EV Concentration Research Articles

9317 Diabetogenic Stress-Mediated Beta Cell Extracellular Vesicle:Autophagy Pathway Crosstalk

Abstract Disclosure: A. Roy: None. G. Ariyaratne: None. A. Matveyenko: None. N. Javeed: None. Diabetogenic stressors including elevated free fatty acids (lipotoxicity), low-grade systemic inflammation, and proteotoxicity are central to the development of Type 2 Diabetes (T2D). Due to their high protein synthetic burden, β-cells employ autophagy and endo/lysosomal pathways to reduce protein aggregates, and protect β-cell mass and function. Recently, extracellular vesicles (EVs; 30-150 nm sized nanoparticles) have been shown to work in tandem with autophagy to facilitate cellular adaptation and intercellular communication. As diabetogenic stressors have been shown to perturb autophagy via lysosomal defects, our group has noted enhanced EV secretion. Thus, we hypothesize that diabetogenic factors enhance EV secretion through activation of an EV-based secretory autophagy pathway and that re-balancing of both pathways could improve β-cell function in T2D. To test this, MIN6 cells were exposed to free fatty acid palmitate (PAL; 24h), or pro-inflammatory cytokines IL-1β, TNFα, and IFNγ (CYTO; 48h). Expression of autophagic markers, LC3 and p62 were increased in CYTO and PAL conditions suggesting defects in auto/lysosomal function. Upon PAL/CYTO treatment in MIN6 cells, conditioned media EVs were isolated using differential ultracentrifugation. We noted enhanced protein expression of LC3-II and p62 in PAL- and cytoEVs, suggesting a diversion of immature autophagosome components into EVs due to lysosomal inhibition. To restore balance between autophagy:EV flux, we used EV biogenesis inhibitor, GW4869 (GW; 24h) or autophagy inducer, rapamycin (Rapa; 24h) on MIN6 cells exposed to PAL or CYTO. Isolated EVs were subjected to Nanoparticle Tracking Analysis (NTA) which showed a significant reduction in EV concentration with GW or Rapa addition (p<.05). Isolated mouse islet exposed to CYTO+GW/Rapa or PAL+GW/Rapa showed significant improvements in glucose stimulated insulin secretion (vs. CYTO or PAL; p<.05). Taken together, these data implicate activation of EV-based secretory autophagy in response to diabetogenic stressors and thus, potential targets to improve functional β-cell mass. Presentation: 6/2/2024

EV-miRNAs from breast cancer patients of plasma as potential prognostic biomarkers of disease recurrence

BackgroundExtracellular vesicles (EVs), ubiquitously released by blood cells, facilitate intercellular communication. In cancer, tumor-derived EVs profoundly affect the microenvironment, promoting tumor progression and raising the risk of recurrence. These EVs contain miRNAs (EV-miRNAs), promising cancer biomarkers. Characterizing plasma EVs and identifying EV-miRNAs associated with breast cancer recurrence are crucial aspects of cancer research since they allow us to discover new biomarkers that are effective for understanding tumor biology and for being used for early detection, disease monitoring, or approaches to personalized medicine. This study aimed to characterize plasma EVs in breast cancer (BC) patients and identify EV-miRNAs associated with BC recurrence. MethodsThis retrospective observational study included 24 BC patients divided into recurrence (n= 11) and non-recurrence (n= 13) groups. Plasma EVs were isolated and characterized. Total RNA from EVs was analyzed for miRNA expression using NanoString's nCounter® miRNA Expression Assays panel. MicroRNA target prediction used mirDIP, and pathway interactions were assessed via Reactome. ResultsA stronger presence of circulating EVs was found to be linked with a less favorable prognosis (p = 0.0062). We discovered a distinct signature of EV-miRNAs, notably including miR-19a-3p and miR-130b-3p, which are significantly associated with breast cancer recurrence. Furthermore, miR-19a-3p and miR-130b-3p were implicated in the regulation of PTEN and MDM4, potentially contributing to breast cancer progression.A notable association emerged, indicating a high concentration of circulating EVs predicts poor prognosis (p = 0.0062). Our study found a distinct EV-miRNA signature involving miR-19a-3p and miR-130b-3p, strongly associated with disease recurrence. We also presented compelling evidence for their regulatory roles in PTEN and MDM4 genes, contributing to BC development. ConclusionThis study revealed that increased plasma EV concentration is associated with BC recurrence. The prognostic significance of EVs is closely tied to the unique expression profiles of miR-19a-3p and miR-130b-3p. These findings underscore the potential of EV-associated miRNAs as valuable indicators for BC recurrence, opening new avenues for diagnosis and treatment exploration.

