Ultracentrifugation Research Articles

Optimization of ultracentrifugation-based method to enhance the purity and proteomic profiling depth of plasma-derived extracellular vesicles and particles.

Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of EVPs isolated from peripheral blood plasma has posed a challenge of in-depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)-based protocols, phosphatidylserine-Tim4 interaction-based affinity capture (referred to as "PS"), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC-based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC-based protocols for circulating EVP isolation, which enable further in-depth proteomic analysis for biomarker discovery.

Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications.

Anti-diabetic active constituents of pomegranate peel-derived extracellular nanovesicles

Pomegranate peel-derived extracellular nanovesicles(PPENs) were isolated and purified by ultra-high speed centrifugation and sucrose density gradient centrifugation. Their morphology and structure were characterized. In vitro α-glucosidase inhibition assay and model test of insulin resistance(IR) in HepG2 cells showed that PPENs had good anti-diabetic activity. The IC_(50) value of α-glucosidase inhibition was(35.3±1.1) μg·mL~(-1), significantly better than the positive drug acarbose. At a concentration of 100 μg·mL~(-1), PPENs could increase the glucose absorption of IR cells significantly. Lipidome, proteome, and metabolite analysis of PPENs were performed using chromatography-mass spectrometry. MicroRNA(miRNA) sequences were identified, and target genes of miRNA were predicted. The analysis results indicated that PPENs contained abundant lipids and transport proteins, providing a material basis for the transportation and distribution of PPENs in tissue. Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis suggested that lipids and miRNAs may be the key components of PPENs to exert anti-diabetic activity.

Extracellular vesicles from primary human macrophages stimulated with VIP or PACAP mediate anti-SARS-CoV-2 activities in monocytes through NF-κB signaling pathway

Infection by SARS-CoV-2 is associated with uncontrolled inflammatory response during COVID-19 severe disease, in which monocytes are one of the main sources of pro-inflammatory mediators leading to acute respiratory distress syndrome. Extracellular vesicles (EVs) from different cells play important roles during SARS-CoV-2 infection, but investigations describing the involvement of EVs from primary human monocyte-derived macrophages (MDM) on the regulation of this infection are not available. Here, we describe the effects of EVs released by MDM stimulated with the neuropeptides VIP and PACAP on SARS-CoV-2-infected monocytes. MDM-derived EVs were isolated by differential centrifugation of medium collected from cells cultured for 24 h in serum-reduced conditions. Based on morphological properties, we distinguished two subpopulations of MDM-EVs, namely large (LEV) and small EVs (SEV). We found that MDM-derived EVs stimulated with the neuropeptides inhibited SARS-CoV-2 RNA synthesis/replication in monocytes, protected these cells from virus-induced cytopathic effects and reduced the production of pro-inflammatory mediators. In addition, EVs derived from VIP- and PACAP-treated MDM prevented the SARS-CoV-2-induced NF-κB activation. Overall, our findings suggest that MDM-EVs are endowed with immunoregulatory properties that might contribute to the antiviral and anti-inflammatory responses in SARS-CoV-2-infected monocytes and expand our knowledge of EV effects during COVID-19 pathogenesis.

Gradual change and mosaic pattern of macrofaunal assemblages along depth and sediment gradients in a tropical oceanic island

Gradual change and mosaic pattern of macrofaunal assemblages along depth and sediment gradients in a tropical oceanic island

Comparing the Proteomic Profiles of Extracellular Vesicles Isolated using Different Methods from Long-term Stored Plasma Samples

BackgroundThe lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes.ResultsEVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusion chromatography (SEC), or SEC and UC combined (SEC + UC). The EV isolates were then characterized by transmission electron microscopy imaging, nanoparticle tracking analysis (NTA) and western blotting. Proteomic procedures were used to analyze protein contents. The presence of EV markers in all isolates was confirmed by western blotting yet this analysis revealed higher albumin expression in EVs-UC, suggesting plasma protein contamination. Proteomic analysis identified 542 proteins, SEC + UC yielding the most complex proteome at 364 proteins. Through gene ontology enrichment, we observed differences in the cellular components of EVs and plasma in that SEC + UC isolates featured higher proportions of EV proteins than those derived from the other two methods. Analysis of proteins unique to each isolation method served to identify 181 unique proteins for the combined approach, including those normally appearing in low concentrations in plasma. This indicates that with this combined method, it is possible to detect less abundant plasma proteins by proteomics in the resultant isolates.ConclusionsOur findings reveal that the SEC + UC approach yields highly pure and diverse EVs suitable for comprehensive proteomic analysis with applications for the detection of new biomarkers in biobank-stored plasma samples.

