Tracking Analysis Research Articles

Deep reinforcement learning-based variable impedance control for grinding workpieces with complex geometry

Purpose This paper aims to design a deep reinforcement learning (DRL)-based variable impedance control policy that supports stability analysis for robot force tracking in complex geometric environments. Design/methodology/approach The DRL-based variable impedance controller explores and pre-learns the optimal policy for impedance parameter tuning in simulation scenarios with randomly generated workpieces. The trained results are then used as feedforward inputs to improve the force-tracking performance of the robot during contact. Based on Lyapunov’s theory, the stability of the proposed control policy is analysed to illustrate the interpretability of the results. Findings Simulations and experiments are performed on different types of complex environments. The results show that the proposed method is not only theoretically feasible but also has better force-tracking effects in practice. Originality/value Compared with most other DRL-based control policies, the proposed method possesses stability and interpretability, effectively avoids the overfitting phenomenon and thus has better simulation-to-real deployment results.

Platelet membrane-modified exosomes targeting plaques to activate autophagy in vascular smooth muscle cells for atherosclerotic therapy.

Atherosclerosis is one of the leading causes of ischemic cardiovascular disease worldwide. Recent studies indicated that vascular smooth muscle cells (VSMCs) play an indispensable role in the progression of atherosclerosis. Exosomes derived from mesenchymal stem cells (MSCs) have demonstrated promising clinical applications in the treatment of atherosclerosis. However, there are still challenges and limitations persist in targeted therapy. This study aims to develop a bionic nano-delivery system by fusing platelet membranes with exosomes (MSC-ExoP) and explore the anti-atherosclerosis effect of MSC-ExoP by improving the targeting efficiency and participating in regulating the pathophysiological processes associated with VSMCs. The morphology, particle size, stability, and fusion efficiency of MSC-ExoP were assessed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), immunofluorescence staining, and Western blotting, respectively. MSC-ExoP was administered intravenously into ApoE-/- mice via the tail vein. In vivo, immunofluorescence staining was used to assess the targeting efficacy of MSC-ExoP. The ORO staining, H&E staining, Masson staining, aortic root immunofluorescence staining, and Western blot were utilized to evaluate the VSMC autophagy and anti-atherosclerosis effects of MSC-ExoP. In vitro, the autophagy activation of MSC-ExoP on VSMCs was further assessed by immunofluorescence staining and Western blotting. The effects of MSC-ExoP on VSMCs proliferation, migration, and foam cell formation were detected by EdU experiment, Transwell experiment, wound healing experiment, ORO staining, and BODIPY staining. The TEM revealed that MSC-ExoP retained a ring nanostructure, which was similar to MSC-Exo in morphology. NTA analysis indicated the MSC-ExoP exhibited a slight increase after cell membrane fusion. Besides, the stability analysis of exosomes and MSC-ExoP resulted in no significant changes in particle size. Western blot analysis confirmed that MSC-ExoP simultaneously expressed platelet-specific markers (GPVI, GPIbα, CD62P) and exosome-specific markers (CD81, TSG101, and Alix). In ApoE-/- mice, the immunofluorescence of aorta and its roots was significantly enhanced after injection of DiI-labeled MSC-ExoP, indicating enhanced targeting of MSC-Exo to atherosclerotic plaques by platelets. In vivo experiments demonstrated that MSC-ExoP could significantly suppress the progression of atherosclerosis and reduce the area of atherosclerotic plaques by reducing lipid deposition and necrotic nucleus area and increasing collagen content. In vitro experiments further revealed that the uptake of MSC-ExoP by foam cells significantly increased, and their proliferation, migration, and foam formation were inhibited by autophagy activation. This study demonstrated successful fusion of platelet membranes with exosomes derived from MSCs. MSC-ExoP could significantly improve the targeting efficiency of atherosclerosis and play an anti-atherosclerosis effect by activating VSMC autophagy.

