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Journal of Extracellular Biology Jan 2024Extracellular vesicles (EVs) are membranous structures released by cells into the extracellular space and are thought to be involved in cell-to-cell communication. While...
Extracellular vesicles (EVs) are membranous structures released by cells into the extracellular space and are thought to be involved in cell-to-cell communication. While EVs and their cargo are promising biomarker candidates, sorting mechanisms of proteins to EVs remain unclear. In this study, we ask if it is possible to determine EV association based on the protein sequence. Additionally, we ask what the most important determinants are for EV association. We answer these questions with explainable AI models, using human proteome data from EV databases to train and validate the model. It is essential to correct the datasets for contaminants introduced by coarse EV isolation workflows and for experimental bias caused by mass spectrometry. In this study, we show that it is indeed possible to predict EV association from the protein sequence: a simple sequence-based model for predicting EV proteins achieved an area under the curve of 0.77 ± 0.01, which increased further to 0.84 ± 0.00 when incorporating curated post-translational modification (PTM) annotations. Feature analysis shows that EV-associated proteins are stable, polar, and structured with low isoelectric point compared to non-EV proteins. PTM annotations emerged as the most important features for correct classification; specifically, palmitoylation is one of the most prevalent EV sorting mechanisms for unique proteins. Palmitoylation and nitrosylation sites are especially prevalent in EV proteins that are determined by very strict isolation protocols, indicating they could potentially serve as quality control criteria for future studies. This computational study offers an effective sequence-based predictor of EV associated proteins with extensive characterisation of the human EV proteome that can explain for individual proteins which factors contribute to their EV association.
PubMed: 38938677
DOI: 10.1002/jex2.120 -
Journal of Extracellular Biology Jan 2024Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between...
Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between cells. EVs contain microRNAs and various other types of non-coding RNA, of which Y RNA is among the most abundant types. Studies on how RNAs and their binding proteins are sorted into EVs have mainly focused on comparing intracellular (cytoplasmic) levels of these RNAs to the extracellular levels in EVs. Besides overall transcriptional levels that may regulate sorting of RNAs into EVs, the process may also be driven by local intracellular changes in RNA/RBP concentrations. Changes in extracellular Y RNA have been linked to cancer and cardiovascular diseases. Although the loading of RNA cargo into EVs is generally thought to be influenced by cellular stimuli and regulated by RNA binding proteins (RBP), little is known about Y RNA shuttling into EVs. We previously reported that immune stimulation alters the levels of Y RNA in EVs independently of cytosolic Y RNA levels. This suggests that Y RNA binding proteins, and/or changes in the local Y RNA concentration at EV biogenesis sites, may affect Y RNA incorporation into EVs. Here, we investigated the subcellular distribution of Y RNA and Y RNA binding proteins in activated and non-activated THP1 macrophages. We demonstrate that Y RNA and its main binding protein Ro60 abundantly co-fractionate in organelles involved in EV biogenesis and in EVs. Cellular activation led to an increase in Y RNA concentration at EV biogenesis sites and this correlated with increased EV-associated levels of Y RNA and Ro60. These results suggest that Y RNA incorporation into EVs may be controlled by local intracellular changes in the concentration of Y RNA and their protein binding partners.
PubMed: 38938676
DOI: 10.1002/jex2.123 -
Journal of Extracellular Biology Jan 2024Human milk extracellular vesicles (HM EVs) are proposed to protect against disease development in infants. This protection could in part be facilitated by the bioactive...
Human milk extracellular vesicles (HM EVs) are proposed to protect against disease development in infants. This protection could in part be facilitated by the bioactive EV cargo of proteins and RNA. Notably, mothers birth infants of different gestational ages with unique needs, wherein the EV cargo of HM may diverge. We collected HM from lactating mothers within two weeks of a term or preterm birth. Following purification of EVs, proteins and mRNA were extracted for proteomics and sequencing analyses, respectively. Over 2000 protein groups were identified, and over 8000 genes were quantified. The total number of proteins and mRNA did not differ significantly between the two conditions, while functional bioinformatics of differentially expressed cargo indicated enrichment in immunoregulatory cargo for preterm HM EVs. In term HM EVs, significantly upregulated cargo was enriched in metabolism-related functions. Based on gene expression signatures from HM-contained single cell sequencing data, we proposed that a larger portion of preterm HM EVs are secreted by immune cells, whereas term HM EVs contain more signatures of lactocyte epithelial cells. Proposed differences in EV cargo could indicate variation in mother's milk based on infants' gestational age and provide basis for further functional characterisation.
PubMed: 38938674
DOI: 10.1002/jex2.128 -
Journal of Extracellular Biology Sep 2023Extracellular vesicles (EVs) are potentially useful biomarkers for disease detection and monitoring. Development of a label-free technique for imaging and distinguishing...
