Sucrose Density Gradient Ultracentrifugation Research Articles

A review on separation and application of plant-derived exosome-like nanoparticles.

Exosomes-like nanoparticles (ELNs) (exosomes or extracellular vesicles) are vesicle-like bodies secreted by cells. Plant ELNs (PENs) are membrane vesicles secreted by plant cells, with a lipid bilayer as the basic skeleton, enclosing various active substances such as proteins and nucleic acids, which have many physiological and pathological functions. Recent studies have found that the PENs are widespread within different plant species and their biological functions are increasingly recognized. The effective separation method is also necessary for its function and application. Ultracentrifugation, sucrose density gradient ultracentrifugation, ultrafiltration, polymer-based precipitation methods, etc., are commonly used methods for plant exosome-like nanoparticle extraction. In recent years, emerging methods such as size exclusion chromatography, immunoaffinity capture-based technique, and microfluidic technology have shown advancements compared to traditional methods. The standardized separation process for PENs continues to evolve. In this review, we summarized the recent progress in the biogenesis, components, separation methods, and some functions of PENs. When the research on the separation method of PENs and their unique biological structure is further studied. A brand-new idea for the efficient separation and utilization of PENs can be provided in the future, which has a very broad prospect.

Proteome-Wide Identification of RNA-dependent proteins and an emerging role for RNAs in Plasmodium falciparum protein complexes

Ribonucleoprotein complexes are composed of RNA, RNA-dependent proteins (RDPs) and RNA-binding proteins (RBPs), and play fundamental roles in RNA regulation. However, in the human malaria parasite, Plasmodium falciparum, identification and characterization of these proteins are particularly limited. In this study, we use an unbiased proteome-wide approach, called R-DeeP, a method based on sucrose density gradient ultracentrifugation, to identify RDPs. Quantitative analysis by mass spectrometry identifies 898 RDPs, including 545 proteins not yet associated with RNA. Results are further validated using a combination of computational and molecular approaches. Overall, this method provides the first snapshot of the Plasmodium protein-protein interaction network in the presence and absence of RNA. R-DeeP also helps to reconstruct Plasmodium multiprotein complexes based on co-segregation and deciphers their RNA-dependence. One RDP candidate, PF3D7_0823200, is functionally characterized and validated as a true RBP. Using enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq), we demonstrate that this protein interacts with various Plasmodium non-coding transcripts, including the var genes and ap2 transcription factors.

Expression and Function of Connexins in Extracellular Vesicles.

Extracellular vesicles (EVs) are recognized as major vehicles for exchange of information across distant cells and tissues, which have been extensively explored for diagnosis and therapeutic purposes. The presence of multiple connexin (Cx) proteins has been described in EVs, where they might facilitate EV-cell communication. However, quantitative changes in Cx levels and functional assessment of Cx channels have only been established for Cx43. In present work, we provide a detailed description of the protocols we have optimized to assess the expression and permeability of Cx43 channels in EVs derived from cultured cells and human peripheral blood. Particularly, we include some modifications to improve quantitative analysis of EV-Cx43 by enzyme-linked immunosorbent assay (ELISA) and assessment of channel functionality by sucrose-density gradient ultracentrifugation, which can be easily adapted to other Cx family members, leveraging the development of diagnostic and therapeutic applications based on Cx-containing EVs.

Large-scale column-free purification of bovine F-ATP synthase

Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at a quantity, stability and purity that meets the demand of the planned experiment. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favoring IF1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the glyco-diosgenin (GDN) to lauryl maltose neopentyl glycol (LMNG) ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.

Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses.

Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing.

3,4-Dihydroxybenzalacetone Inhibits the Propagation of Hydrogen Peroxide-Induced Oxidative Effect via Secretory Components from SH-SY5Y Cells.

The polyphenol derivative 3,4-dihydroxybenzalacetone (DBL) is the primary antioxidative component of the medicinal folk mushroom Chaga (Inonotus obliquus (persoon) Pilat). In this study, we investigated whether the antioxidative effect of DBL could propagate to recipient cells via secreted components, including extracellular vesicles (EVs), after pre-exposing SH-SY5Y human neuroblastoma cells to DBL. First, we prepared EV-enriched fractions via sucrose density gradient ultracentrifugation using conditioned medium from SH-SY5Y cells exposed to 100 µM hydrogen peroxide (H2O2) for 24 h, with and without 1 h of 5 µM DBL pre-treatment. CD63 immuno-dot blot analysis demonstrated that fractions with density of 1.06-1.09 g/cm3 had CD63-like immuno-reactivities. Furthermore, the 2,2-diphenyl-1-picrylhydrazyl assay revealed that the radical scavenging activity of fraction 11 (density of 1.06 g/cm3), prepared after 24-h H2O2 treatment, was significantly increased compared to that in the control group (no H2O2 treatment). Notably, 1 h of 5 µM DBL pre-treatment or 5 min of heat treatment (100 °C) diminished this effect, although concentrating the fraction by 100 kDa ultrafiltration enhanced it. Overall, the effect was not specific to the recipient cell types. In addition, the uptake of fluorescent Paul Karl Horan-labeled EVs in concentrated fraction 11 was detected in all treatment groups, particularly in the H2O2-treated group. The results suggest that cell-to-cell communication via bioactive substances, such as EVs, in conditioned SH-SY5Y cell medium, propagates the H2O2-induced radical scavenging effect, whereas pre-conditioning with DBL inhibits it.

Different culture media and purification methods unveil the core proteome of Propionibacterium freudenreichii-derived extracellular vesicles.

Bacterial extracellular vesicles (EVs) are natural lipidic nanoparticles implicated in intercellular communication. Although EV research focused mainly on pathogens, the interest in probiotic-derived EVs is now rising. One example is Propionibacterium freudenreichii, which produces EVs with anti-inflammatory effects on human epithelial cells. Our previous study with P. freudenreichii showed that EVs purified by size exclusion chromatography (SEC) displayed variations in protein content according to bacterial growth conditions. Considering these content variations, we hypothesized that a comparative proteomic analysis of EVs recovered in different conditions would elucidate whether a representative vesicular proteome existed, possibly providing a robust proteome dataset for further analysis. Therefore, P. freudenreichii was grown in two culture media, and EVs were purified by sucrose density gradient ultracentrifugation (UC). Microscopic and size characterization confirmed EV purification, while shotgun proteomics unveiled that they carried a diverse set of proteins. A comparative analysis of the protein content of UC- and SEC-derived EVs, isolated from cultures either in UF (cow milk ultrafiltrate medium) or YEL (laboratory yeast extract lactate medium), showed that EVs from all these conditions shared 308 proteins. This EV core proteome was notably enriched in proteins related to immunomodulation. Moreover, it showed distinctive features, including highly interacting proteins, compositional biases for some specific amino acids, and other biochemical parameters. Overall, this work broadens the toolset for the purification of P. freudenreichii-derived EVs, identifies a representative vesicular proteome, and enumerates conserved features in vesicular proteins. These results hold the potential for providing candidate biomarkers of purification quality, and insights into the mechanisms of EV biogenesis and cargo sorting.

Proteome-Wide Identification of RNA-Dependent Proteins in Lung Cancer Cells.

Following the concept of RNA dependence and exploiting its application in the R-DeeP screening approach, we have identified RNA-dependent proteins in A549 lung adenocarcinoma cells. RNA-dependent proteins are defined as proteins whose interactome depends on RNA and thus entails RNA-binding proteins (RBPs) as well as proteins in ribonucleoprotein complexes (RNPs) without direct RNA interaction. With this proteome-wide technique based on sucrose density gradient ultracentrifugation and fractionation followed by quantitative mass spectrometry and bioinformatic analysis, we have identified 1189 RNA-dependent proteins including 170 proteins which had never been linked to RNA before. R-DeeP provides quantitative information on the fraction of a protein being RNA-dependent as well as it allows the reconstruction of protein complexes based on co-segregation. The RNA dependence of three newly identified RNA-dependent proteins, DOCK5, ELMO2, also known as CED12A, and ABRAXAS1, also known as CCDC98, was validated using western blot analysis, and the direct RNA interaction was verified by iCLIP2 for the migration-related protein DOCK5 and the mitosis-related protein ABRAXAS1. The R-DeeP 2.0 database provides proteome-wide and cell line-specific information from A549 and HeLa S3 cells on proteins and their RNA dependence to contribute to understanding the functional role of RNA and RNA-binding proteins in cancer cells.

Method of obtaining purified SARS-CoV-2 antigen for structural and immunological studies in whole-virion vaccines against coronavirus infection

BACKGROUND: Despite the large-scale development of vaccines against coronavirus infection, there is still no complete information about the antigenic structure of the SARS-CoV-2 virus particles. This article describes a method of obtaining a pure concentrated whole-virion sample that can be used in various studies.
 AIM: The goal is to develop an optimal method of purification of the inactivated SARS-CoV-2 virus in order to obtain a standard with parameters of purity and antigen content sufficient for structural and immunological studies.
 MATERIALS AND METHODS: To obtain a pure concentrated virus SARS-CoV-2 we used the sucrose density gradient ultracentrifugation. Fractions with the highest content of viral particles were evaluated based on the concentration of nucleocapsid N and glycoprotein S. The purity of the pooled fractions (the purified sample) was evaluated by the presence of impurity proteins, toxins and bovine serum albumin.
 RESULTS: The optimal conditions for obtaining the inactivated purified SARS-CoV-2 whole virus were determined. A standard sample of the inactivated SARS-CoV-2 virus was isolated and characterized.
 CONCLUSIONS: The obtained standard sample can be used in enzyme immunoassay to measure the amount of antigen in whole-virion vaccines against coronavirus infection, as well as in various structural studies of SARS-CoV-2 virus particle.

Production of Neuraminidase Virus Like Particles by Stably Transformed Insect Cells: A Simple Process for NA-Based Influenza Vaccine Development.

Neuraminidase (NA) is a second major surface protein of the influenza virus and has recently been suggested as a supplemental antigen to the major immunodominant hemagglutinin (HA) antigen in the influenza vaccine. NA is less affected by antigenic drift compared to the HA, induces strong anti-neuraminidase immune responses, and provides broader protection against many influenza strains. However, the NA amount in currently licensed influenza virus vaccines is much lower than that of HA, and not standardized. A platform to produce NA antigen, in the form of virus-like particles (VLPs), was thus developed, to facilitate supplementation of NA antigen in the influenza vaccine formula. Stably transformed Sf9 insect cells had been engineered to express the influenza A virus (H5N1) NA gene under a baculovirus OpMNPV IE2 promoter. Recombinant NA protein was synthesized and assembled into VLPs, in the intact cellular environment provided by insect cells. Approximately 150 µg/ml of NA-VLPs was obtained in the culture medium. Purification of the NA-VLPs was achieved by a sucrose density gradient ultracentrifugation. The purified NA-VLPs effectively induced anti-NA antibodies with neuraminidase inhibition activities in mice. This work demonstrates a simple process to produce an immunocompetent NA-VLPs antigen, exclusively made of only neuraminidase, by insect cells.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12033-022-00519-8.

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer's disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

Comparison of High-Performance Liquid Chromatography with Sucrose Density Gradient Ultracentrifugation for the Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Foot-and-mouth disease (FMD) causes substantial economic losses in the livestock industry. The protective immunizing component of the FMD virus (FMDV) is a ribonucleoprotein particle with a sedimentation coefficient of 146S. Size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace sucrose density gradient ultracentrifugation (SDG), which is the gold standard for the quantification of FMDV 146S particles. SE-HPLC showed a pattern similar to that of SDG; however, the two methods resulted in different quantities for the same amount of 146S particles. This study aimed to identify the reason for this disparity and adjust the difference between the two methods by employing a standard material. While SE-HPLC displayed all the virus particles in the peak fraction by SDS-PAGE and Western blotting, the virus particles were widely dispersed in multiple fractions, including peak fractions in the SDG. To adjust the difference between the two methods, a stable surrogate virus, bovine enterovirus, was devised to draw a standard curve, and the gap was reduced to <10%. To our knowledge, this is the first report to provide experimental evidence on the difference between SDG and SE-HPLC for the quantification of FMDV particles.

Сравнение методов очистки фаговых лизатов грамотрицательных бактерий для персонализированной терапии

Phage therapy is a promising method of treating antibiotic-resistant infections. To obtain a safe therapeutic formulation, bacterial cell components, including endotoxins, must be removed from the phage lysate. This study was aimed at comparing the efficacy of purification methods for phage lysates intended for therapeutic use. Phages vB_KpnM_Seu621 (Myoviridae) and vB_KpnP_Dlv622 (Autographiviridae) were grown using the KP9068 strain of Klebsiella pneumoniae as a host. The obtained lysates were purified using phage precipitation with polyethylene glycol, CsCl density gradient ultracentrifugation, sucrose density gradient ultracentrifugation, precipitation with 100 kDa centrifugal filter units, and phage concentration on 0.22 µm cellulose filters in the presence of MgSO4. Endotoxin concentrations were determined by LAL testing. The obtained lysates contained 1.25 × 1012 ± 7.46 × 1010 and 2.25 × 1012 ± 1.34 × 1011 PFU/ml of vB_KpnM_Seu621 and vB_KpnP_Dlv622, respectively, and had endotoxin concentrations of 3,806,056 ± 429,410 and 189,456 ± 12,406 EU/ml, respectively. CsCl gradient ultracentrifugation was found to be the optimal conventional purification method in terms of reducing endotoxin concentrations and maintaining phage titers (303 ± 20 — 313 ± 35 EU/ml, 1.5–2.75 × 1012 ± 1.71 × 1011 PFU/ml). Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 were found to be the optimal non-traditional purification methods. A method for phage lysate purification should be selected for each phage preparation individually. Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 hold promise as purification methods that can produce phage preparations suitable for intravenous administration.

Preparation of nuclease-resistant RNA standard for hepatitis C virus quantification

Positive control (or standard) is an indispensable ingredient in molecular biology assays widely used for the quantification of nucleic acid. The commonly used standards are plasmid DNA, cDNA, or naked RNA, which are unstable and easily degraded by nucleases in the surrounding environment; this might affect the accuracy of quantitative results. In this study, the authors designed and created a positive control for the hepatitis C virus (HCV) quantification based on armored RNA technology. The 5’UTR non-encoding sequence of HCV was cloned into the BH20 plasmid. Armored RNA HCV (AR-HCV) was induced for expression in the E. coli BL21 (DE3) by the addition of an IPTG inducer. AR-HCV was collected by sucrose density gradient ultracentrifugation followed by gel filtration chromatography using Superdex 75 column. Created AR-HCV was determined the concentration and examined the formation of pseudo viral particles by transmission electron microscopy (TEM). Stability assessment of AR-HCV to DNase and RNase treatment simultaneously has demonstrated its ability to resist these nucleases. Moreover, AR-HCV is stable over time and storage conditions. Strikingly, AR-HCV can be directly added to the specimen, allowing better and more accurate control of the whole quantitative procedure of HCV.

Accessing the Human Pluripotent Stem Cell Translatome by Polysome Profiling.

Polysome profiling is a technique that uses sucrose density gradient ultracentrifugation to separate complexes of mRNAs associated with one or more ribosomes. Here we describe polysome profiling analysis in human pluripotent stem cells (hPSCs) using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR or large-scale sequencing analysis, used to establish the translational status of specific mRNAs and identify the role of noncoding RNA in translation.

Biomanufacturing of Tomato-Derived Nanovesicles.

Micro- and nano-sized vesicles (MVs and NVs, respectively) from edible plant resources are gaining increasing interest as green, sustainable, and biocompatible materials for the development of next-generation delivery vectors. The isolation of vesicles from complex plant matrix is a significant challenge considering the trade-off between yield and purity. Here, we used differential ultracentrifugation (dUC) for the bulk production of MVs and NVs from tomato (Solanum lycopersicum L.) fruit and analyzed their physical and morphological characteristics and biocargo profiles. The protein and phospholipid cargo shared considerable similarities between MVs and NVs. Phosphatidic acid was the most abundant phospholipid identified in NVs and MVs. The bulk vesicle isolates were further purified using sucrose density gradient ultracentrifugation (gUC) or size-exclusion chromatography (SEC). We showed that SEC using gravity column efficiently removed co-purifying matrix components including proteins and small molecular species. dUC/SEC yielded a high yield of purified vesicles in terms of number of particles (2.6 × 1015 particles) and protein quantities (6.9 ± 1.5 mg) per kilogram of tomato. dUC/gUC method separated two vesicle populations on the basis of buoyant density. Proteomics and in silico studies of the SEC-purified MVs and NVs support the presence of different intra- and extracellular vesicles with highly abundant lipoxygenase (LOX), ATPases, and heat shock proteins (HSPs), as well as a set of proteins that overlaps with that previously reported in tomato chromoplast.

Analysis of cell‐type specific exosomes isolated from cryopreserved human brain

AbstractBackgroundExosomes are small extracellular vesicles (EVs) originated from multivesicular endosomes which carry biochemical information about their cell‐of‐origin and signals for surrounding cells. EVs may also play a role in spread of proteopathic tau and amyloid beta seeds in Alzheimer’s disease (AD). Thus, brain exosomes may be a valuable therapeutic target for AD treatment. Ability of brain exosomes to cross the blood brain barrier make them a potential diagnostic and monitoring tool. Despite recent progress in development of EV isolation methods, our knowledge of human brain exosomal subpopulations and their roles in disease progression is very limited.MethodCryopreserved parietal cortex from 5 late stage AD (Braak V‐VI) and 3 control (NL) cases were used for the experiment. Brain exosomes/EVs were purified by sucrose density gradient ultracentrifugation after gentle enzymatic and mechanical dissociation of the brain tissue. Microglia‐derived EVs were isolated from the brain EV fraction by immunoprecipitation with anti‐CD11b antibodies. We performed analysis of miRNA transcriptome and proteome using NanoString and quantitative proteomics respectively.ResultWe identified 105 miRNAs which are present in more than 80% of analyzed human cases. The pathways controlled by five significantly upregulated miRNAs can be converged to control microglia activation (TGFβ, TLRs, and chemokine signaling pathways) and neuronal survival and function (LTD and LTP, NGF and neurotrophin signaling pathways). One of the upregulated exosomal miRNAs (miR‐188‐5p) is known to be downregulated in human AD brain and it can restore synaptic and cognitive deficits in 5xFAD mice. MiR‐381‐3p can promote recovery of spinal cord injury in rats. On the protein level we found a moderate increase (around 50%) in exosomal markers CD9 and CD81 and decrease in CHMP4B (ESCRT‐III) protein in the AD group. Levels of mitochondrial proteins and annexins were around 2 times higher in AD group compared to NL controls. ApoE protein was also significantly upregulated in AD microglial EVs.ConclusionOur data revealed the presence of anti‐inflammatory and neuroprotective miRNA signature in CD11b‐positive microglia‐derived exosomes. Proteomics data suggests upregulation of ESCRT‐independent pathway of exosome biogenesis in AD microglia. Increased association of microglial EVs with ApoE maybe related to amyloid packaging and release by exosome‐mediated mechanism.