"Modeling Diffusive Search by Non-Adaptive Sperm: Empirical and Computational Insights".

During fertilization, mammalian sperm undergo a winnowing selection process that reduces the candidate pool of potential fertilizers from ~106-1011 cells to 101-102 cells (depending on the species). Classical sperm competition theory addresses the positive or 'stabilizing' selection that acts on sperm phenotypes within populations of organisms but does not strictly address the developmental consequences of sperm traits among individual organisms that are under purifying selection during fertilization. It is the latter that is of utmost concern for improving assisted reproductive technologies (ART) because 'low fitness' sperm may be inadvertently used for fertilization during interventions that rely heavily on artificial sperm selection, such as intracytoplasmic sperm injection (ICSI). Importantly, some form of sperm selection is used in nearly all forms of ART (e.g., differential centrifugation, swim-up, or hyaluronan binding assays, etc.). To date, there is no unifying quantitative framework (i.e., theory of sperm selection) that synthesizes causal mechanisms of selection with observed natural variation in individual sperm traits. In this report, we reframe the physiological function of sperm as a collective diffusive search process and develop multi-scale computational models to explore the causal dynamics that constrain sperm 'fitness' during fertilization. Several experimentally useful concepts are developed, including a probabilistic measure of sperm 'fitness' as well as an information theoretic measure of the magnitude of sperm selection, each of which are assessed under systematic increases in microenvironmental selective pressure acting on sperm motility patterns.

Variation of sperm quality and circular RNA content in men exposed to environmental contamination with heavy metals in 'Land of Fires', Italy.