Facilitatory effect of low-pulse repetition frequency ultrasound on release of extracellular vesicles from cultured myotubes

PurposeExtracellular vesicles (EVs) serve as carriers of intracellular factors with therapeutic effects, including tissue regeneration and attenuation of inflammatory responses. The majority of EVs in vivo are derived from skeletal muscle, which is reported to have anti-inflammatory effects. While high-intensity pulsed ultrasound (US) irradiation has been shown to promote EV secretion from myotubes, the impact of pulse repetition frequency, a US parameter affecting pulse length, on EV release remains unclear. This study aimed to investigate the impact of pulse repetition frequency of US on the release of EVs from myotubes.MethodsC2C12 myoblasts were used in this study. After differentiation into C2C12 myotubes, US was performed for 5 min at an intensity of 3.0 W/cm2, duty cycle of 20%, acoustic frequency of 1 MHz, and different pulse repetition frequencies (100 Hz, 10 Hz, or 1 Hz). After 12 h, EVs and cells were collected for subsequent analyses.ResultsUS did not cause a reduction in cell viability across all US groups compared to the control. The concentration of EVs was significantly higher in all US groups compared to the control group. In particular, the highest increase was observed in the 1-Hz group on EV concentration as well as intracellular Ca2+ level.ConclusionThis study investigated the effect of three different pulse repetition frequencies of US on the release of EVs from cultured myotubes. It is concluded that a low-pulse repetition frequency of 1 Hz is the most effective for enhancing EV release from cultured myotubes with pulsed ultrasound.

Analysing the suitability of CaTiO3/Ca1–xSrxTiO3/SrTiO3 perovskite for fabrication of optoelectronic devices using QuantumATK tool: a study for electronic and optical properties

Calcium Titanate (CaTiO3) as a material has been researched for various applications like those in capacitors, piezoelectric devices and ceramic applications. Same has been possible owing to the exhibition of various characteristics of this material like high melting point (1,625 °C) and dielectric properties. However, only limited studies have been conducted to understand the impact of Strontium (Sr) doping and possibility of adopting this material into any kind of optoelectronic applications. This paper carries on an in-depth analysis on CaTiO3 and study effect of Sr as a dopant on its properties. Analysis has been carried out using Quantum ATK tool based on DFT approximations. CaTiO3 perovskite properties were studied by using the GGA (Generalized Gradient Approximation) and PBE (Perdew–Burke–Ernzerhof) functionals, which are used with the LCAO calculator. As a result of the inclusion of Sr at the Ca site, the electronic band structure of CaTiO3 was changed by the introduction of a gamma point. Bandgap of CaTiO3 was analysed to be dependent on the Sr site concentration and increased from 2.3 eV (Direct bandgap) to 2.3612 eV (Indirect bandgap), when Sr and Ca site concentration were equal, but it decreased with further increase in Sr site concentration to 1.7155 eV. Pure CaTiO3’s projected density of states changed after doping, providing evidence for the effects of the dopant on the system. As a result of an examination of the optical properties of both systems, it is found that CaTiO3 at 380 nm (close to infrared) exhibits an effective extinction coefficient (k) value of 0.78, whereas at 600 nm, the attenuation of light decreases to zero. This makes Sr doping in CaTiO3 another attractive candidate for optical devices in addition to its ability to change optical properties.

