Search Results (404)

Microglia-mediated neuroinflammation is a key player in the pathogenesis of amyotrophic lateral sclerosis (ALS) as it can contribute to the progressive degeneration of motor neurons (MNs). Here, we investigated the role of mSOD1 NSC-34 MN-like cell-derived extracellular vesicles (EVs) in inducing the activation of BV2 microglial cells. NSC-34-released EVs were isolated by culture medium differential ultracentrifugation to obtain two fractions, one containing small EVs (diameter < 200 nm) and the other containing large EVs (diameter > 200 nm). BV2 cells were incubated with the two EV fractions for 12, 24, and 48 h to evaluate 1) the state of microglial inflammation through RT-PCR of IL-1β, IL-6, IL-4, and IL-10 and 2) the expression of proteins involved in inflammasome activation (IL-β and caspase 1), cell death (caspase 3), and glial cell recruitment (CXCR1), and presence of the TGFβ cytokine receptor (TGFβ-R2). The obtained results suggest a mSOD1 type-dependent polarization of BV2 cells towards an early neurotoxic phenotype and a late neuroprotective status, with an appearance of mixed M1 and M2 microglia subpopulations. A significant role in driving microglial cell activation is played by the TGFβ/CX3CR1 axis. Therefore, targeting the dysregulated microglial response and modulating neuroinflammation could hold promise as a therapeutic strategy for ALS. Full article

Outer membrane vesicles (OMVs) are nanostructures derived from the outer membrane of Gram-negative bacteria. We previously demonstrated that vaccination with endotoxin-free OMVs isolated from an Acinetobacter baumannii strain lacking lipooligosaccharide (LOS) biosynthesis, due to a mutation in lpxD, provides full protection in a murine sepsis model. The present study characterizes the protein content of highly-purified OMVs isolated from LOS-replete and LOS-deficient strains. Four purification methods were evaluated to obtain highly purified OMV preparations: ultracentrifugation, size exclusion chromatography (SEC), ultracentrifugation followed by SEC, and Optiprep™. OMVs from each method were characterized using nanoparticle tracking analysis and electron microscopy. OMVs from LOS-deficient and LOS-replete strains purified using the Optiprep™ method were subjected to LC-MS/MS analysis to determine protein content. Significant differences in protein composition between OMVs from LOS-deficient and LOS-replete strains were found. Computational analyses using Bepipred 3.0 and SEMA 2.0 indicated that the lack of LOS led to the overexpression of immunogenic proteins found in LOS-containing OMVs and the presence of immune-stimulating proteins absent in LOS-replete OMVs. These findings have important implications for developing OMV-based vaccines against A. baumannii, using both LOS-containing and LOS-free OMVs preparations. Full article

This study investigated the therapeutic effects of exosomes derived from human-induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) on corneal epithelial wound healing. Exosomes were isolated from the culture medium of the hiPSC-derived ROs (Exo-ROs) using ultracentrifugation, and then they were characterized by a nanoparticle tracking analysis and transmission electron microscopy. In a murine model of corneal epithelial wounds, these exosomes were topically applied to evaluate their healing efficacy. The results demonstrated that the exosome-treated eyes showed significantly enhanced wound closures compared with the controls at 24 h post-injury. The 5-ethyl-2′-deoxyuridine assay and quantitative reverse transcription polymerase chain reaction revealed a substantial increase in cell proliferation and a decrease in inflammatory marker contents in the exosome-treated group. The RNA sequencing and exosomal microRNA analysis revealed that the Exo-RO treatment targeted various pathways related to inflammation and cell proliferation, including the PI3K-Akt, TNF, MAPK, and IL-17 signaling pathways. Moreover, the upregulation of genes related to retinoic acid and eicosanoid metabolism may have enhanced corneal epithelial healing in the eyes treated with the Exo-ROs. These findings suggest that hiPSC-derived RO exosomes could be novel therapeutic agents for promoting corneal epithelial wound healing. Full article

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development. Full article

Sensory neurons densely innervate the myocardium. The role of their sensing and response to acute and prolonged ischemia is largely unclear. In a cellular model of ischemia-reperfusion injury, the presence of sensory neurons increases cardiomyocyte survival. Here, after the exclusion of classical neurotransmitter release, and measurement of cytokine release, we modified the experiment from a direct co-culture of primary murine cardiomyocytes and sensory neurons to a transfer of the supernatant. Sensory neurons were exposed to ischemia and the resulting conditioned supernatant was transferred onto cardiomyocytes. This approach largely increased the tolerance of cardiomyocytes to ischemia and reperfusion. Towards the identification of the mechanism, it was demonstrated that after ten-fold dilution, the conditioned solution lost its protective effect. The effect remained after removal of extracellular vesicles by ultracentrifugation, and was not affected by exposure to protease activity, and fractionation pointed towards a hydrophilic agent. Solutions conditioned by HEK293t cells or 3T3 fibroblasts also increase cardiomyocyte survival, but to a lower degree. A metabolomic search identified 64 at least two-fold changed metabolites and lipids. Many of these could be identified and are involved in essential cellular functions. In the presented model for ischemia-reperfusion, sensory neurons secrete one or more cardioprotective substances that can improve cardiomyocyte survival. Full article