Neuropilin1-dependent paracrine signaling of cancer cells mediated by miRNA exosomal cargo

BackgroundNeuropilin-1 (NRP1) is a transmembrane protein involved in surface receptor complexes for a variety of extracellular signals. NRP1 expression in human cancers is associated with prominent angiogenesis and advanced progression stage. However, the molecular mechanisms underlying NRP1 activity in the tumor microenvironment remain unclear. Notably, diffusible forms of NRP1 in the extracellular space have been reported, but their functional role is poorly understood.MethodsExtracellular vesicles (EV) were isolated from conditioned media of diverse cancer cells. The quality of exosome-enriched preparations was validated by the presence of specific markers in western blotting, as well as by light scattering and nanoparticle tracking analysis. Wound healing, transwell, and digital real-time migration assays were carried out to assess the activity of cancer cell-derived exosomes in the regulation of endothelial cells. RNA interference was applied to obtain NRP1 knock-down, and cDNA transfer to achieve its overexpression, in exosome-releasing cells. The micro-RNA profile carried by exosomes was investigated by Next Generation Sequencing. miRNA-Scope in situ hybridization was used to assess the transfer of miRNA exosome cargo to target cells, and immunofluorescence analysis revealed expression regulation of targeted proteins. miRNA activity was blocked by the use of specific antago-miRs.ResultsIn this study, we show that diverse human cancer cells release NRP1 embedded in exosome-like small extracellular vesicles, which mediate a previously unknown NRP1-dependent paracrine signaling mechanism regulating endothelial cell migration. By transcriptomic analysis of the cargo of NRP1-loaded exosomes, we found a significant enrichment of miR-210-3p, known to promote tumor angiogenesis. Gene knock-down and overexpression experiments demonstrated that the loading of miR-210-3p into exosomes is dependent on NRP1. Data furthermore indicate that the exosomes released through this NRP1-driven mechanism effectively transfer miR-210-3p to human endothelial cells, causing paracrine downregulation of the regulatory cue ephrin-A3 and promotion of cell migration. The mechanistic involvement of miR-210-3p in this pathway was confirmed by applying a specific antago-miR.ConclusionsIn sum, we unveiled a previously unknown NRP1-dependent paracrine signaling mechanism, mediated by the loading of pro-angiogenic miR-210-3p in exosomes released by cancer cells, which underscores the relevance of NRP1 in controlling the tumor microenvironment.

Analysis of small extracellular vesicles from dried blood spots

This protocol paper describes how to extract small extracellular vesicles (sEVs) from dried blood spots (DBS). The methodology is described in detail and offers further evidence that the extracted particles are sEVs using western blotting (anti-CD9, CD63 and CD81) and fluorescence nanoparticle tracking analysis (fNTA). In addition, we present evidence that approximately 40% of the sEVs were recovered from DBS compared with EVs analyzed from plasma directly. The protocol proves to be robust, reliable and displays very interesting performances even after several weeks (up to 3 weeks) of storage of the DBS when analyzing the sEVs using protein microarray for the presence of the markers CD9, CD63, CD81, EpCAM, Flotilin-1, CD62E/P, CD142 and CD235a. These findings have important implications for using sEVs as future potential diagnostic tools by supporting the validity of less-invasive methods that can be implemented within vulnerable populations or in the field.

Mental Health Through Music Therapy

This project, Mental Health Through Music Therapy, focuses on leveraging the therapeutic power of music to support mental well-being while integrating a robust user management system. It offers a secure and interactive platform where users can engage with therapy-specific music tailored to their needs. The system features user registration with age verification, ensuring appropriate access, and securely stores user details in structured files and a CSV database for efficient session tracking and future analysis. Users can explore categorized music folders, read therapeutic text files, and enjoy a seamless music playback experience, fostering a relaxing and personalized environment. Designed with a neon-themed interface, the platform incorporates custom animations and error-handling mechanisms for an engaging and user-friendly experience. By combining the emotional benefits of music therapy with modern technological advancements, this project aims to provide an innovative and practical solution for improving mental health and enhancing emotional well-being

Decline in Water Treatment Efficiency of an Estuarine Constructed Wetland over Its Operating Years

Estuarine constructed wetlands (ECWs) play a role as ecological barriers in the control of external pollution in lakes. Usually, ECWs show reduced water treatment efficiency after many years of operation compared to their initial performance. However, it is unclear how the water purification efficiency of an ECW changes over time. After over a decade of tracking analysis on an ECW, this study found that it indeed played a significant role in achieving water quality improvement effects. The average removal rates for total nitrogen (TN) and total phosphorus (TP) and the permanganate index (CODMn) were 36.2%, 26.7%, and 30.7%, respectively, with annual reductions of 1.6 t/a, 20.8 t/a, and 44.6 t/a. The surface hydraulic load is a critical indicator for the design and operational management of ECWs. The reduction loads of TP, TN, and CODMn increased with the rise in surface hydraulic load, indicating that this ECW project had certain advantages in treating large-volume water bodies. However, when strict CODMn treatment is needed, the surface hydraulic load should be reduced. During the high-efficiency period (2010–2015), the treatment effects on TN and TP were more than twice those during the degradation period (2016–2021), and the effect on CODMn was about 1.5 times greater. With increased operation years, the TN removal rate declined most rapidly due to pollutant accumulation and sediment release.