Trap States and Carrier Diffusion in Czochralski (100) Single Crystal β-Ga2O3

Deep trap spectra and carrier diffusion lengths were measured for unintentionally doped β-Ga2O3 bulk crystals with (100) orientation. The 20-mm diameter, 15-mm length boule was pulled by the Czochralski method from gallium oxide in (010) direction. It is found that the net density of shallow donors in (100) plates cleaved from the crystal was 2.6 × 1017 cm−3, with ionization energies of 0.05 eV measured from admittance spectra. Three deep electron traps with respective ionization energies of 0.6 eV (concentration 1.1 × 1014 cm−3), 0.8 eV (concentration 3.9 × 1016 cm−3) and 1.1 eV (concentration 8.9 × 1015 cm−3) were detected by Deep Level Transient Spectroscopy. The dominant 0.8 eV trap is associated with the E2 centers due to Fe acceptors, the two other traps are the well documented E1 and E3 centers. The major deep acceptors in the lower half of the bandgap have optical ionization threshold of 2.3 eV and concentration of 4 × 1015 cm−3 and are believed to be due to the split Ga vacancies acceptors. The diffusion length of non-equilibrium charge carriers was 90 nm. The electrical properties of these (100) oriented crystals grown by Czochralski are quite similar to those synthesized by the undoped Edge-defined Film-Fed Growth technique.

Multilayer Characterization of Plasma EV Concentration and Cargo Allows to Diagnose and Monitor Glioblastoma by Liquid Biopsy

Multilayer Characterization of Plasma EV Concentration and Cargo Allows to Diagnose and Monitor Glioblastoma by Liquid Biopsy

Vapor transport deposited Sb2(S,Se)3 thin film: Effect of deposition temperature and Sb2S3/Sb2Se3 mass ratio

The vapor transport deposition (VTD) processing is an effective and low-cost techniques to fabricate antimony selenosulfide (Sb2(S,Se)3) photovoltaic materials with large grains and preferred crystal orientations. Currently, the influences of various single deposition conditions of VTD on performances of Sb2(S,Se)3 solar cells have been studied, but which parameter should be optimized and how it affects is a noteworthy issue. Herein, the effects of two important deposition parameters, deposition temperature and mass ratio of Sb2S3/Sb2Se3 on the structure and photo-electrochemical properties of Sb2(S,Se)3 were investigated, and the mechanism of their impact on the growth of thin film was also discussed. It found that the (hk1) orientation growth of Sb2(S,Se)3 was dominated by Sb-Se bonds at deposition temperatures ranging from 460 °C to 500 °C. As the temperature increased, the S content in the film decreased. When the deposition temperature was fixed at 480 °C, it was found that the effect of the mass ratio of Sb2S3/Sb2Se3 on the growth was more about adjusting the concentration of Se than increasing the bonding of Sb-S. The introduced S can make the film smooth and dense, and also induce the growth of film in the (hk1) orientation when mass ratio is coupled with deposition temperature. Furthermore, the results of photo-electrochemical measurement indicated that photocurrent density is related to S content and preferred orientation. By adjusting the mass ratio, flat Sb2(S,Se)3 films were obtained with band gaps ranging from 1.24 eV to 1.42 eV and carrier concentrations ranging from 9.62 × 1014 cm−3 to 1.62 × 1015 cm−3, exhibiting good absorption layer characteristics.

