, which are typically not easily detectable by more conventional methods such as UV-visible detectors. In many HPLC-ELSD methods to analyze lipids, a volatile buffer is included in the mobile phase to control the pH and facilitate separation between lipid species. Here, we report an unintended effect that buffer choice can have in HPLC-ELSD analysis of lipids - the identity and concentration of the buffer can substantially influence the resulting ELSD peak areas.

Viral Size Modulates Sendai Virus Binding to Cholesterol-Stabilized Receptor Nanoclusters.

Binding to the host membrane is the initial infection step for animal viruses. Sendai virus (SeV), the model respirovirus studied here, utilizes sialic-acid-conjugated glycoproteins and glycolipids as receptors for binding. In a previous report studying single virus binding to supported lipid bilayers (SLBs), we found a puzzling mechanistic difference between the binding of SeV and influenza A virus (strain X31, IAV).

Single-virus assay reveals membrane determinants and mechanistic features of Sendai virus binding.

Sendai virus (SeV, formally murine respirovirus) is a membrane-enveloped, negative-sense RNA virus in the Paramyxoviridae family and is closely related to human parainfluenza viruses. SeV has long been utilized as a model paramyxovirus and has recently gained attention as a viral vector candidate for both laboratory and clinical applications. To infect host cells, SeV must first bind to sialic acid glycolipid or glycoprotein receptors on the host cell surface via its hemagglutinin-neuraminidase (HN) protein.

Modulating the Influenza A Virus-Target Membrane Fusion Interface With Synthetic DNA-Lipid Receptors.

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell-surface glycans are highly heterogeneous in length and glycosylation density, which leads to variations in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors.

Effects of long-term exercise and a high-fat diet on synovial fluid metabolomics and joint structural phenotypes in mice: an integrated network analysis.

Objective: To explore how systemic factors that modify knee osteoarthritis risk are connected to 'whole-joint' structural changes by evaluating the effects of high-fat diet and wheel running exercise on synovial fluid (SF) metabolomics.

Methods: Male mice were fed a defined control or high-fat (60% kcal fat) diet from 6 to 52 weeks of age, and half the animals were housed with running wheels from 26 to 52 weeks of age (n = 9-13 per group). Joint tissue structure and osteoarthritis pathology were evaluated by histology and micro-computed tomography.

Introducing the ArsR-Regulated Arsenic Stimulon.

The microbial operon encodes the primary bacterial defense response to the environmental toxicant, arsenic. An important component of this operon is the gene, which encodes ArsR, a member of the family of proteins categorized as DNA-binding transcriptional repressors. As currently documented, ArsR regulates its own expression as well as other genes in the same operon.

Kinetic Modeling of West Nile Virus Fusion Indicates an Off-Pathway State.

West Nile virus (WNV) is a prominent mosquito-borne flavivirus that causes febrile illness in humans. To infect host cells, WNV virions first bind to plasma membrane receptors, then initiate membrane fusion following endocytosis. The viral transmembrane E protein, triggered by endosomal pH, catalyzes fusion while undergoing a dimer-to-trimer transition.

Metabolic Responses to Arsenite Exposure Regulated through Histidine Kinases PhoR and AioS in 5A.

Arsenite (As) oxidation is a microbially-catalyzed transformation that directly impacts arsenic toxicity, bioaccumulation, and bioavailability in environmental systems. The genes for As oxidation () encode a periplasmic As sensor AioX, transmembrane histidine kinase AioS, and cognate regulatory partner AioR, which control expression of the As oxidase AioBA. The genes are under ultimate control of the phosphate stress response via histidine kinase PhoR.

Influenza hemagglutinin drives viral entry via two sequential intramembrane mechanisms.

Enveloped viruses enter cells via a process of membrane fusion between the viral envelope and a cellular membrane. For influenza virus, mutational data have shown that the membrane-inserted portions of the hemagglutinin protein play a critical role in achieving fusion. In contrast to the relatively well-understood ectodomain, a predictive mechanistic understanding of the intramembrane mechanisms by which influenza hemagglutinin drives fusion has been elusive.

Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis.

BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging.

Detecting and Controlling Dye Effects in Single-Virus Fusion Experiments.

Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner.

Characterization of synovial fluid metabolomic phenotypes of cartilage morphological changes associated with osteoarthritis.

Objective: Osteoarthritis (OA) is a multifactorial disease with etiological heterogeneity. The objective of this study was to classify OA subgroups by generating metabolomic phenotypes from human synovial fluid.