Further research of multi-scale assemble and matrix reassemble method for efficient analysis of railway track–substructure system subjected to a moving train

Abstract For a large-scale dynamic system, the efficiency of computation becomes a vital work sometimes in engineering practices. As a layered structural system, ballastless track and substructure occupy most part of the degrees of freedom of the whole system. It is, therefore, rather important to optimize the structural models in dynamic equation formulations. In this work, a three-dimensional and coupled model for multi-rigid-body of train and finite elements of track and substructures is presented by multi-scale assemble and matrix reassemble method. The matrix reassembling tactic is based on the multi-scale assemble method, through which the finite element matrix bandwidth is greatly narrowed, and the Cholesky factorization, iterative and multi-time-step solution have been introduced to efficiently obtain the train, track and substructure responses. The subgrade and its subsoil works as a typical substructural system, and comparisons with the previous model without matrix reassembling, SIMPACK and ABAQUS have been conducted to fully validate the efficiency and accuracy of this train–track–subgrade dynamic interaction model.

Lipopolysaccharide (LPS) induces sclerostin secretion by extracellular vesicle via TLR4/miR-92a-3p/PTEN/NF-κB signalling pathway in murine macrophage.

Sclerostin (SOST) is traditionally regarded as an osteocyte-derived secreted glycoprotein that regulates bone mineralization. Recent studies reported that SOST is also released from non-skeletal sources, especially during inflammation. However, the cellular source and regulatory mechanisms governing SOST generation in inflammation remain unclear. This study investigated whether macrophages produce SOST in response to inflammatory stimuli and determined associated regulatory pathways. The effect of lipopolysaccharide (LPS)-induced inflammation in SOST generation and its underlying regulatory mechanism was examined on mouse macrophage RAW 264.7 by western blot and immunofluorescent staining. Transfection with miR-92a-3p mimic and inhibitor were used to validate its role in SOST production. The role of NF-κB and TLR4 were studied using pharmacological inhibitors BAY 11-7085 and TAK242, respectively. The involvement of NF-κB and TLR4 in LPS-induced SOST production was further validated through nuclear NF-κB p65 immunoprecipitation and TLR4 small interfering RNA (siRNA) experiments, respectively.GW4869 and manumycin A (extracellular vesicles (EV) biogenesis inhibitors) were used to examine the associated of SOST and EV. Finally, SOST expression and characteristics of the isolated EV were assessed by Western blot and nanoparticle tracking analysis (NTA). LPS significantly induced SOST protein expression and secretion in RAW 264.7. MiR-92a-3p was upregulated by LPS stimulation in macrophages. Transfection of miR-92a-3p mimic increased SOST generation in RAW 264.7. Inhibition of TLR4 and NF-κB signalling pathways using pharmacological inhibitors significantly suppressed LPS-induced SOST in RAW 264.7. Similarly, TLR4 siRNA effectively suppressed LPS-induced SOST level. However, the LPS-induced upregulation of miR-92a-3p was only regulated by TLR4, but not by NF-κB. NF-κB was found to directly bind to the mouse sost promoter, thereby activating sost transcription. Additionally, SOST secretion was found predominantly associated with EV from LPS-stimulated cells, and inhibition of EV biogenesis suppressed SOST production in RAW 264.7 cells. In conclusion, our study showed, for the first time, that LPS induced SOST generation and secretion via TLR4/miR-92a-3p/PTEN/NF-κB singling pathway in murine macrophage RAW 264.7 cells. Moreover, we showed that SOST is secreted from the RAW 264.7 cells in the form of extracellular vesicle. This study identified macrophage as a novel source of SOST, highlighting its potential role in inflammatory diseases.

Extracellular vesicles in ageing cold-stored whole blood may not compensate for the decreasing haemostatic function in vitro.