Extracellular vesicles during the three trimesters of pregnancy

ObjectivesExtracellular vesicles (EVs) are cell-derived particles released during different pathophysiological processes and emerging as relevant players in inter-cellular crosstalk. Previous studies have highlighted the role of EVs as potential biomarkers for several pregnancy complications, including miscarriage, pre-eclampsia and gestational diabetes. Despite that, the actual distribution of EVs through gestation has not been reported yet. The aim of this study was to report the concentration of different sub-types of EVs in the first, second and third trimester of pregnancy and to correlate them with different pregnancy and ultrasound characteristics. Study designsProspective observational study including uncomplicated pregnancies in the first, second and third trimester of pregnancy. The first aim of the study was to report the concentration of the EVs derived from endothelial, epithelial, platelet and leukocyte cells of maternal peripheral blood samples in the first, second and third trimester pregnancy using polychromatic flow cytometry. The secondary aim was to correlate EVs with neonatal birthweight and fetal Dopplers, including uterine and umbilical arteries. Un and multivariate analyses were used to compute the data. Results64 women (20 in the first, 22 in the second and 22 in the third trimester of pregnancies) were included in the analysis. There was no difference in the median concentration of either platelet, leukocyte and endothelial EVs between the first, second and third trimester of pregnancy. The concentration of epithelial derived EVs was higher in the third compared to first and second trimester of pregnancy. When analyzing the percentage of EV vesicles through gestation, there was no difference in the percentage of either leukocyte or endothelial EVs through gestation. Conversely, the median percentage of platelet derived vesicles was higher in the first (48.7 %, IQR 34.1–58.5) compared to second (34.0 %, IQR 22.7–44.9) and third (9.13 %, IQR 5.01–12.1) trimester of pregnancy, while the median percentage of third trimester (6.01, IQR 2.42–7.34) epithelial derived vesicles was higher than that of the second (1.53 %, IQR 0.65–2.98), but not of the first (4.45 %, IQR 1.44–6.07) trimester. Finally, we found no association between the median concentration or percentage of endothelial, epithelial, leukocyte vesicles, neonatal birthweight and fetal or maternal Dopplers. ConclusionsDistribution of EVs examined does not change during the three trimesters of pregnancy and is not influenced by neonatal birthweight or maternal and fetal Dopplers. The findings from this study allows a more objective interpretation of studies comparing EVs in pregnancies with compared to those without obstetric complication. EVs in future can be used for "liquid biopsy" for the early diagnosis of pathological pregnancies up to the development of possible screening protocols.

Plasma extracellular vesicle test sample to standardize flow cytometry measurements

BackgroundExtracellular vesicles (EVs) in body fluids are explored as disease biomarkers, but EV concentrations measured by flow cytometers (FCMs) are incomparable. ObjectivesTo improve data comparability, new reference materials with physical properties resembling EVs and reference procedures are being developed. The validation of new reference materials and procedures requires biological test samples. We developed a human plasma EV test sample (PEVTES) that i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry–compatible, and iv) is stable. MethodsThe PEVTES was prepared from human plasma of 3 fasting donors. EVs were immunofluorescently stained with antibodies against platelet-specific (CD61) and erythrocyte-specific (CD235a) antigens or lactadherin. To reduce the concentration of soluble proteins, lipoproteins, and unbound reagents, stained EVs were isolated from plasma by size-exclusion chromatography. After isolation, the PEVTES was filtered to remove remnant platelets. PEVTESs were diluted in cryopreservation agents, dimethyl sulfoxide, glycerol, or trehalose and stored at −80 °C for 12 months. After thawing, stained EV concentrations were measured with a calibrated FCM (Apogee A60-Micro). ResultsWe demonstrate that the developed PEVTES resembles subcellular particles in human plasma when measured using FCM and that the concentrations of prestained platelet-derived, erythrocyte-derived, and lactadherin+ EVs in the PEVTES are stable during storage at −80 °C for 12 months when stored in trehalose. ConclusionThe PEVTES i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry–compatible, and iv) is stable. Therefore, the developed PEVTES is an ideal candidate to validate newly developed reference materials and procedures.

Extracellular RNA splice events in cerebrospinal fluid as candidate biomarkers of myotonic dystrophy type 1 (N4.004)