Extracellular vesicles (EVs) are potentially useful biomarkers for disease detection and monitoring. Development of a label-free technique for imaging and distinguishing small volumes of EVs from different cell types and cell states would be of great value. Here, we have designed a method to explore the chemical changes in EVs associated with neuroinflammation using Time-of-Flight Secondary Ion Mass spectrometry (ToF-SIMS) and machine learning (ML). Mass spectral imaging was able to identify and differentiate EVs released by microglia following lipopolysaccharide (LPS) stimulation compared to a control group. This process requires a much smaller sample size (1 µL) than other molecular analysis methods (up to 50 µL). Conspicuously, we saw a reduction in free cysteine thiols (a marker of cellular oxidative stress associated with neuroinflammation) in EVs from microglial cells treated with LPS, consistent with the reduced cellular free thiol levels measured experimentally. This validates the synergistic combination of ToF-SIMS and ML as a sensitive and valuable technique for collecting and analysing molecular data from EVs at high resolution.
PubMed: 38938371
DOI: 10.1002/jex2.110 -
International Journal of Analytical... 2024An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to determine the concentrations of Rhodojaponin II and Rhodojaponin...
An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to determine the concentrations of Rhodojaponin II and Rhodojaponin III in rat plasma, and their pharmacokinetic profiles were investigated. A UPLC HSS T3 (2.1 mm × 50 mm, 1.8 m) chromatographic column was employed at a temperature of 40°C. The mobile phase consisted of acetonitrile-0.1% formic acid in water, and a gradient elution method with an elution time of 6 min and flow rate of 0.4 mL/min was utilized for analysis purposes. Methodological investigations were conducted accordingly. The plasma concentrations of Rhodojaponin II and Rhodojaponin III exhibited excellent linearity within the range of 2 ng/mL-1250 ng/mL. Moreover, both intraday and interday precision were below 15%, while accuracy ranged from 88% to 115%. Additionally, matrix effect fell within the range of 90%-110%, and recoveries ranged from 78% to 87%. These results comply with relevant regulations for drug analysis in biological samples. Therefore, this method is deemed suitable for quantifying Rhodojaponin II and Rhodojaponin III levels in rats.
PubMed: 38938264
DOI: 10.1155/2024/6386570 -
Military Medical Research Jun 2024Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with...
BACKGROUND
Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet β cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed.
METHODS
db/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein-protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays.
RESULTS
In livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers.
CONCLUSIONS
PANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A's suppressive effects on hepatic gluconeogenesis and lipogenesis.
Topics: Animals; Connexins; Mice; Gluconeogenesis; Nerve Tissue Proteins; Adenosine Triphosphate; Lipogenesis; Liver; Mice, Knockout; Male; Humans; Diet, High-Fat; Cytokines
PubMed: 38937853
DOI: 10.1186/s40779-024-00543-6 -
Biotechnology For Biofuels and... Jun 2024Sophorolipids (SLs) are a class of natural, biodegradable surfactants that found their way as ingredients for environment friendly cleaning products, cosmetics and...
BACKGROUND
Sophorolipids (SLs) are a class of natural, biodegradable surfactants that found their way as ingredients for environment friendly cleaning products, cosmetics and nanotechnological applications. Large-scale production relies on fermentations using the yeast Starmerella bombicola that naturally produces high titers of SLs from renewable resources. The resulting product is typically an extracellular mixture of acidic and lactonic congeners. Previously, we identified an esterase, termed Starmerella bombicola lactone esterase (SBLE), believed to act as an extracellular reverse lactonase to directly use acidic SLs as substrate.
RESULTS
We here show based on newly available pure substrates, HPLC and mass spectrometric analysis, that the actual substrates of SBLE are in fact bola SLs, revealing that SBLE actually catalyzes an intramolecular transesterification reaction. Bola SLs contain a second sophorose attached to the fatty acyl group that acts as a leaving group during lactonization.
CONCLUSIONS
The biosynthetic function by which the Starmerella bombicola 'lactone esterase' converts acidic SLs into lactonic SLs should be revised to a 'transesterase' where bola SL are the true intermediate. This insights paves the way for alternative engineering strategies to develop designer surfactants.
PubMed: 38937850
DOI: 10.1186/s13068-024-02533-1 -
Journal of Experimental & Clinical... Jun 2024Triple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are...
BACKGROUND
Triple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore.
METHODS
We investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan-Meier plotter database and clinical breast cancer tissue samples.
RESULTS
We demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3β-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo.
CONCLUSIONS
Our findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.
Topics: Humans; Triple Negative Breast Neoplasms; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Mice; Animals; Organelle Biogenesis; Phosphorylation; Glycogen Synthase Kinase 3 beta; Naphthoquinones; Female; Cancer-Associated Fibroblasts; Cell Line, Tumor; Mice, SCID; Neoplasm Metastasis; Mice, Inbred NOD; Mitochondria; Xenograft Model Antitumor Assays
PubMed: 38937832
DOI: 10.1186/s13046-024-03101-z -
Lipids in Health and Disease Jun 2024Traumatic brain injury (TBI) causes neuroinflammation and can lead to long-term neurological dysfunction, even in cases of mild TBI (mTBI). Despite the substantial...