Extracellular vesicles (EVs) have procoagulative properties. As EVs are known to accumulate in stored blood products, we compared the EV content and coagulation capacity of leukoreduced cold-stored whole blood (CSWB) with current prehospital and in-hospital component therapies to understand the role of EVs in the haemostatic capacity of ageing CSWB. Blood was obtained from 12 O RhD-positive male donors. CSWB was compared with in-hospital component therapy of red blood cells (RBCs), OctaplasLG and buffy-coat platelets and prehospital component therapy of RBC and lyophilized plasma. Samples were drawn on Days 1 and 14 of CSWB and RBC cold storage. Blood count, haemolysis markers, rotational thromboelastometry, sonorheometry and thrombin generation were analysed. EVs were analysed using nanoparticle tracking analysis and cellular origin was determined using imaging flow cytometry. There was a trend towards increased production of both platelet and RBC-derived EVs during CSWB storage. Particle count increased during storage, whereas thrombin generation slowed down and in viscoelastic assays, clotting times prolonged, clot formation became impaired, and stiffness of the resulting clot decreased. Both platelet and RBC-derived EVs increased in number in CSWB during storage. This did not appear to compensate for the in vitro decreasing haemostatic capacity of ageing CSWB, suggesting EVs produced during storage may not have active procoagulative effects, but rather reflect the ageing of blood cells.

Wavelength-Dependent Calcium Signaling Response to Photobiomodulation in Pancreatic Cells

Diabetes mellitus is a metabolic disorder that is rapidly growing across the world. Our laboratory has recently demonstrated that photobiomodulation (PBM) can couple to its metabolic pathways by modulating calcium dynamics in islet cells, including α- and β-cells. Using computer vision algorithms, changes in PBM-induced calcium dynamics can be verified, and, more importantly, this led us to propose hypotheses that will likely advance our understanding of photostimulatory effects in islet cells. In our previous paper, we determined changes in calcium spiking in response to PBM at 810 nm by manually segmenting the cells and the calcium spiking patterns. We have since developed a computer vison pipeline to automate cell segmentation and subsequent image analyses. By using automated methods for segmentation, registration, tracking, and statistical analysis, we were able to improve the accuracy of previously observed changes in calcium spiking in response to PBM in both cell types. Moreover, this pipeline was applied to elucidate the wavelength-dependent modulation of calcium dynamics at 1064 nm. The extent of increase in calcium spiking appears to have been overestimated by manual analysis, and the machine learning pipeline was able to capture and segment nearly 3-fold more cells, suggesting improved accuracy in the analysis of calcium spiking in islet cells. Detailed calcium analysis also indicates a biphasic dose response among α- and β-cells in response to PBM therapy at different wavelengths. The current findings offer a novel hypothesis and may facilitate the use of translational PBM as a potential therapy for diabetes mellitus.

Corneal Stromal Stem Cell-Derived Extracellular Vesicles Attenuate ANGPTL7 Expression in the Human Trabecular Meshwork.

Regulating intraocular pressure (IOP), mainly via the trabecular meshwork (TM), is critical in developing glaucoma. Whereas current treatments aim to lower IOP, directly targeting the dysfunctional TM tissue for therapeutic intervention has proven challenging. In our study, we utilized Dexamethasone (Dex)-treated TM cells as a model to investigate how extracellular vesicles (EVs) from immortalized corneal stromal stem cells (imCSSCs) could influence ANGPTL7 and MYOC genes expression within TM cells. Human TM cell lines were isolated and cultured from donor corneoscleral rims. EVs were purified from imCSSC conditioned media (CM) using size exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy (TEM), and ExoView technology. TM cells were treated with either Dex alone or with EVs for 5 days. Quantitative polymerase chain reaction (PCR) was carried out to quantify the mRNA level of MYOC and ANGPTL7. A notable increase in the expression levels of MYOC and ANGPTL7 genes was observed compared with untreated TM cells (control). Furthermore, upon comparing Dex-treated TM cells with those receiving both Dex and EV treatments, a statistically significant reduction in ANGPTL7 expression (P < 0.05) was detected. The present study demonstrates that imCSSCs-derived EVs can effectively decrease the expression of ANGPLT7, a gene associated with fibrosis and implicated in the abnormal elevation of IOP in patients with glaucoma. Our study shows that imCSSC-derived EVs can specifically target ANGPTL7 expression, making them a promising preclinical therapy for glaucoma.