<h3>Objective:</h3> To evaluate extracellular RNA (exRNA) in cerebrospinal fluid (CSF) as a source of CNS-derived splice events that may serve as biomarker of DM1 disease activity. <h3>Background:</h3> Alternative splicing is mis-regulated in post-mortem DM1 central nervous system (CNS) tissue. In past few years Cerebrospinal Fluid (CSF) biomarkers have been used to characterize disease severity and prognosis of the disease and helpful tool for to measure changes after treatment. exRNA in CSF provides a source of CNS-derived splice events in living individuals. <h3>Design/Methods:</h3> We examined fresh CSF samples from unaffected controls (N=14) from the MGH CSF biobank and DM1 individuals with mild CNS involvement (N=2). To separate EVs from cells contained in CSF, we used low speed centrifugation followed by filtration of the supernatant. EV size and concentration were measured using microscopy and particle tracking analysis software (NanoSight). We extracted exRNA from the EV pellet, produced cDNA, and quantified gene expression and splice events (% exon inclusion) by droplet digital PCR. <h3>Results:</h3> Mean particle diameter is about 175 nm in CSF and serum vs. 220 nm in urine. In CSF cells, total RNA content is 10 – 100-fold higher than exRNA. Normalized expression of both <i>DMPK</i> and <i>CNBP</i> was 50 – 60% higher in CSF exRNA than in CSF cells. Splicing is significantly different in CSF exRNA vs CSF cells. Alternative splicing of GOLGA4 exon 23 inclusion was 65% in DM1 and 40% in UA (P&lt;0.01). Differential splicing of NUMA1, NCOR2 and MBNL2 was non-significant between groups. <h3>Conclusions:</h3> Quantification of CNS-derived alternative splice products in CSF exRNA is feasible. Removal of leukocytes and erythrocytes normally found in CSF will enhance accurate quantification of CNS-derived splice events. GOLGA4 is a candidate biomarker of early CNS involvement in DM1, while NUMA1, NCOR2, and MBNL2 splice events are candidate biomarkers of disease severity. <b>Disclosure:</b> Dr. Kumari has nothing to disclose. Mr. Hu has nothing to disclose. Mr. Sizemore has nothing to disclose. Ms. Sullivan has nothing to disclose. Mr. McKee has nothing to disclose. Mr. Conquest has nothing to disclose. Dr. Wheeler has nothing to disclose.

Abstract 4352: Leveraging deep proteome profiling of plasma- and serum-derived extracellular vesicles for melanoma biomarker discovery and disease dissection

Abstract Extracellular vesicles (EV) play an important role in melanoma progression but their potential as clinical biomarkers has yet to be realized. EVs can be found in most liquid biopsies (e.g., blood, urine, CSF) and exosomes are the most prominent subcategory of EVs. Exosomes are 50-200 nm lipid-bilayer enclosed particles secreted by all body cells, including tumor cells, and serve as mediators of metastasis formation and typically contain several classes of bioactive molecules such as RNA, proteins, lipids, and metabolites. Blood and its liquid components plasma/serum are the most frequently used matrix for biomarker discovery due to the ease of collection. However, most proteomic platforms for plasma/serum profiling are unable to profile EV proteins due to the high dynamic range of protein concentrations in EV preparations. This is due to 1) EV isolation methods that vary in their potential to separate EVs from free proteins, and 2) the presence of a natural corona of high-abundant blood proteins attached to the EV surface. To tackle this challenge, we developed SEC-DIA-MS an integrated workflow combining size-exclusion chromatography, EV concentration, and optimized mass spectrometry to enable deep profiling of the proteome content of the enriched vesicles. From 200 µl of plasma or serum from a test melanoma patient cohort (6 patients and 3 matched controls), we quantified 2,242 exosome-associated proteins, achieving a 2.5-fold increase in depth compared to previous melanoma studies. To gain a better understanding of the exosome enrichment efficiency, we extensively characterized the plasma/serum proteome by analyzing native, depleted, and EV-enriched blood from the same donors. We successfully validated well-known exosome markers such as CD9, CD63, CD81, PDCD6IP, and TSG101, and found that EV samples are significantly enriched in intact membrane proteins and those related to T cell biology, further underlining the uniqueness of the EV proteome composition. We further assessed the differences between plasma and serum EVs and suggest the use of plasma samples for future studies due to better separation of healthy and melanoma samples. Furthermore, known melanoma markers such as MCAM, TNC, and TGFBI were upregulated in melanoma plasma-derived EV but not depleted plasma samples, highlighting the specific information contained in EVs. Taken together, we demonstrated the applicability and potential of an EV-based proteomic workflow for biomarker discovery in plasma and serum. The ease of automating and scaling up such an approach enables a broader application to other indications and biological matrices. Citation Format: Evelyn Lattmann, Luca Räss, Marco Tognetti, Julia Martinez Gomez, Valérie Lapaire, Roland Bruderer, Lukas Reiter, Yuehan Feng, Lars M. Steinmetz, Mitchell P. Levesque. Leveraging deep proteome profiling of plasma- and serum-derived extracellular vesicles for melanoma biomarker discovery and disease dissection. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4352.