Can illegal discharge of toxic waste into the environment induce a new condition of morpho-epigenetic pathozoospermia in normozoospermic young men? Toxic environmental contaminants promote the onset of a new pathozoospermic condition in young normozoospermic men, consisting of morpho-functional defects and a sperm increase of low-quality circular RNA (circRNA) cargo, tightly linked to contaminant bioaccumulation in seminal plasma. Epidemiological findings have reported several reproductive anomalies depending on exposure to contaminants discharged into the environment, such as germ cell apoptosis, steroidogenesis defects, oxidative stress induction, blood-testis barrier dysfunctions, and poor sperm quality onset. In this scenario, a vast geographical area located in Campania, Italy, called the 'Land of Fires', has been associated with an excessive illegal discharge of toxic waste into the environment, negatively impacting human health, including male reproductive functions. Semen samples were obtained from healthy normozoospermic men divided into two experimental groups, consisting of men living in the 'Land of Fires' (LF; n = 80) or not (CTRL; n = 80), with age ranging from 25 to 40 years. The study was carried out following World Health Organization guidelines. Quality parameters of semen from CTRL- and LF-normozoospermic men were evaluated by computer-assisted semen analysis; high-quality spermatozoa from CTRL and LF groups (n = 80 for each experimental group) were obtained using a 80-40% discontinuous centrifugation gradient. Seminal plasma was collected following centrifugation and used for the dosage of chemical elements, dioxins and steroid hormones by liquid chromatography with tandem mass spectrometry. Sperm morpho-functional investigations (cellular morphology, acrosome maturation, IZUMO1 fertility marker analysis, plasma membrane lipid state, oxidative stress) were assessed on the purified high-quality spermatozoa fraction by immunochemistry/immunofluorescence and western blot analyses. Sperm circRNA cargo was evaluated by quantitative RT-PCR, and the physical interaction among circRNAs and fused in sarcoma (FUS) protein was detected using an RNA-binding protein immunoprecipitation assay. Protein immunoprecipitation experiments were carried out to demonstrate FUS/p-300 protein interaction in sperm cells. Lastly, in vitro lead (Pb) treatment of high-quality spermatozoa collected from normozoospermic controls was used to investigate a correlation between Pb accumulation and onset of the morpho-epigenetic pathozoospermic phenotype. Several morphological defects were identified in LF-spermatozoa, including: a significant increase (P < 0.05 versus CTRL) in the percentage of spermatozoa characterized by structural defects in sperm head and tail; and a high percentage (P < 0.01) of peanut agglutinin and IZUMO1 null signal cells. In agreement with these data, abnormal steroid hormone levels in LF seminal plasma suggest a premature acrosome reaction onset in LF-spermatozoa. The abnormal immunofluorescence signals of plasma membrane cholesterol complexes/lipid rafts organization (Filipin III and Flotillin-1) and of oxidative stress markers [3-nitrotyrosine and 3-nitrotyrosine and 4-hydroxy-2-nonenal] observed in LF-spermatozoa and associated with a sperm motility reduction (P < 0.01), demonstrated an affected membrane fluidity, potentially impacting sperm motility. Bioaccumulation of heavy metals and dioxins occurring in LF seminal plasma and a direct correlation between Pb and deregulated circRNAs related to high- and low-sperm quality was also revealed. In molecular terms, we demonstrated that Pb bioaccumulation promoted FUS hyperacetylation via physical interaction with p-300 and, in turn, its shuttling from sperm head to tail, significantly enhancing (P < 0.01 versus CTRL) the endogenous backsplicing of sperm low-quality circRNAs in LF-spermatozoa. Participants were interviewed to better understand their area of origin, their eating habits as well as their lifestyles, however any information incorrectly communicated or voluntarily omitted that could potentially compromise experimental group determination cannot be excluded. A possible association between seminal Pb content and other heavy metals in modulating sperm quality should be explored further. Future investigations will be performed in order to identify potential synergistic or anti-synergistic effects of heavy metals on male reproduction. Our study provides new findings regarding the effects of environmental contaminants on male reproduction, highlighting how a sperm phenotype classified as normozoospermic may potentially not match with a healthy morpho-functional and epigenetic one. Overall, our results improve the knowledge to allow a proper assessment of sperm quality through circRNAs as biomarkers to select spermatozoa with high morpho-epigenetic quality to use for ART. This study was supported by 'Convenzione Azienda Sanitaria Locale (ASL) Caserta, Regione Campania' (ASL CE Prot. N. 1217885/DIR. GE). The authors have no conflict of interest to declare. N/A.

Effect of Sediment on the Population of Macrobrachium nipponense (De Haan, 1849) in Anzali Lagoon.

&lt;b&gt;Background and Objective:&lt;/b&gt; The role of sediment in water bodies is significant because sediment can affect water quality and benthic organisms. This study investigated sediment's effect on the &lt;i&gt;Macrobrachium nipponense&lt;/i&gt; population in the Anzali Lagoon. &lt;b&gt;Materials and Methods:&lt;/b&gt; The samples were taken at three different sites of Anzali Lagoon in 2015. All the prawns cached by traps and sediment samples were taken exactly next to the traps. All samples were transported to the laboratory for analysis. Canonical correspondence analysis (CCA) was used to analyze the total catch for prawns across the sediment gradients measured. &lt;b&gt;Results:&lt;/b&gt; It showed clear separation in all sites. Site 2 is more affected by clay; in Site 2, the number and the weight of &lt;i&gt;M. nipponense&lt;/i&gt; were lower than in the other sites. In Sites 1 and 3, fine sand and medium sand were more affected, respectively. The results showed a relationship between the prawn population and sediment in the Anzali Lagoon. &lt;b&gt;Conclusion:&lt;/b&gt; In some parts of the Anzali Lagoon, the quality and quantity of sediments were different, which directly affected the population of &lt;i&gt;M. nipponense&lt;/i&gt;. Furthermore, because some parts of the lagoon are in good condition according to sediments and water quality, the Anzali Lagoon has a good potential for growing and living &lt;i&gt;M. nipponense&lt;/i&gt;.

Delivery of neurotrophin-3 by RVG-Lamp2b-modified mesenchymal stem cell-derived exosomes alleviates facial nerve injury.