P1341 Probing the interaction of probiotic bacterial extracellular vesicles with primary colonic epithelium using an ex-vivo organoid platform

Abstract Background Bacterial extracellular vesicles (BEV) are increasingly recognized as a relevant mode of contactless host-microbiota communication. BEV are nanoparticles pinched off from the membrane of multiple bacteria and carry nucleic acid snippets and functional proteins inherited from their parental bacteria. While evidence suggests that BEV act as transmitters to modulate the immune response, only scarce data exist as to whether and how efficiently BEV are internalized by colonic epithelium as the host‘s front line in the gut. This study aims to quantify BEV internalization into primary colonic epithelium and explore structural and physicochemical properties of BEV potentially influencing uptake. Methods BEV were isolated from Limosilactobacillus reuteri (LR) and Lactiplantibacillus plantarum (LP) and incubated with either colonoids or colonic epithelial monolayers from C57BL/6 mice. BEV were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), zeta potential (ZP) analysis, as well as lipidomic and proteomic analysis. Uptake of BEV into colonic epithelium was assessed by confocal microscopy and flow cytometry. Results BEV of either species displayed a uniform appearance with a lipid bilayer on TEM. NTA showed similar spectra of sizes with a dominant peak around 100 nm whereas with DLS particle size readings were higher and showed a bimodal distribution. The ZP of BEV measured in buffer with physiological salinity did not differ between LR and LP. Gas chromatographic mass spectrometry analysis revealed neither qualitative nor quantitative differences in the composition of long chain fatty acid in BEV membranes. As opposed to LP, LR pellets showed proteomic enrichment of pathways linked to glycerolipid metabolism, fatty acid biosynthesis, and terpenoid backbone synthesis, being precursor steps of lipoteichoic acid synthesis (LTA). LTA could be detected in BEV of both species, but appeared to more abundant in LR-BEV. A dose-dependent increment in BEV uptake rates was noted in both species, with LR-BEV being more readily internalized by colonic epithelium. Epithelial cells exposed to LR-BEV were characterized by irregular nuclei and partial detachment from the surface whilst cells incubated with LP-BEV appeared morphologically normal. The quantity of viable cells was reduced in colonoids treated with LR-BEV, but not LP-BEV. In the presence of LR-BEV, colonoids were less likely to undergo apoptosis. Conclusion LR- and LP-BEV differ in their ability to enter colonic epithelium and interfere with cellular homeostasis. A variable degree of LTA decoration of BEV membranes might account for differential uptake into the host cell.

P1310 Role of gut pathobiont-derived membrane vesicles for biliary epithelium in PSC-IBD

Abstract Background Primary sclerosing cholangitis (PSC), a cholestatic liver disease highly associated with inflammatory bowel disease (IBD), is characterized by progressive inflammatory destruction of bile ducts. Bacterial membrane vesicles (bEVs) derived from gut pathobionts may play a significant role in the pathogenesis of PSC-IBD by disrupting the biliary barrier, promoting liver inflammation and contributing to liver fibrosis. A deeper understanding of how bEVs influence bile duct homeostasis is crucial for unraveling the mechanisms behind PSC-IBD and developing targeted therapeutic strategies. Methods Bile duct organoids were generated from WT mice and PSC-IBD patients. BEVs were isolated from commensals or PSC-associated gut pathobionts following MISEV guidelines. To assess the role of bEVs to ductal barrier dysfunction and inflammation, we employed an organoid based permeability assay, immunofluorescence staining, qPCR and bulk RNA sequencing. Furthermore, to investigate whether the presence and amount of bEVs in the systemic circulation were associated with PSC-IBD, we analyzed serum-derived EVs from either healthy control, as well as PSC and PSC-IBD patients using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and a TLR4-Receptor based reporter assay. Results Our results demonstrate that particularly bEVs isolated from gut pathobionts trigger signaling pathways associated with inflammation in both murine and human biliary epithelial cells. Furthermore, we observed a disease and strain specific fingerprint after bEV treatment, which was characterized by several significantly enriched terms related to PSC-related inflammation, including genes involved in T-helper 17 cell differentiation, and tumor necrosis factor (TNF) signaling. However, we did not observe a direct impact of bEVs on biliary barrier function. Consistent with this, we detected increased levels of bEVs in the systemic circulation of PSC-IBD patients, but not in those with PSC alone or in healthy controls, suggesting that these bacterial components may translocate across a compromised intestinal barrier into the bloodstream. Conclusion Overall, our study suggests that bEVs may contribute to biliary inflammation, but not barrier dysfunction and thereby may represent a novel player in the pathogenesis of PSC-IBD. Additionally, it underscores the critical role of bEVs in host-microbe communication, suggesting their potential as diagnostic biomarkers and highlighting, that therapeutic strategies targeting bEVs could offer promising avenues for managing PSC-IBD.