Investigation of the surface temperature in contact with plasma by two-color pyrometry

A high-speed two-color pyrometer has been developed that allows measuring the intensity of thermal radiation in temperature range of 100-3500oC with 2 μs time resolution. The analysis of the main factors affecting the measurement of surface temperature in the conditions of its interaction with plasma is carried out. It is shown that at plasma temperature of more than 10 eV and moderate concentration of particles, the wall plasma can be considered transparent to thermal radiation. Experimental studies on non-contact measurement of temperature of surface interacting with plasma on Globus-M2 tokamak and plasma gun test bench have been carried out. Sawtooth temperature fluctuations were detected on the wall of tokamak chamber near the exit of outer separatrix branch. The period of these flashes was 3-4 ms, and duration was 2 ms. The maximum wall temperature measured by a high-speed pyrometer exceeded the temperature measured by infrared camera. Measurements of temperature of the front surface of W-Li sample were carried out at plasma gun test bench under conditions of deuterium plasma jet cyclic action under loads simulating regimes of plasma current disruption in tokamak. The sample temperature reached more than 3000^oC during &amp;lt;1 ms. The thermal conductivity of the sample degraded with an increase in number of plasma exposure cycles, which was expressed in progressive decrease in target cooling rate after the pulse. Using a high-speed pyrometer, it is proposed to control the formation of heat-insulating foils on surface interacting with plasma Keywords: two-color pyrometer, thermal radiation, surface temperature, materials irradiation.

Исследование температуры поверхности, контактирующей с плазмой, методом двухцветной пирометрии

A high-speed two-color pyrometer has been developed that allows measuring the intensity of thermal radiation in temperature range of 100-3500 0C with 2 µs time resolution. The analysis of the main factors affecting the measurement of surface temperature in the conditions of its interaction with plasma is carried out. It is shown that at plasma temperature of more than 10 eV and moderate concentration of particles, the wall plasma can be considered transparent to thermal radiation. Experimental studies on non-contact measurement of temperature of surface interacting with plasma on Globus-M2 tokamak and plasma gun test bench have been carried out. Sawtooth temperature fluctuations were detected on the wall of tokamak chamber near the exit of outer separatrix branch. The period of these flashes was 3-4 ms, and duration was 2 ms. The maximum wall temperature measured by a high-speed pyrometer exceeded the temperature measured by infrared camera. Measurements of temperature of the front surface of W-Li sample were carried out at plasma gun test bench under conditions of deuterium plasma jet cyclic action under loads simulating regimes of plasma current disruption in tokamak. The sample temperature reached more than 3000 0C during &lt; 1 ms. The thermal conductivity of the sample degraded with an increase in number of plasma exposure cycles, which was expressed in progressive decrease in target cooling rate after the pulse. Using a high-speed pyrometer, it is proposed to control the formation of heat-insulating foils on surface interacting with plasma.