We aim to investigate the effect of RVG-Lamp2b-modified exosomes (exos) loaded with neurotrophin-3 (NT-3) on facial nerve injury. Exos were collected from control cells (Ctrl Exo) or bone marrow mesenchymal stem cells co-transfected with RVG-Lamp2b and NT-3 plasmids (RVG-NT-3 Exo) by gradient centrifugation and identified by western blotting, transmission electron microscopy, and nanoparticle tracking analysis. Effect of RVG-NT-3 Exo on oxidative stress damage was determined by analysis of the morphology, viability, and ROS production of neurons. Effect of RVG-NT-3 Exo on facial nerve axotomy (FNA) was determined by detecting ROS production, neuroinflammatory reaction, microglia activation, facial motor neuron (FMN) death, and myelin sheath repair. Loading NT-3 and modifying with RVG-Lamp2b did not alter the properties of the exos. Moreover, RVG-NT-3 Exo could effectively target neurons to deliver NT-3. Treatment with RVG-NT-3 Exo lowered H2O2-induced oxidative stress damage in primary neurons and Nsc-34 cells. RVG-NT-3 Exo treatment significantly decreased ROS production, neuroinflammatory response, FMN death, and elevated microglia activation and myelin sheath repair in FNA rat models. Our findings suggested that RVG-NT-3 Exo-mediated delivery of NT-3 is effective for the treatment of facial nerve injury.

Proteomic profiling of mesenchymal stem cell-derived extracellular vesicles: Impact of isolation methods on protein cargo.

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are secreted by cells and play a critical role in cell-to-cell communication. Despite the promising reports regarding their diagnostic and therapeutic potential, the utilization of EVs in the clinical setting is limited due to insufficient information about their cargo and a lack of standardization in isolation and analysis methods. Considering protein cargos in EVs as key contributors to their therapeutic potency, we conducted a tandem mass tag (TMT) quantitative proteomics analysis of three subpopulations of mesenchymal stem cell (MSC)-derived EVs obtained through three different isolation techniques: ultracentrifugation (UC), high-speed centrifugation (HS), and ultracentrifugation on sucrose cushion (SU). Subsequently, we checked EV marker expression, size distribution, and morphological characterization, followed by bioinformatic analysis. The bioinformatic analysis of the proteome results revealed that these subpopulations exhibit distinct molecular and functional characteristics. The choice of isolation method impacts the proteome of isolated EVs by isolating different subpopulations of EVs. Specifically, EVs isolated through the high-speed centrifugation (HS) method exhibited a higher abundance of ribosomal and mitochondrial proteins. Functional apoptosis assays comparing isolated mitochondria with EVs isolated through different methods revealed that HS-EVs, but not other EVs, induced early apoptosis in cancer cells. On the other hand, EVs isolated using the sucrose cushion (SU) and ultracentrifugation (UC) methods demonstrated a higher abundance of proteins primarily involved in the immune response, cell-cell interactions and extracellular matrix interactions. Our analyses unveil notable disparities in proteins and associated biological functions among EV subpopulations, underscoring the importance of meticulously selecting isolation methods and resultant EV subpopulations based on the intended application.

Removal of empty capsids from high-dose adeno-associated virus 9 gene therapies.

Recombinant adeno-associated virus, serotype 9 (rAAV9) has shown promise as a gene therapy vector for muscle and central nervous diseases. High-dose requirements of these therapies present critical safety considerations and biomanufacturing challenges. Notably, the reduction of empty capsids (ECs), which lack therapeutic transgene, from rAAV9 products is critical to maximize efficacy. Removal of rAAV ECs from full capsids is a major downstream challenge because of their highly similar biophysical characteristics. Ultracentrifugation (UC) reduces ECs but is laborious and difficult to scale. In this paper, to replace a poorly scalable UC process, we developed an anion exchange (AEX) chromatography for rAAV9 EC reduction from full capsids. AEX load preparation by dilution incurred major product loss. The addition of histidine and surfactants to dilution buffers increased yield and reduced aggregation. Elution salts were screened and sodium acetate was found to maximize yield and EC reduction. The most promising load dilution buffer and elution salt were used in combination to form an optimized AEX method. The process reduced ECs three-fold, demonstrated robustness to a broad range of EC load challenges, and was scaled for large-scale manufacture. Compared to UC, the AEX method simplified scale-up, reduced ECs to comparable levels (20%), afforded similar purity and product quality, and increased yield by 14%.

Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

#302 Proteomic study reveals indoxyl sulfate induced changes in endothelial cells: role of extracellular vesicles in cardiovascular disease

Abstract Background and Aims Chronic kidney disease (CKD) lead to the accumulation of uremic toxins in the bloodstream, among which indoxyl sulfate (IS) is the most prominent. IS causes damage to endothelial cells lining blood vessels, ultimately leading to cardiovascular diseases in patients with CKD. Under physiological conditions, extracellular vesicles (EVs) are released by cells as intercellular communicators; however, after damage, the cargo of the EVs can be modified. This makes EV potential markers of damage, possibly carrying proteins of interest. This study aimed to investigate the impact of IS on endothelial cells and the release of EVs to identify potential therapeutic targets for preventing endothelial injury. Method Human umbilical vein endothelial cells (HUVEC) were cultured under control conditions and with IS exposure (250 μM IS, 24 h, n = 5). The released EVs were isolated from the supernatants by differential centrifugation and characterized by TEM and flow cytometry to confirm their identity as EVs (following the MISEV2018 guidelines). Proteome profiles of both cells and EVs were analyzed using a mass spectrometer (Orbitrap Exploris™ 240), followed by a Gene Set Enrichment Analysis (GSEA) which revealed altered functions. Results A total of 5871 proteins were identified in the proteomic analysis of cells, and 3614 proteins in EVs (with a False Discovery Rate (FDR) &amp;lt; 1%). In cells, 30 proteins were upregulated and 46 were found downregulated in response to IS, while in EVs, 51 proteins were found upregulated and 47 downregulated (IS vs C). GSEA revealed that IS potentiated adipogenesis (p-value &amp;lt; 0.001), TNF-α signaling via NFкB (p-value = 0.006), and xenobiotic metabolism in cells (p-value = 0.012). In contrast, EVs from cells treated with IS exhibited downregulation of extracellular matrix components (p-value = 0.003), myogenesis (p-value 0.009), genes downregulated in response to ultraviolet radiation (p-value = 0.014) and TNF-α signaling via NFкB (p-value = 0.041). Conclusion IS induces a change in protein expression in cells and their EVs, potentiating inflammation and atherosclerotic plaque formation with extracellular matrix loss. All these modifications contribute to endothelial dysfunction, which is the first step in the development of cardiovascular disease in patients with CKD. This study shed light on novel targets for intervention strategies to mitigate cardiovascular complications in patients with CKD. Funding Grants from the Instituto de Salud Carlos III (ISCIII) and cofounded by Fondos Europeo de Desarrollo Regional (FEDER): “PI19/00240” and “PI20/01321” and “FI20/00018” contract by the Instituto de Salud Carlos III and Ayuda de la Línea de Actuación “Excelencia para el Profesorado Universitario de la UAH” (EPU-INV-UAH/2022/001) from Alcala University.

#407 Human platelet lysate enhanced angiogenic potential of extracellular vesicles derived from mesenchymal stem cells

Abstract Background and Aims Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as a promising regenerative therapy. However, their clinical application is hampered by the presence of xenogeneic components in the medium. In the present study, we aimed to decipher the regenerative properties of MSC-EVs generated under xeno-free conditions to favor the clinical translation of MSC-EVs. Method MSCs were isolated from human umbilical cord and expanded in fetal bovine serum (FBS) and clinical-grade human platelet lysate (HPL) respectively. MSC-EVs were isolated by differential centrifugation combined with size exclusion chromatography and characterized through transmission electron microscopy, nanoparticle tracking analysis, and exoview. Subsequently, a multi-omics approach, comprising transcriptomics, proteomics, and metabolomics, was employed to investigate the molecular cargo of the MSC-EVs. Finally, in vitro and in vivo functional assays were performed to assess the regenerative potential of MSC-EVs in acute kidney injury. Results Our study demonstrated that HPL promoted MSC-EVs production without compromising EVs characteristics. Multi-omics sequencing revealed the stability of HPL cultured MSC-EVs (H-EVs) from different umbilical cord donors and global functional alterations for MSC-EVs under different culture conditions. In comparison to FBS cultured MSC-EVs (F-EVs), the regenerative potential of H-EVs was enhanced especially angiogenesis associated with increased cargo expression. The in vitro studies demonstrated that H-EVs induced more tube-like structure formation and migration. Further, it significantly reduced kidney capillary sparsity in mice with renal ischemic-reperfusion model. Conclusion Our data provided a comprehensive understanding about H-EVs and demonstrated their potential as a safe and effective regenerative therapy, especially targeting angiogenesis-related disorders.

#348 Neutrophil membrane-derived nanovesicles deliver IL-37 to repair renal endothelial cells in ischemia‒reperfusion injury

Abstract Background and Aims Renal ischemia‒reperfusion injury (IRI) is one of the most important causes of acute kidney injury (AKI). Interleukin (IL)-37 is a novel anti-inflammatory factor, but its application is still limited by its low stability and delivery efficiency. In recent years, engineered extracellular vesicles (EVs) have attracted substantial attention as an emerging drug delivery system. The aim of this study was to investigate the reparative effect of neutrophil membranes-derived nanovesicle (N-MV)-delivered IL-37 on renal IRI and its mechanism of action. Method The density gradient centrifugation method was used to extract peripheral blood neutrophils from rats, and the differential centrifugation method was used to extract neutrophil membranes. Cell membranes and IL-37 were then extruded with polycarbonate porous membranes with different pore sizes using a mini extruder to prepare IL-37 that was coated with neutrophil membrane (N-MV@IL-37). Subsequently, the physical and chemical properties and in vitro biological functions were identified. The in vivo distribution of N-MV@IL-37 as well as the renal function and inflammatory response in rats were further evaluated in a rat renal IRI model, and the targeting and anti-inflammatory mechanisms of N-MV@IL-37 were further explored. Results N-MV@IL-37 not only enhanced the stability of IL-37 but also targeted endothelial cells via P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophil membrane surfaces. In vitro experiments showed that N-MV@IL-37 inhibited endothelial cell apoptosis and inflammatory factor production and promoted endothelial cell viability and angiogenesis. In the rat renal IRI model, N-MV@IL-37 inhibited renal cell apoptosis and alleviated inflammatory responses, thereby improving renal function in IRI rats. Conclusion N-MVs provide an effective method of delivering IL-37, thereby alleviating the inflammatory response due to renal IRI and repairing renal endothelial cells; these vesicles could be a potential system for the treatment of renal IRI. Meanwhile, N-MVs also provided a drug delivery platform to provide a new strategy for the treatment of other IRI-like diseases.

POST-COVID SYNDROME - A MODEL OF PATHOGENESIS

Post- COVID is characterized by a decrease in the patients’ quality of life, a tendency to hemocoagulation. Symptoms of post- COVID are diverse and difficult to classify. Purpose : Study of neutrophil extracellular traps (NETs) in post-COVID patients to clarify the mechanisms of damage. Patients and methods : Two groups of patients with postCOVID were examined. In the first group (21 patients), COVID-19 treatment was carried out in a hospital, in the second group (20 patients) - on an outpatient clinic. The comparison group consisted of 10 patients with acute appendicitis, the control group consisted of 20 healthy people. Neutrophils were isolated using gradient centrifugation. NETs was visualized using fluorescence microscopy with SYBR Green dye (Evrogen; Russia), which specifically interacts with double-stranded DNA. Purine nitrogenous bases (PNB) were determined by the color reaction method based on their interaction with silver nitric acid.