Hepatic fibrosis, arising from prolonged liver injury, entails the activation of hepatic stellate cells (HSCs) into myofibroblast-like cells expressing alpha-smooth muscle actin (α-SMA), thereby driving extracellular matrix deposition and fibrosis progression. Strategies targeting activated HSC reversal and hepatocyte regeneration show promise for fibrosis management. Previous studies suggest that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) can suppress HSC activation, but ensuring EV purity is essential for clinical use. This study investigated the effects of MSC-derived EVs cultured in chemically defined conditions on liver spheroids and activated HSCs. Umbilical cord- and bone marrow-derived MSCs were expanded in chemically defined media, and EVs were isolated using filtration and differential ultracentrifugation. The impact of MSC-EVs was evaluated on liver spheroids generated in Sphericalplate 5D™ and on human HSCs, both activated by transforming growth factor beta 1 (TGF-β1). MSC-EVs effectively reduced the expression of profibrotic markers in liver spheroids and activated HSCs induced by TGF-β1 stimulation. These results highlight the potential of MSC-EVs collected under chemically defined conditions to mitigate the activated phenotype of HSCs and liver spheroids, suggesting MSC-EVs as a promising treatment for hepatic fibrosis. Full article

Prussian Blue nanoparticles (PBnps) are now popular in nanomedicine thanks to the FDA approval of PB. Despite the numerous papers suggesting or describing the in vivo use of PBnps, no studies have been carried out on the formation of a protein corona on the PBnp surface and its stabilizing role. In this paper, we studied qualitatively and quantitatively the corona formed by the most abundant protein of blood, human serum albumin (HSA). Cubic PBnps (41 nm side), prepared in citric acid solution at PB concentration 5 × 10⁻⁴ M, readily form a protein corona by redissolving ultracentrifuged PBnp pellets in HSA solutions, with C_HSA ranging from 0.025 to 7.0 mg/mL. The basic decomposition of PBnp@HSA was studied in phosphate buffer at the physiological pH value of 7.4. Increased stability with respect to uncoated PBnps was observed at all concentrations, but a minimum C_HSA value of 3.0 mg/mL was determined to obtain stability identical to that observed at serum-like HSA concentrations (35–50 mg/mL). Using a modified Lowry protocol, the quantity of firmly bound HSA in the protein corona (hard corona) was determined for all the C_HSA used in the PBnp@HSA synthesis, finding increasing quantities with increasing C_HSA. In particular, an HSA/PBnp number in the 1500–2300 range was found for C_HSA 3.0–7.0 mg/mL, largely exceeding the 180 HSA/PBnp value calculated for an HSA monolayer on a PBnp. Finally, the stabilization brought by the HSA corona allowed us to carry out pH-spectrophotometric titrations on PBnp@HSA in the 3.5-9-0 pH range, revealing a pKa value of 6.68 for the water molecules bound to the Fe³⁺ centers on the PBnp surface, whose deprotonation is responsible for the blue-shift of the PBnp band from 706 nm (acidic solution) to 685 nm (basic solution). Full article

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal. Full article

Recombinant adeno-associated viruses (rAAVs) play a pivotal role in the treatment of genetic diseases. However, current production and purification processes yield AAV-based preparations that often contain unwanted empty, partially filled or damaged viral particles and impurities, including residual host cell DNA and proteins, plasmid DNA, and viral aggregates. To precisely understand the composition of AAV preparations, we systematically compared four different single-stranded AAV (ssAAV) and self-complementary (scAAV) fractions extracted from the CsCl ultracentrifugation gradient using established methods (transduction efficiency, analytical ultracentrifugation (AUC), quantitative and digital droplet PCR (qPCR and ddPCR), transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA)) alongside newer techniques (multiplex ddPCR, multi-angle light-scattering coupled to size-exclusion chromatography (SEC-MALS), multi-angle dynamic light scattering (MADLS), and high-throughput sequencing (HTS)). Suboptimal particle separation within the fractions resulted in unexpectedly similar infectivity levels. No single technique could simultaneously provide comprehensive insights in the presence of both bioactive particles and contaminants. Notably, multiplex ddPCR revealed distinct vector genome fragmentation patterns, differing between ssAAV and scAAV. This highlights the urgent need for innovative analytical and production approaches to optimize AAV vector production and enhance therapeutic outcomes. Full article