BIOM-34. MULTIPLEX PHENOTYPING OF EXTRACELLULAR VESICLES FOR ANALYSIS OF POTENTIAL BIOMARKERS IN GLIOBLASTOMA PATIENTS

Abstract INTRODUCTION Extracellular vesicles (EVs) carry biological information from their cell of origin that is useful for non-invasive detection of tumor biomarkers and disease monitoring. In glioblastoma (GBM), blood circulating EVs are elevated and carry GBM-associated proteins. However, it is still challenging to analyze tumor derived EVs for translational purposes. Here, we used imaging flow cytometry (IFCM) as a robust strategy to perform phenotyping of EVs with GBM related surface markers in human plasma. METHODS EVs were isolated via differential ultracentrifugation from plasma of (a) 40 GBM patients, pre- and post-surgery, (b) 11matched GBM relapses and (c) 12 healthy donors (HD). EV sizes and concentrations were evaluated by NTA. EV markers (CD9,CD63 and CD81) together with glioma-related markers (integrin beta-1 [ITGB1], tenascin C [TNC], Profilin-1 [PFN1], CD44,GPNMB, SPARC, HLA-II or CD133) were analyzed by IFCM. EV percentages and objects/mL plasma were compared among the groups and correlated with clinical parameters. RESULTS CD9 was the predominant tetraspanin in all groups (15-96%), while CD63 had the lowest levels (0-33%) and the strongestdecrease in GBM patients after surgery (fold change [FC]=-5.4, p&amp;lt;0.01). Among the glioma-related markers, ITGB1 and TNC displayed the most significant differences between the analyzed groups, especially the double positives ITGB1+/CD63+and TNC+/CD63+, which decreased in patients after tumor removal (FC=-3.5 and -12, respectively; p&amp;lt;0.001). Meanwhile,ITGB1+/CD9+and TNC+/CD9+EVs exhibited the highest levels in GBM when compared to HD subjects (FC=8.6 and 17.4;p&amp;lt;0.001) and upon tumor recurrence (FC=3.7 and 10.9, respectively; p&amp;lt;0.01). SUMMARY/CONCLUSION We identified EV surface antigens with potential clinical utility as GBM biomarkers. Among them, we highlight ITGB1 and TNC as the most promising markers.

Extracellular vesicles in heart failure - A study in patients with heart failure with preserved ejection fraction or heart failure with reduced ejection fraction characteristics undergoing elective coronary artery bypass grafting.

Extracellular vesicles (EVs) were investigated as potential biomarkers associated with heart failure (HF) pathophysiology in patients undergoing elective coronary artery bypass surgery characterized by HF phenotype. Patients with preoperative proxy-diagnoses of HF types i.e., preserved (HFpEF; n = 19) or reduced ejection fraction (HFrEF; n = 20) were studied and compared to patients with normal left ventricular function (n = 42). EVs in plasma samples collected from the coronary sinus, an arterial line, and from the right atrium were analyzed by flow cytometry. We studied EVs of presumed cardiomyocyte origin [EVs exposing Connexin-43 + Caveolin-3 (Con43 + Cav3) and Connexin-43 + Troponin T (Con43 + TnT)], of endothelial origin [EVs exposing VE-Cadherin (VE-Cad)] and EVs exposing inflammatory markers [myeloperoxidase (MPO) or pentraxin3 (PTX3)]. Median concentrations of EVs exposing Con43 + TnT and Con43 + Cav3 were approximately five to six times higher in coronary sinus compared to radial artery indicative of cardiac release. Patients with HFrEF had high trans-coronary gradients of both Con43 + TnT and Con43 + Cav3 EVs, whereas HFpEF had elevated gradients of Con43 + Cav3 EVs but lower gradients of Con43 + TnT. Coronary sinus concentrations of both Con43 + TnT and Con43 + Cav3 correlated significantly with echocardiographic and laboratory measures of HF. MPO-EV concentrations were around two times higher in the right atrium compared to the coronary sinus, and slightly higher in HFpEF than in HFrEF. EV concentrations of endothelial origin (VE-Cad) were similar in all three patient groups. Con43 + TnT and Con43 + Cav3 EVs are released over the heart indicating cardiomyocyte origin. In HFrEF the EV release profile is indicative of myocardial injury and myocardial stress with elevated trans-coronary gradients of both Con43 + TnT and Con43 + Cav3 EVs, whereas in HFpEF the profile indicates myocardial stress with less myocardial injury.

Acute changes in free and extracellular vesicle-associated circulating miRNAs and myokine profile in professional sky-runners during the Gran Sasso d'Italia vertical run.