Endothelial Cell-Derived Exosomes Inhibit Osteoblast Apoptosis and Steroid-Induced Necrosis of Femoral Head Progression by Activating the PI3K/Akt/Bcl-2 Pathway

The aim of the study was to investigate the therapeutic potential of exosomes secreted by endothelial cells (EC-exos) on steroid-induced osteonecrosis of femoral head (SNFH). First, we successfully obtained EC-exos through differential centrifugation. Then, the effects of EC-exos on mouse embryo osteoblast precursor (MC3T3-E1) cells under high concentration of dexamethasone (Dex) were analysed in vitro, which included cell migration, viability, and apoptosis. In vivo, a SNFH rat model was successfully established and treated with EC-exos. Micro-computed tomography (micro-CT) and haematoxylin and eosin (H&amp;E) were used to observe femoral trabeculae. Our in vitro results showed that EC-exos improved cell viability and migration of osteoblasts and reduced the apoptotic effect of high concentration of Dex on osteoblasts in vitro. Phosphoinositide 3-kinase (PI3K)/Akt/Bcl-2 signalling pathway was activated in MC3T3-E1 cells under the response to EC-exos. In vivo, increased bone volume per tissue volume (BV/TV) p=0.031, trabecular thickness (Tb.Th) p=0.020, and decreased separation (Tb.Sp) p=0.040 were observed in SNFH rats treated with EC-exos. H&amp;E staining revealed fewer empty lacunae and pyknotic osteocytes in trabeculae. The expression of Bcl-2 and Akt in EC-exos group was significantly increased in trabeculae tissue. Overall, our finding indicated that EC-exos could attenuate SNFH by inhibiting osteoblast apoptosis via the PI3K/Akt/Bcl-2 pathway.

Study on liver tissue derived-extracellular vesicles regulating the osteogenic differentiation ability of mesenchymal stem cells and promoting the healing of jaw bone defects

Objective: To explore the biological process of liver tissue-derived extracellular vesicle (LT-EV) in promoting osteogenic differentiation of mesenchymal stem cells and healing of jaw defects to provide a feasible treatment method for the clinical treatment of jaw bone defects. Methods: Enzymatic hydrolysis and differential centrifugation were used to extract LT-EV, scanning electron microscopy, Western blotting, and nanoparticle tracking analyzers were used to identify and characterize LT-EV, and further to explore the biological functions of LT-EV through proteomics and Kyoto Encyclopedia of Genes and Genomes. Flow cytometry was used to detect LT-EV plasma concentration and to calculate the plasma half-life of LT-EV. Small animal in vivo imaging system was used to detect the biological distribution of LT-EV 24 hours after injection. Six C57BL/6 mice were divided into control group and LT-EV group (3 mice in each group) by simple random sampling method. All mice underwent jaw bone defect surgery and tail vein injection every 7 days (the control group was injected with phosphoric buffer saline, LT-EV group was injected with LT-EV), micro-CT was used to evaluate the degree of mouse jaw bone healing 28 days after surgery, HE staining was used to analyze the multi-organ biosafety of LT-EV, and immunofluorescence staining was used to detect the jaw bone expression of osteogenic marker proteins in the defect area. Human jaw bone mesenchymal stem cells (hJBMSC) induced by osteogenic differentiation were treated with LT-EV (obtained from orthognathic surgery patients provided by the Department of Traumatology and Orthognathic Surgery of School of Stomatology of The Fourth Military Medical University resected normal jaw bone fragments), and the difference in osteogenic differentiation ability between the hJBMSC group and the control group (phosphate buffer saline treatment) was compared, and the in vitro bone differentiation promoting effect of LT-EV was verified through alkaline phosphatase (ALP) staining and real-time fluorescence quantitative PCR. Results: The yield of LT-EV was high, and proteomics and Kyoto Encyclopedia of Genes and Genomes showed that LT-EV contained a series of proteins that regulated cell biological functions. LT-EV injected into the tail vein could reach the mouse jaw bone defect area and promote the regeneration and repair of the jaw bone defect [the bone volume fractions of the LT-EV group and the control group were (36.06±4.20)% and (18.58±5.61)%, respectively; t=4.32, P=0.013], and had good biosafety. LT-EV could promote osteogenic differentiation of hJBMSC in vitro. Compared to the control group, ALP staining and osteogenic gene expression levels were significantly enhanced after osteogenic differentiation of hJBMSC (P<0.05). Conclusions: LT-EV exhibits a high yield, ease of acquisition, high biological safety, and excellent bone-promoting effects. It holds promise as a novel cell-free therapy strategy for regenerating craniofacial bone defects.

A new effective method for isolating single spermatozoa from surgically obtained material in azoospermia

A new effective method for isolating single spermatozoa from surgically obtained material in azoospermia