Exposure to Group B Streptococcus-induced chorioamnionitis alters the proteome of placental extracellular vesicles.

Group B Streptococcus (GBS) is an opportunistic pathogen that can induce chorioamnionitis (CA), increasing the risk of neurodevelopmental disorders (NDDs) in the offspring. The placenta facilitates maternal-fetal communication through the release of extracellular vesicles (EVs), which may carry inflammatory molecules such as interleukin (IL)-1. Although the role of EVs in immune modulation is well established, their specific characterization in the context of GBS-induced CA has not yet been investigated. Understanding placental-derived EVs could further define how IL-1 and other inflammatory factors contribute to NDDs. We used an established rat model of GBS-induced CA. EVs from control and GBS infected dams were isolated from placentas and characterized using nanoparticle tracking analysis and transmission electron microscopy. The protein content was assessed via mass spectrometry, followed by subsequent pathway analysis. ELISA was used to quantify cytokine levels. GBS-infected placentas exhibited calcification and increased weight, while fetal weight decreased. Analysis of the proteome from control versus GBS placental EVs revealed distinct profiles, with many proteins involved in the innate immune response, including alarmins (S100A8/9), complement pathways, and cytokine signaling pathways. Pathway analysis highlighted IL-1α and IL-1β identified as key upstream regulators. Notably, EVs from GBS-infected males showed a 44-fold increase in intracellular IL-1β compared to controls. These findings indicate that GBS-induced CA alters the protein content of EVs from placental cells. Our findings of increased IL-1β-associated EVs highlight the need for further investigation into the role of these cytokines from GBS-exposed placentas and their role in brain injuries leading to NDDs.

Molecular Characterization of Whole Genome Sequencing of Salmonella spp. in Shapingba District, Chongqing, China, 2016-2023.

In recent years, Salmonella infection is a major global public health concern, particularly in food safety. This study analyzed the genomes of 102 Salmonella strains isolated between 2016 and 2023 from food, foodborne disease patients, and food poisoning incidents, focusing on their molecular characteristics, antibiotic resistance genes (ARGs), and virulence genes. S. enterica serovar Enteritidis (37.3%) and S. enterica serovar Typhimurium (21.6%, including its monophasic variant 1,4,[5],12:i:-) were the main strains among 22 serotypes. Multilocus sequence typing revealed 23 sequence types (STs), with ST11, ST19, and ST34 as the most prevalent. All strains carried at least 24 ARGs. Detection rates for aac(6')-Iy, blaTEM-1, and sul2 ranged from 44.1% to 63.7%, mainly in S. Enteritidis and S. Typhimurium. Rates for qnrS1, sul1, and aadA were 12.8% to 16.7%, while mcr-1 appeared in one ST34 S. Typhimurium strain. All strains contained at least 98 virulence genes. The genes pefABCD, mig-5, and spvBCD were in 48.0% of strains, while rck was found in 36.3%, mainly linked to S. Enteritidis and S. Typhimurium. The tssM gene was found in 37.3% of the strains, exclusively in S. Enteritidis. Core genome single nucleotide polymorphisms (cgSNPs) analysis grouped the strains into nine clusters, with 75.5% belonging to three major groups. Food poisoning event 1 was correlated with cluster 3, while events 2 and 3 were linked to cluster 1. Across events, SNP differences among strains were ≤6. Strains with SNP differences ≤10 were also found in other clusters. This method is promising for tracking sporadic cases and identifying potential foodborne safety incidents. Salmonella strains in the region exhibit substantial genetic diversity, demonstrating the efficacy of cgSNPs analysis for source tracking. Ongoing surveillance is essential given the prevalence of ARGs and virulence genes. This study provides a data foundation for local Salmonella epidemiology.