Design: Post mortem synovial fluids (n = 75) were analyzed by high performance-liquid chromatography mass spectrometry (LC-MS) to measure changes in the global metabolome.

Transcriptomics analysis defines global cellular response of Agrobacterium tumefaciens 5A to arsenite exposure regulated through the histidine kinases PhoR and AioS.

In environments where arsenic and microbes coexist, microbes are the principal drivers of arsenic speciation, which directly affects bioavailability, toxicity and bioaccumulation. Speciation reactions influence arsenic behaviour in environmental systems, directly affecting human and agricultural exposures. Arsenite oxidation decreases arsenic toxicity and mobility in the environment, and therefore understanding its regulation and overall influence on cellular metabolism is of significant interest.

Global metabolomic profiling of human synovial fluid for rheumatoid arthritis biomarkers.

Objectives: The objective of this study was to analyse the metabolomic profiles of rheumatoid arthritis synovial fluid to test the use of global metabolomics by liquid chromatography-mass spectrometry for clinical analysis of synovial fluid.

Methods: Metabolites were extracted from rheumatoid arthritis (n=3) and healthy (n=5) synovial fluid samples using 50:50 water: acetonitrile. Metabolite extracts were analysed in positive mode by normal phase liquid chromatography-mass spectrometry for global metabolomics.

pH Dependence of Zika Membrane Fusion Kinetics Reveals an Off-Pathway State.

The recent spread of Zika virus stimulated extensive research on its structure, pathogenesis, and immunology, but mechanistic study of entry has lagged behind, in part due to the lack of a defined reconstituted system. Here, we report Zika membrane fusion measured using a platform that bypasses these barriers, enabling observation of single-virus fusion kinetics without receptor reconstitution. Surprisingly, target membrane binding and low pH are sufficient to trigger viral hemifusion to liposomes containing only neutral lipids.

Application of global metabolomic profiling of synovial fluid for osteoarthritis biomarkers.

Osteoarthritis affects over 250 million individuals worldwide. Currently, there are no options for early diagnosis of osteoarthritis, demonstrating the need for biomarker discovery. To find biomarkers of osteoarthritis in human synovial fluid, we used high performance liquid-chromatography mass spectrometry for global metabolomic profiling.

Cholesterol enhances influenza binding avidity by controlling nanoscale receptor clustering.

Influenza virus infects cells by binding to sialylated glycans on the cell surface. While the chemical structure of these glycans determines hemagglutinin-glycan binding affinity, bimolecular affinities are weak, so binding is avidity-dominated and driven by multivalent interactions. Here, we show that membrane spatial organization can control viral binding.

Multi-omics analysis provides insight to the Ignicoccus hospitalis-Nanoarchaeum equitans association.

Background: Studies of interspecies interactions are inherently difficult due to the complex mechanisms which enable these relationships. A model system for studying interspecies interactions is the marine hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans. Recent independently-conducted 'omics' analyses have generated insights into the molecular factors modulating this association.

Influenza viral membrane fusion is sensitive to sterol concentration but surprisingly robust to sterol chemical identity.

Influenza virions are enriched in cholesterol relative to the plasma membrane from which they bud. Previous work has shown that fusion between influenza virus and synthetic liposomes is sensitive to the amount of cholesterol in either the virus or the target membrane. Here, we test the chemical properties of cholesterol required to promote influenza fusion by replacing cholesterol with other sterols and assaying viral fusion kinetics.

Disentangling Viral Membrane Fusion from Receptor Binding Using Synthetic DNA-Lipid Conjugates.

Enveloped viruses must bind to a receptor on the host membrane to initiate infection. Membrane fusion is subsequently initiated by a conformational change in the viral fusion protein, triggered by receptor binding, an environmental change, or both. Here, we present a strategy to disentangle the two processes of receptor binding and fusion using synthetic DNA-lipid conjugates to bind enveloped viruses to target membranes in the absence of receptor.

Choose your label wisely: water-soluble fluorophores often interact with lipid bilayers.

Water-soluble organic fluorophores are widely used as labels in biological systems. However, in many cases these fluorophores can interact strongly with lipid bilayers, influencing the interaction of the target with the bilayer and/or leading to misleading fluorescent signals. Here, we quantify the interaction of 32 common water-soluble dyes with model lipid bilayers to serve as an additional criterion when selecting a dye label.

Individual vesicle fusion events mediated by lipid-anchored DNA.

Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk.