Density Gradient Ultracentrifugation Research Articles

Neonatal Human Enteroids Take Up Human Milk Extracellular Vesicles That Survive Neonatal Human Digestion

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. HM contains thousands of bioactive components, including extracellular vesicles (HMEVs). HMEVs are lipid bilayer-encased nanoparticles released from mammary epithelial cells that carry biological cargo to the infant. Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aims to develop a neonatal enteroid model and EV isolation pipeline to study HMEV-intestinal epithelial cell (IEC) interactions with the ultimate goal of therapy development. Hypothesis: HMEVs survive in vivo neonatal digestion to be taken up by infant IECs. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from post-pyloric tubes. HMEVs were isolated from raw and pasteurized human milk and digesta (n=3 each) by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. HMEV sub-populations were examined by super-resolution microscopy. HMEV uptake was tested using apical-out neonatal human enteroids isolated from a 2-month-old patient undergoing surgery for ileal atresia and validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid uptake was assessed with CMTPX dye-labeled-HMEVs, dynamin inhibitor, and confocal microscopy. Results: Our HMEV isolation pipeline reliably isolates HMEVs exhibiting classic EV morphology and enriched in extracellular vesicle markers CD81 and CD9, but depleted of b-casein and lactalbumin from HM and digesta. NTA data indicates that only 17.8 ± 2.9% (p>0.0001) of input HMEVs survive to reach the neonatal intestine. Human mammary gland-derived protein BTN1A1 is present in digesta EVs. Neonatal apical-out human enteroids represent a less proliferative and more differentiated epithelium, e.g. down-regulated KI67 (0.05 ± 0.03-fold relative to basal-out, p<0.0001, n=4-6) and elevated CHGA (14.22 ± 9.30-fold relative to basal-out, p=0.0072, n=4-6) as measured by qRT-PCR and normalized to housekeeping gene RPLP0. Neonatal enteroids take up digested HMEVs largely via dynamin-mediated endocytosis, as measured by CMTPX dye intensity normalized to enteroid area using Fiji (2.05 x 105 ± 2.95 x 104 vs 5.38 x 104 ± 6.08 x 103, p<0.0001, n=62-115 enteroids, HMEV-treatment alone and HMEVs + dynamin inhibitor, respectively).Conclusion: Our data suggest that HMEVs survive to the neonatal intestine and can be absorbed by neonatal enteroids largely via dynamin-mediated endocytosis. These data are an important first step to leveraging HMEVs and their cargo to treat neonatal intestinal inflammation. Funding: NIH K01DK129401, NIH R01HD097367, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Using molecular fine structure to identify optimal methods of extracting fungal glycogen

Glycogen is a highly branched glucose polymer that is an energy storage material in fungi and animals. Extraction of glycogen from its source in a way that minimizes its molecular degradation is essential to investigate its native structure. In this study, the following extraction methods were compared: sucrose gradient density ultracentrifugation, thermal alkali, hot alcohol and hot water extractions. Molecular-size and chain-length distributions of glycogen were measured by size-exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis, respectively. These two fine-structure features are the most likely structural characteristics to be degraded during extraction. The results show that the thermal alkali, hot alcohol and hot water extractions degrade glycogen molecular size and/or chain-length distributions, and that sucrose gradient density ultracentrifugation with neither high temperature nor alkaline treatment is the most suitable method for fungal glycogen extraction.

Isolation of Adeno-associated Viral Vectors Through a Single-step and Semi-automated Heparin Affinity Chromatography Protocol.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid.

Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.

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.

High-throughput and proteome-wide discovery of endogenous biomolecular condensates.

Phase separation inside mammalian cells regulates the formation of the biomolecular condensates that are related to gene expression, signalling, development and disease. However, a large population of endogenous condensates and their candidate phase-separating proteins have yet to be discovered in a quantitative and high-throughput manner. Here we demonstrate that endogenously expressed biomolecular condensates can be identified across a cell's proteome by sorting proteins across varying oligomeric states. We employ volumetric compression to modulate the concentrations of intracellular proteins and the degree of crowdedness, which are physical regulators of cellular biomolecular condensates. The changes in degree of the partition of proteins into condensates or phase separation led to varying oligomeric states of the proteins, which can be detected by coupling density gradient ultracentrifugation and quantitative mass spectrometry. In total, we identified 1,518 endogenous condensate proteins, of which 538 have not been reported before. Furthermore, we demonstrate that our strategy can identify condensate proteins that respond to specific biological processes.

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.

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.

Identification of specific markers for human pluripotent stem cell-derived small extracellular vesicles.

Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs.

Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization.

JOURNAL/nrgr/04.03/01300535-202410000-00027/figure1/v/2024-02-06T055622Z/r/image-tiff Spinal cord injury is a disabling condition with limited treatment options. Multiple studies have provided evidence suggesting that small extracellular vesicles (SEVs) secreted by bone marrow mesenchymal stem cells (MSCs) help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury. Strikingly, hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs (HSEVs) exhibit increased therapeutic potency. We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair. SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation. HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation. HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro. MicroRNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that miR-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1. Reducing miR-146a-5p expression in HSEVs partially attenuated macrophage polarization. Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting miR-146a-5p, which alters macrophage polarization. This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Human placenta releases extracellular vesicles carrying corticotrophin releasing hormone mRNA into the maternal blood

The human placenta releases diverse extracellular vesicles (EVs), including microvesicles (100–1000 nm) and exosomes (30–150 nm), into the maternal blood for feto-maternal communication. Exosomes and microvesicles contribute to normal pregnancy physiology and major pregnancy pathologies. Differences in miRNA expressions and protein content in placental exosomes have been reported in complicated pregnancies. During human pregnancy, Corticotropin-Releasing Hormone (CRH) is produced and released by the placenta into the maternal blood. CRH is involved in regulating gestational length and the initiation of labour. CRH mRNA levels in the maternal plasma rise with gestation. High levels of CRH mRNA are reported to be associated with preeclamptic and preterm pregnancies. However, the underlying mechanism of placental CRH mRNA secretion remains to be elucidated. We hypothesise that the placenta releases CRH mRNA packaged within extracellular vesicles (EVs) into the maternal blood. In this study, placental EVs (microvesicles and exosomes) were isolated from human term healthy placentas via villus washes and from explant culture media by differential centrifugation and purified by density gradient ultracentrifugation using a continuous sucrose gradient (0.25–2.5 M). Western blotting using placenta- and exosome-specific markers and electron microscopy confirmed exosomes and microvesicles in the placental wash and explant media samples. Real-time quantitative RT-PCR data detected CRH mRNA in placenta-derived EVs from placental washes and explants. We also sorted placenta-secreted EVs in maternal plasma samples (≥37 weeks) by high-resolution flow cytometry using a fluorescent-labelled PLAP antibody. CRH mRNA was demonstrated in placental EVs obtained from maternal blood plasma. We therefore show that human placental EVs carry CRH mRNA into the maternal blood. Our study implies that measuring CRH mRNA in placental EVs in the maternal plasma could beused for monitoring pregnancy.

Applications and Limitations of Equilibrium Density Gradient Analytical Ultracentrifugation for the Quantitative Characterization of Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are produced as a mixture of the desired particle (full particle, FP), which is filled with the designed DNA, product-related impurities such as particle without DNA (empty particle, EP), and aggregates. Cesium chloride or iodixanol equilibrium density gradient ultracentrifugation (DGE-UC) has been used for the purification of AAV vectors. DGE-UC can separate FP from impurities based on the difference in their buoyant densities. Here, we report the applications and limitations of equilibrium density gradient analytical ultracentrifugation (DGE-AUC) using a modern AUC instrument that employs DGE-UC principles for the characterization and quantitation of AAV vectors. We evaluated the quantitative ability of DGE-AUC in comparison with sedimentation velocity AUC (SV-AUC) or band sedimentation AUC (BS-AUC) using AAVs with different DNA lengths and different serotypes. DGE-AUC enabled the accurate quantification of the ratio of FP to EP when the AAV vector primarily contains these particles. Furthermore, we developed a new workflow to identify the components of separated peaks in addition to FP and EP. Ultraviolet absorption spectra obtained by multiwavelength detection can also support peak assignment following component identification. DGE-AUC experiments for AAV vectors have limitations with regard to minor components with low absorption at the detected wavelength or those with a density similar to that of major components of AAV vectors. DGE-AUC is the only analytical method that can evaluate particle density heterogeneity; therefore, SV-AUC or BS-AUC and DGE-AUC are complementary methods for reliable assessment of the purity of AAV vectors.

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.

Acute myocardial infarction preferentially alters low-abundant, long-chain unsaturated phospholipid and sphingolipid species in plasma high-density lipoprotein subpopulations

AimHigh-density lipoprotein (HDL) particles in ST-segment elevation myocardial infarction (STEMI) are deficient in their anti-atherogenic function. Molecular determinants of such deficiency remain obscure. MethodsFive major HDL subpopulations were isolated using density-gradient ultracentrifugation from STEMI patients (n = 12) and healthy age- and sex-matched controls (n = 12), and 160 species of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, sphingomyelin and ceramide were quantified by LC-MS/MS. ResultsMultiple minor species of proinflammatory phosphatidic acid and lysophosphatidylcholine were enriched by 1.7–27.2-fold throughout the majority of HDL subpopulations in STEMI. In contrast, minor phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, sphingomyelin and ceramide species were typically depleted up to 3-fold in STEMI vs. control HDLs, while abundances of their major species did not differ between the groups. Intermediate-to-long-chain phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol species were more affected by STEMI than their short-chain counterparts, resulting in positive correlations between their fold decrease and the carbon chain length. Additionally, fold decreases in the abundances of multiple lipid species were positively correlated with the double bond number in their carbon chains. Finally, abundances of several phospholipid and ceramide species were positively correlated with cholesterol efflux capacity and antioxidative activity of HDL subpopulations, both reduced in STEMI vs controls. KEGG pathway analysis tied these species to altered glycerophospholipid and linoleic acid metabolism. ConclusionsMinor unsaturated intermediate-to-long-chain phospholipid and sphingolipid species in HDL subpopulations are most affected by STEMI, reflecting alterations in glycerophospholipid and linoleic acid metabolism with the accumulation of proinflammatory lysolipids and maintenance of homeostasis of major phospholipid species.

Optimizing Conditions for the Production of Bacterial Extracellular Vesicles of Vibrio vulnificus and Analysis of the Inner Small RNA Compositions.

Chemical and physical elements affecting the production of bacterial extracellular vesicles (BEVs) of the human pathogen Vibrio vulnificus were quantitatively assessed to optimize the conditions for the BEV production by using the western blot quantification for an outer membrane porin OmpU and by fluorescent dye FM4-64. When cells were cultured at 37°C in an enriched medium (2 × Luria Bertani; 2 × LB) in the presence of EDTA, they produced about 70% more BEVs. BEVs were purified from the cells cultured in the established optimal conditions by the density gradient ultracentrifugation. The dynamic light scattering measurement of the purified BEVs showed that the diameter of them ranged from approximately 25 nm to 161 nm. We hypothesized that there may be some features in nucleotide sequences specific to RNAs packaged in BEVs compared to those in cellular RNA molecules. We compared the nucleotide sequences and abundance of sRNAs between in the cellular fraction and in BEVs through next-generation sequencing (NGS). While no distinct feature was observed in the nucleotide sequences of sRNAs between two groups, the length of sRNA fragments from BEVs were significantly shorter than those in cytoplasm.

Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes.

Lysosomes are degradative organelles that facilitate the removal and recycling of potentially cytotoxic materials and mediate a variety of other cellular processes, such as nutrient sensing, intracellular signaling, and lipid metabolism. Due to these central roles, lysosome dysfunction can lead to deleterious outcomes, including the accumulation of cytotoxic material, inflammation, and cell death. We previously reported that cationic amphiphilic drugs, such as imipramine, alter pH and lipid metabolism within macrophage lysosomes. Therefore, the ability for imipramine to induce changes to the lipid content of isolated macrophage lysosomes was investigated, focusing on sphingomyelin, cholesterol, and glycerophospholipid metabolism as these lipid classes have important roles in inflammation and disease. The lysosomes were isolated from control and imipramine-treated macrophages using density gradient ultracentrifugation, and mass spectrometry was used to measure the changes in their lipid composition. An unsupervised hierarchical cluster analysis revealed a clear differentiation between the imipramine-treated and control lysosomes. There was a significant overall increase in the abundance of specific lipids mostly composed of cholesterol esters, sphingomyelins, and phosphatidylcholines, while lysophosphatidylcholines and ceramides were overall decreased. These results support the conclusion that imipramine's ability to change the lysosomal pH inhibits multiple pH-sensitive enzymes in macrophage lysosomes.

Proteome and immune responses of extracellular vesicles derived from macrophages infected with the periodontal pathogen Tannerella forsythia.

Periodontitis is a chronic inflammatory disease caused by periodontal pathogens in subgingival plaque and is associated with systemic inflammatory diseases. Extracellular vesicles (EVs) released from host cells and pathogens carry a variety of biological molecules and are of interest for their role in disease progression and as diagnostic markers. In the present study, we analysed the proteome and inflammatory response of EVs derived from macrophages infected with Tannerella forsythia, a periodontal pathogen. The EVs isolated from the cell conditioned medium of T. forsythia-infected macrophages were divided into two distinct vesicles, macrophage-derived EVs and T. forsythia-derived OMVs, by size exclusion chromatography combined with density gradient ultracentrifugation. Proteome analysis showed that in T. forsythia infection, macrophage-derived EVs were enriched with pro-inflammatory cytokines and inflammatory mediators associated with periodontitis progression. T. forsythia-derived OMVs harboured several known virulence factors, including BspA, sialidase, GroEL and various bacterial lipoproteins. T. forsythia-derived OMVs induced pro-inflammatory responses via TLR2 activation. In addition, we demonstrated that T. forsythia actively released OMVs when T. forsythia encountered macrophage-derived soluble molecules. Taken together, our results provide insight into the characterisation of EVs derived from cells infected with a periodontal pathogen.

Rapid plasma membrane isolation via intracellular polymerization-mediated biomolecular confinement

Plasma membrane isolation is a foundational process in membrane proteomic research, cellular vesicle studies, and biomimetic nanocarrier development, yet separation processes for this outermost layer are cumbersome and susceptible to impurities and low yield. Herein, we demonstrate that cellular cytosol can be chemically polymerized for decoupling and isolation of plasma membrane within minutes. A rapid, non-disruptive in situ polymerization technique is developed with cell membrane-permeable polyethyleneglycol-diacrylate (PEG-DA) and a blue-light-sensitive photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The photopolymerization chemistry allows for precise control of intracellular polymerization and tunable confinement of cytosolic molecules. Upon cytosol solidification, plasma membrane proteins and vesicles are rapidly derived and purified as nucleic acids and intracellular proteins as small as 15 kDa are stably entrapped for removal. The polymerization chemistry and membrane derivation technique are broadly applicable to primary and fragile cell types, enabling facile membrane vesicle extraction from shorted-lived neutrophils and human primary CD8 T cells. The study demonstrates tunable intracellular polymerization via optimized live cell chemistry, offers a robust membrane isolation methodology with broad biomedical utility, and reveals insights on molecular crowding and confinement in polymerized cells. Statement of significanceIsolating the minute fraction of plasma membrane proteins and vesicles requires extended density gradient ultracentrifugation processes, which are susceptible to low yield and impurities. The present work demonstrates that the membrane isolation process can be vastly accelerated via a rapid, non-disruptive intracellular polymerization approach that decouples cellular cytosols from the plasma membrane. Following intracellular polymerization, high-yield plasma membrane proteins and vesicles can be derived from lysis buffer and sonication treatment, respectively. And the intracellular content entrapped within the polymerized hydrogel is readily removed within minutes. The technique has broad utility in membrane proteomic research, cellular vesicle studies, and biomimetic materials development, and the work offers insights on intracellular hydrogel-mediated molecular confinement.

Multi-omics analyses reveal the importance of chromoplast plastoglobules in carotenoid accumulation in citrus fruit.

Chromoplasts act as a metabolic sink for carotenoids, in which plastoglobules serve as versatile lipoprotein particles. PGs in chloroplasts have been characterized. However, the features of PGs from non-photosynthetic plastids are poorly understood. We found that the development of chromoplast plastoglobules (CPGs) in globular and crystalloid chromoplasts of citrus is associated with alterations in carotenoid storage. Using Nycodenz density gradient ultracentrifugation, an efficient protocol for isolating highly purified CPGs from sweet orange (Citrus sinensis) pulp was established. Forty-four proteins were defined as likely comprise the core proteome of CPGs using comparative proteomics analysis. Lipidome analysis of different chromoplast microcompartments revealed that the nonpolar microenvironment within CPGs was modified by 35 triacylglycerides, two sitosterol esters, and one stigmasterol ester. Manipulation of the CPG-localized gene CsELT1 (esterase/lipase/thioesterase) in citrus calli resulted in increased lipids and carotenoids, which is further evidence that the nonpolar microenvironment of CPGs contributes to carotenoid accumulation and storage in the chromoplasts. This multi-feature analysis of CPGs sheds new light on the role of chromoplasts in carotenoid metabolism, paving the way for manipulating carotenoid content in citrus fruit and other crops.

Validating the NIH LDL-C equation for provincial implementation in Alberta

BackgroundLDL-C, a cardiovascular disease risk assessment biomarker, is commonly calculated using the Friedewald equation. The NIH equation overcomes several limitations of the Friedewald equation. Consistent with the Canadian Society of Clinical Chemists (CSCC) lipid reporting recommendations, we assessed the NIH LDL-C equation in Alberta prior to its provincial implementation. Methods1-year (01/01/2021–12/31/2021) of lipid results (n = 1,486,584 after data cleaning) were obtained from five analytical instrument groups used across Alberta. Analyses were performed on all data and after separating by age, analytical instrument group, and fasting status. The correlation between Friedewald- and NIH-calculated LDL-C and between Friedewald- and NIH-calculated LDL-C difference and each lipid parameter, was determined. The frequency of unreportable/inaccurate LDL-C results was compared between the two equations. The concordance between the two equations and with non-HDL-C was determined at LDL-C thresholds. Lastly, LDL-C calculated by Friedewald, NIH, and Martin-Hopkins equations was compared to density-gradient ultracentrifugation. ResultsFriedewald- and NIH-calculated LDL-C exhibit the strongest correlation when triglycerides ≤ 4.52 mmol/L. The difference between Friedewald- and NIH-calculated LDL-C increases with decreasing LDL-C concentration. The NIH equation yields fewer inaccurate results (0.35 % vs. 22.0 %). The percent agreement between equations was > 96 % at all LDL-C thresholds, suggesting most patients will not require treatment changes. NIH-calculated LDL-C exhibited better agreement with non-HDL-C when triglycerides ≤ 9.04 mmol/L and better correlated with LDL-C measured by ultracentrifugation (r2 = 0.926 vs. 0.775 (Friedewald) and 0.863 (Martin-Hopkins)). Results were consistent across age, analytical instrument group, and fasting status. ConclusionsOur findings demonstrate the benefits of implementing the NIH equation across Alberta.