Small extracellular vesicles derived from sequential stimulation of canine adipose-derived mesenchymal stem cells enhance anti-inflammatory activity

BackgroundSmall extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) are recognized for their therapeutic potential in immune modulation and tissue repair, especially in veterinary medicine. This study introduces an innovative sequential stimulation (IVES) technique, involving low-oxygen gas mixture preconditioning using in vitro fertilization gas (IVFG) and direct current electrical stimulation (ES20), to enhance the anti-inflammatory properties of sEVs from canine adipose-derived MSCs (cAD-MSCs). Initial steps involved isolation and comprehensive characterization of cAD-MSCs, including morphology, gene expression, and differentiation potentials, alongside validation of the electrical stimulation protocol. IVFG, ES20, and IVES were applied simultaneously with a control condition. Stimulated cAD-MSCs were evaluated for morphological changes, cell viability, and gene expressions. Conditioned media were collected and purified for sEV isolation on Day1, Day2, and Day3. To validate the efficacy of IVES for sEV production, various analyses were conducted, including microscopic examination, surface marker assessment, zeta-potential measurement, protein quantification, nanoparticle tracking analysis, and determination of anti-inflammatory activity.ResultsWe found that IVES demonstrated non-cytotoxicity and induced crucial genotypic changes associated with sEV production in cAD-MSCs. Interestingly, IVFG influenced cellular adaptation, while ES20 induced hypoxia activation. By merging these stimulations, IVES enhanced sEV stability and quality profiles. The cAD-MSC-derived sEVs exhibited anti-inflammatory activity in lipopolysaccharide-induced RAW264.7 macrophages, emphasizing their improved effectiveness without cytotoxicity or immunogenicity. These effects were consistent across day 3 collection, indicating the establishment of an effective protocol for sEV production.ConclusionsThis research established an innovative sequential stimulation method with positive impact on sEV characteristics including stability, quality, and anti-inflammatory activity. This study not only contributes to the enhancement of sEV production but also sheds light on their functional aspects for therapeutic interventions.

Klebsiella pneumoniae-derived extracellular vesicles impair endothelial function by inhibiting SIRT1

BackgroundThe potential role of Klebsiella pneumoniae (K.pn) in hypertension development has been emphasized, although the specific mechanisms have not been well understood. Bacterial extracellular vesicles (BEVs) released by Gram-negative bacteria modulate host cell functions by delivering bacterial components to host cells. Endothelial dysfunction is an important early event in the pathogenesis of hypertension, yet the impact of K.pn-secreted EVs (K.pn EVs) on endothelial function remains unclear. This study aimed to investigate the effects of K.pn EVs on endothelial function and to elucidate the underlying mechanisms.MethodsK.pn EVs were purified from the bacterial suspension using ultracentrifugation and characterized by transmission electron microscopy nanoparticle tracking analysis, and EV marker expression. Endothelium-dependent relaxation was measured using a wire myograph after in vivo or ex vivo treatment with K.pn EVs. Superoxide anion production was measured by confocal microscopy and HUVEC senescence was assessed by SA-β-gal activity. SIRT1 overexpression or activator was utilized to investigate the underlying mechanisms.ResultsOur data showed that K.pn significantly impaired acetylcholine-induced endothelium-dependent relaxation and increased superoxide anion production in endothelial cells in vivo. Similarly, in vivo and ex vivo studies showed that K.pn EVs caused significant endothelial dysfunction, endothelial provocation, and increased blood pressure. Further examination revealed that K.pn EVs reduced the levels of SIRT1 and p-eNOS and increased the levels of NOX2, COX-2, ET-1, and p53 in endothelial cells. Notably, overexpression or activation of SIRT1 attenuated the adverse effects and protein changes induced by K.pn EVs on endothelial cells.ConclusionThis study reveals a novel role of K.pn EVs in endothelial dysfunction and dissects the relevant mechanism involved in this process, which will help to establish a comprehensive understanding of K.pn EVs in endothelial dysfunction and hypertension from a new scope.

STIPS algorithm enables tracking labyrinthine patterns and reveals distinct rhythmic dynamics of actin microridges.

Tracking and motion analyses of semi-flexible biopolymer networks from time-lapse microscopy images are important tools that enable quantitative measurements to unravel the dynamic and mechanical properties of biopolymers in living tissues, crucial for understanding their organization and function. Biopolymer networks are challenging to track due to continuous stochastic transitions, such as merges and splits, which cause local neighbourhood rearrangements over short time and length scales. To address this, we propose the STIPS algorithm (Spatio Temporal Information on Pixel Subsets) to track these events by creating pixel subsets that link trajectories across frames. Using this method, we analysed actin-enriched protrusions, or 'microridges,' which form dynamic labyrinthine patterns on squamous cell epithelial surfaces, mimicking 'active Turing-patterns.' Our results reveal two distinct actomyosin-based rhythmic dynamics in neighbouring cells: a common pulsatile mechanism between 2 and 6.25 minutes period governing both fusion and fission events contributing to pattern maintenance, and cell area pulses predominantly exhibiting 10-minutes period.