Multimeric and monomeric photosystem II supercomplexes represent structural adaptations to low- and high-light conditions

An intriguing molecular architecture called the "semi-crystalline photosystem II (PSII) array" has been observed in the thylakoid membranes in vascular plants. It is an array of PSII-light-harvesting complex II (LHCII) supercomplexes that only appears in low light, but its functional role has not been clarified. Here, we identified PSII-LHCII supercomplexes in their monomeric and multimeric forms in low light-acclimated spinach leaves and prepared them using sucrose-density gradient ultracentrifugation in the presence of amphipol A8-35. When the leaves were acclimated to high light, only the monomeric forms were present, suggesting that the multimeric forms represent a structural adaptation to low light and that disaggregation of the PSII-LHCII supercomplex represents an adaptation to high light. Single-particle EM revealed that the multimeric PSII-LHCII supercomplexes are composed of two ("megacomplex") or three ("arraycomplex") units of PSII-LHCII supercomplexes, which likely constitute a fraction of the semi-crystalline PSII array. Further characterization with fluorescence analysis revealed that multimeric forms have a higher light-harvesting capability but a lower thermal dissipation capability than the monomeric form. These findings suggest that the configurational conversion of PSII-LHCII supercomplexes may serve as a structural basis for acclimation of plants to environmental light.

Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes.

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40–150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.

Soyasaponin A1 Inhibits the Lipid Raft Recruitment and Dimerization of TLR4, MyD88 and TRIF in Palmitate-Stimulated Inflammatory Macrophages

Abstract Objectives Soyasaponin A1 (SSA1) was previously shown to inhibit the palmitate (PA)-induced inflammation via regulating the toll-like receptor 4 (TLR4) signaling in macrophages. Since the lipid raft recruitment and dimerization of TLR4 and its downstream adaptor molecules are vital for PA-initiated TLR4 signaling, we explored whether this process would be modulated by SSA1. Methods Murine macrophage RAW264.7 were stimulated with PA (200 μmol/L) in the presence or absence of SSA1 (40 μmol/L). The lipid raft fractions were separated by sucrose density gradient ultracentrifugation and immunoblotted with anti-flotillin-1, anti-TLR4, anti-myeloid differentiation primary response protein 88 (MyD88), or anti-Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF) antibody. Lipid rafts, TLR4, MyD88 and TRIF were fluorescently labeled and analyzed by confocal microscopy to visualize the recruitment of molecules into lipid rafts and investigate the clustering and size of lipid rafts. The complexes of TLR4/MyD88 and TLR4/TRIF were immunoprecipitated by anti-TLR4 antibody first and then immunoblotted by anti-MyD88 or anti-TRIF antibody. Results PA-induced recruitment of TLR4, MyD88 and TRIF into fractions enriched with lipid rafts marker flotillin-1 was inhibited (P &amp;lt; 0.05) by SSA1. Meanwhile, the PA-induced co-localization of TLR4, MyD88, and TRIF with lipid rafts was also decreased (P &amp;lt; 0.05) by SSA1 as visualized by confocal immunofluorescence microscopy. Furthermore, the PA-induced clustering of lipid rafts was diminished (P &amp;lt; 0.05) by SSA1. However, the PA-decreased size of lipid rafts was increased (P &amp;lt; 0.05) by SSA1. The formation of TLR4/MyD88 and TLR4/TRIF complexes was suppressed (P &amp;lt; 0.05) by SSA1, whereas the protein expressions of TLR4, MyD88 and TRIF were not changed (P &amp;gt; 0.05) by SSA1 in PA-stimulated macrophages. Conclusions SSA1 inhibits the recruitment of TLR4 and its adaptor molecules (MyD88 and TRIF) into lipid raft as well as their dimerization (TLR4/MyD88 and TLR4/TRIF) in PA-stimulated inflammatory macrophages. Funding Sources This work was supported by grants from National Natural Science Foundation of China (NSFC).