BACKGROUND
Traumatic brain injury (TBI) causes neuroinflammation and can lead to long-term neurological dysfunction, even in cases of mild TBI (mTBI). Despite the substantial burden of this disease, the management of TBI is precluded by an incomplete understanding of its cellular mechanisms. Sphingolipids (SPL) and their metabolites have emerged as key orchestrators of biological processes related to tissue injury, neuroinflammation, and inflammation resolution. No study so far has investigated comprehensive sphingolipid profile changes immediately following TBI in animal models or human cases. In this study, sphingolipid metabolite composition was examined during the acute phases in brain tissue and plasma of mice following mTBI.
METHODS
Wildtype mice were exposed to air-blast-mediated mTBI, with blast exposure set at 50-psi on the left cranium and 0-psi designated as Sham. Sphingolipid profile was analyzed in brain tissue and plasma during the acute phases of 1, 3, and 7 days post-TBI via liquid-chromatography-mass spectrometry. Simultaneously, gene expression of sphingolipid metabolic markers within brain tissue was analyzed using quantitative reverse transcription-polymerase chain reaction. Significance (P-values) was determined by non-parametric t-test (Mann-Whitney test) and by Tukey's correction for multiple comparisons.
RESULTS
In post-TBI brain tissue, there was a significant elevation of 1) acid sphingomyelinase (aSMase) at 1- and 3-days, 2) neutral sphingomyelinase (nSMase) at 7-days, 3) ceramide-1-phosphate levels at 1 day, and 4) monohexosylceramide (MHC) and sphingosine at 7-days. Among individual species, the study found an increase in C18:0 and a decrease in C24:1 ceramides (Cer) at 1 day; an increase in C20:0 MHC at 3 days; decrease in MHC C18:0 and increase in MHC C24:1, sphingomyelins (SM) C18:0, and C24:0 at 7 days. Moreover, many sphingolipid metabolic genes were elevated at 1 day, followed by a reduction at 3 days and an absence at 7-days post-TBI. In post-TBI plasma, there was 1) a significant reduction in Cer and MHC C22:0, and an increase in MHC C16:0 at 1 day; 2) a very significant increase in long-chain Cer C24:1 accompanied by significant decreases in Cer C24:0 and C22:0 in MHC and SM at 3 days; and 3) a significant increase of C22:0 in all classes of SPL (Cer, MHC and SM) as well as a decrease in Cer C24:1, MHC C24:1 and MHC C24:0 at 7 days.
CONCLUSIONS
Alterations in sphingolipid metabolite composition, particularly sphingomyelinases and short-chain ceramides, may contribute to the induction and regulation of neuroinflammatory events in the early stages of TBI, suggesting potential targets for novel diagnostic, prognostic, and therapeutic strategies in the future.
Topics: Animals; Mice; Sphingolipids; Brain; Ceramides; Sphingomyelin Phosphodiesterase; Sphingosine; Disease Models, Animal; Male; Sphingomyelins; Brain Concussion; Mice, Inbred C57BL; Brain Injuries, Traumatic; Lysophospholipids
PubMed: 38937745
DOI: 10.1186/s12944-024-02186-x -
BMC Microbiology Jun 2024This study investigated the influence of bacterial cyclic lipopeptides (LP; surfactins, iturins, fengycins) on microbial interactions. The objective was to investigate...
This study investigated the influence of bacterial cyclic lipopeptides (LP; surfactins, iturins, fengycins) on microbial interactions. The objective was to investigate whether the presence of bacteria inhibits fungal growth and whether this inhibition is due to the release of bacterial metabolites, particularly LP. Selected endophytic bacterial strains with known plant-growth promoting potential were cultured in the presence of Fusarium oxysporum f.sp. strigae (Fos), which was applied as model fungal organism. The extracellular metabolome of tested bacteria, with a focus on LP, was characterized, and the inhibitory effect of bacterial LP on fungal growth was investigated. The results showed that Bacillus velezensis GB03 and FZB42, as well as B. subtilis BSn5 exhibited the strongest antagonism against Fos. Paraburkholderia phytofirmans PsJN, on the other hand, tended to have a slight, though non-significant growth promotion effect. Crude LP from strains GB03 and FZB42 had the strongest inhibitory effect on Fos, with a significant inhibition of spore germination and damage of the hyphal structure. Liquid chromatography tandem mass spectrometry revealed the production of several variants of iturin, fengycin, and surfactin LP families from strains GB03, FZB42, and BSn5, with varying intensity. Using plate cultures, bacillomycin D fractions were detected in higher abundance in strains GB03, FZB42, and BSn5 in the presence of Fos. Additionally, the presence of Fos in dual plate culture triggered an increase in bacillomycin D production from the Bacillus strains. The study demonstrated the potent antagonistic effect of certain Bacillus strains (i.e., GB03, FZB42, BSn5) on Fos development. Our findings emphasize the crucial role of microbial interactions in shaping the co-existence of microbial assemblages.
Topics: Fusarium; Lipopeptides; Bacillus; Antibiosis; Antifungal Agents; Peptides, Cyclic; Microbial Interactions; Burkholderiaceae; Spores, Fungal; Hyphae
PubMed: 38937715
DOI: 10.1186/s12866-024-03386-2