In this study, the protein and salts distribution (Ca, P, Na and Mg) in processed cheese (PC) samples prepared with 180 or 360 mEq/kg of the calcium sequestering salts (CSS) disodium phosphate (DSP), disodium pyrophosphate (DSPP), sodium hexametaphosphate (SHMP) and trisodium citrate (TSC) was studied. For this purpose, a water-soluble extract (WSE) of PC samples was prepared. All PC samples contained 45–46% moisture, 26–27% fat and 20–21% protein and had a pH of 5.2 or 5.7. Ultracentrifugation slightly reduced the protein content of the WSE of PC, indicating that most protein in the WSE was non-sedimentable. At equal concentration of CSS, the protein content of the WSE was higher for PC at pH 5.7 compared to PC at pH 5.2. Approximately 55–85% of the Ca and P in the WSE of samples was 10 kDa-permeable for PC prepared with DSPP and SHMP. This suggests that the formation of non-permeable Ca–polyphosphate–casein complexes. For PC prepared with TSC, >90% of Ca in the WSE was 10 kDa-permeable, indicating that micellar disruption arises from sequestration of micellar Ca. These results indicate that the WSE method is an appropriate method to understand how salts present in PC are distributed. However, the WSE and ultracentrifugal supernatant of the WSE can include both soluble and protein-associated salts. Therefore, determining levels of salts in 10 kDa permeate of ultracentrifugal supernatant of the WSE is most appropriate. Full article

Urine, a common source of biological markers in biomedical research and clinical diagnosis, has recently generated a new wave of interest. It has recently become a focus of study due to the presence of its content of extracellular vesicles (EVs). These uEVs have been found to reflect physiological and pathological conditions in kidney, urothelial, and prostate tissue and can illustrate further molecular processes, leading to a rapid expansion of research in this field In this work, we present the advantages of an immunoaffinity-based method for uEVs’ isolation with respect to the gold standard purification approach performed by differential ultracentrifugation [in terms of purity and antigen presence. The immunoaffinity method was made feasible by combining specific antibodies with a functionalized polymethacrylate polymer. Flow cytometry indicated a significant fluorescence shift, validating the presence of the markers (CD9, CD63, CD81) and confirming the effectiveness of the isolation method. Microscopy evaluations have shown that the morphology of the vesicles remained intact and corresponded to the expected shapes and dimensions of uEVs. The described protocol is inexpensive, fast, easy to process, has good reproducibility, and can be applied to further biological samples. Full article

(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography–mass spectrometry (LC–MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition. Full article

Escherichia coli (E. coli) is commonly utilized as a vehicle for anti-tumor therapy due to its unique tumor-targeting capabilities and ease of engineering modification. To further explore the role of E. coli in tumor treatment, we consider that E. coli outer membrane vesicles (E. coli-OMVs) play a crucial role in the therapeutic process. Firstly, E. coli-OMVs were isolated and partially purified by filtration and ultracentrifugation, and were characterized using techniques such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western Blot (WB). The obtained extracellular nanoparticles, containing OMVs, were found to inhibited the growth of CT26 tumor in mice, while the expression of Bax protein was increased and the expression of Bcl-2 protein decreased. In vitro experiments showed that E. coli-OMVs entered CT26 cells and inhibited cell proliferation, invasion and migration. In addition, in the presence of E. coli-OMVs, we observed an increase in apoptosis rate and a decrease in the ratio of Bcl-2/Bax. These data indicate that E. coli-OMVs inhibits the growth of CT26 colon cancer by inducing apoptosis of CT26 cells. These findings propose E. coli-OMVs as a promising therapeutic drug for colorectal cancer (CRC), providing robust support for further research in related fields. Full article

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His₆-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His₆-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked. Full article

(1) Background: Exosomal PD-L1 has garnered attention owing to its role in instigating systemic immune suppression. The objective of this study is to elucidate whether bone and soft tissue sarcoma cells possess the capacity to secrete functionally active exosomal PD-L1 and whether radiotherapy (RT) induces the exosomal PD-L1 release. (2) Methods: Human osteosarcoma cell line 143B and human fibrosarcoma cell line HT1080 were utilized. Exosomes were isolated from the culture medium and blood via ultracentrifugation. The expression of PD-L1 on both tumor cells and exosomes was evaluated. The inhibitory effect on PBMC was employed to assess the activity of exosomal PD-L1. Post radiotherapy, changes in PD-L1 expression were compared. (3) Results: Exosomal PD-L1 was detected in the culture medium of tumor cells but was absent in the culture medium of PD-L1 knockout cells. Exosomal PD-L1 exhibited an inhibitory effect on PBMC activation. In tumor-bearing mice, human-derived exosomal PD-L1 was detected in the bloodstream. Following radiotherapy, tumor cells upregulated PD-L1, and human-derived exosomal PD-L1 were detected in the bloodstream. (4) Conclusions: Exosomal PD-L1 emanates from bone and soft tissue sarcoma cells and is disseminated into the circulatory system. The levels of PD-L1 in tumor cells and the release of exosomal PD-L1 were augmented after irradiation with RT. Full article