Hypoxic BMSC-derived exosomal miR-210-3p promotes progression of triple-negative breast cancer cells via NFIX-Wnt/β-catenin signaling axis

BackgroundBone marrow mesenchymal stem cells (BMSCs) are a crucial component of the tumor microenvironment (TME), with hypoxic conditions promoting their migration to tumors. Exosomes play a vital role in cell-to-cell communication within the TME. Hypoxic TME have a great impact on the release, uptake and biofunctions of exosomes. This study aims to elucidate the communication between BMSC-derived exosomal miRNA and triple-negative breast cancer (TNBC) in a hypoxic environment.MethodsExosomes were isolated via ultracentrifugation and identified using scanning electron microscopy (SEM), nanoparticle tracking analysis (NTA) and western blot. A range of bioinformatics approaches were used to screen exosomal miRNAs and the target mRNAs of miRNAs and predict the possible signaling pathways. Expression levels of genes and proteins were assessed by quantitative real-time PCR and western blot. Cell proliferation, apoptosis, migration and invasion were analyzed using CCK-8 assay, EDU assay, transwell migration, wound healing assay and invasion assay, respectively. Dual luciferase reporter gene assay was conducted to confirm the binding between miRNAs and the target mRNAs. The impact of hypoxic BMSC-derived exosomal miRNA on TNBC progression in vivo was evaluated using tumor xenograft nude mouse models. Furthermore, the impact of patients’ serum exosomal miRNA on TNBC was implemented.ResultsExosomes derived from hypoxic BMSCs promotes the proliferation, migration, invasion and epithelial-mesenchymal transition of TNBC and suppresses the apoptosis of TNBC. The expression of miR-210-3p in BMSC-derived exosomes is markedly elevated in hypoxic conditions. Exosome-mediated transfer of miR-210-3p from hypoxic BMSCs to TNBC targets NFIX and activates Wnt/β-Catenin signaling in TNBC. Deletion of miR-210-3p in hypoxic BMSC-derived exosomes attenuates TNBC in vivo. Additionally, human exosomal miR-210-3p from the serum of TNBC patients promotes TNBC progression. Moreover, we notably observed a marked downregulation of NFIX expression levels in cancerous tissues compared to paracancerous tissues.ConclusionsHypoxic BMSC-derived exosomal miR-210-3p promotes TNBC progression via NFIX-Wnt/β-catenin signaling axis.

Proteomic Profiling of Extracellular Vesicles in Inflammatory Bowel Diseases.

The proteomic analysis of serum extracellular vesicles (EVs) could be a useful tool for studying the pathophysiology of Crohn's disease (CD) and ulcerative colitis (UC), as well as for biomarker discovery. To characterize the proteomic composition of serum EVs in patients with CD and UC to identify biomarkers and molecular pathways associated with pathogenesis and activity. Methods: Serum EVs were enriched and analyzed in patients with quiescent CD, active CD (aCD), quiescent UC, active UC (aUC), and healthy controls (HCs) (n = 30 per group). All groups were matched for age and sex. Disease activity was assessed by ileocolonoscopy and categorized based on the SES-CD (CD) and the endoscopic sub-score of the Mayo Score (UC). EVs were enriched by ul-tracentrifugation, and their size and concentration were determined by nanoparticle tracking analysis. The expression of CD63, CD81, and CD9 was determined using West-ern blotting. Proteomic analysis was performed by label-free nano-LC MS/MS. A total of 324 proteins were identified; 60 showed differential abundance in CD-HC, 34 in UC-HC, and 21 in CD-UC. Regarding disease activity, the abundance of 58 and 32 proteins was altered in aCD-HC and aUC-HC, respectively. Functional analyses revealed that proteins associated with aCD were involved in immune regulation, whereas those linked to aUC were enriched in oxidative stress. We have identified expressed proteins between EVs from patients with CD and UC, depending on the presence of disease, the disease type, and the disease activity. These proteins are potential candidates as disease biomarkers and open new research avenues to better understand these conditions.