The modification of gene expression profile, a first step in adaptation to exercise, leads to changes in the level of molecules associated with skeletal muscle activity and energy metabolism—such as myokines—as well as those involved in their transcriptional regulation, like microRNA. This study aimed to investigate the influence of strenuous exercise on circulating microRNAs and their possible association with myokine response. Pre-competition and post-competition plasma samples were collected from 14 male athletes participating in a vertical run (+1,000 m gain, 3,600 m length). Circulating total (t-miRNA) and extracellular vesicle-associated (EV-miRNA) miRNAs were extracted from the pooled plasma. Nanoparticle tracking analysis was performed to investigate pre- and post-competition EV concentration and size distribution. A panel of 179 miRNAs was assayed by qPCR and analyzed by Exiqon GenEx v6 normalized on the global mean. t-miRNA and EV-miRNAs whose level was ≥5-fold up- or down-regulated were validated for each single subject. Target prediction on MirWalk v3.0, Gene-Ontology, and pathway enrichment analysis on Panther v17.0 were performed to define the potential biological role of the identified miRNAs. A panel of 14 myokines was assayed in each sample by a multiplex immunoassay. In whole plasma, five miRNAs were upregulated and two were downregulated; in the EV fraction, five miRNAs were upregulated and three were downregulated. Nanoparticle tracking analysis revealed a similar EV size distribution in pre- and post-competition samples and a decreased concentration in post-competition samples related to pre-competition samples. Gene-Ontology and pathway enrichment analysis revealed that the identified t-miRNAs and EV-miRNAs were potentially involved in metabolism regulation in response to exercise. Correlation between fold-change of the post-competition relative to pre-competition plasma level of both t-miRNAs and EV-miRNAs and myokines further confirmed these results. This study provides an example of a systemic response to acute endurance exercise, in which circulating miRNAs play a pivotal role.

Extracellular Vesicles in pregnancy: Their potential role as a liquid biopsy

ObjectiveExtracellular Vesicles (EVs) are cell-derived particles released during different pathophysiological processes, circulating in many body fluids and mediating the inter-cellular crosstalk. We have analyzed, for the first time, different EV phenotypes and concentrations in the peripheral blood of uncomplicated pregnant women. Study designIn this prospective case-control study, uncomplicated singleton pregnant women at term (N = 59) and aged matched non-pregnant women (N = 21) were enrolled. Freshly drowned peripheral blood samples were stained for flow cytometry analyses of EVs. ResultsEVs derived from platelets, leukocytes, endothelial and epithelial cells were identified and counted. Platelet-derived EVs were higher in pregnant compared to non-pregnant women, both in terms of absolute counts (2064.4 ± 1156.3 vs 701.1 ± 378.8; p < 0.0001) and percentages (27.6 ± 17.2 vs 10.7 ± 5.9; p < 0.0001). The opposite pattern was observed both for concentrations of endothelial-EV counts (525.8 ± 499.6 vs 844.7 ± 652.9; p = 0.007) and percentages (6.1 ± 5.5 vs 11.8 ± 8.0; p < 0.0001) and leukocyte-derived EV percentages (10.2 ± 7.4 vs 17.9 ± 11.2; p = 0.002) EVs. ConclusionsUncomplicated pregnancies are characterized by a specific EV signature. These cell-derived particles may therefore represent promising biomarkers of different pathological conditions complicating pregnancies, such as preeclampsia or preterm birth.

Distribution Grid planning by self-designing with reinforcement learning

Decentral energy resources are identified as indispensable for a successful energy transition with the target to reduce greenhouse gas emissions. This leads to amplifying electrification thereby posing new challenges to the distribution grid. In Germany, as in most countries, the electrical grid was designed based on central energy generation principles along with rather static load profiles. Given the two trends of electrification and decentralization, the distribution grid needs to be reinforced more dynamically than before. In this paper, a fully automated pipeline of grid planning is presented. Its central part is a novel (Multi-) Reinforcement Learning scheme operating on the electrical graph. It performs sequential grid planning actions such as switching, cable reinforcement and cable construction. Costs for construction are assigned by a cable route planner based on open street maps. Rewards depending on costs and load flow calculations on real grid data steer the optimization. It is shown that the optimizer develops strategies to solve grid violations while reducing associated costs for the actions. The framework is used to extract grid action sequences which are needed to mitigate increasing EV penetration. Furthermore, the dependency of EV density and associated grid action costs is evaluated quantitatively by running the optimizer for several scenarios. It is shown that costs increase rapidly when exceeding certain limit EV concentrations.

Diagnostic potential of extracellular vesicles in meningioma patients.

Extracellular vesicles (EVs) play an important role in cell-cell communication, and tumor-derived EVs circulating in patient blood can serve as biomarkers. Here, we investigated the potential role of plasma EVs in meningioma patients for tumor detection and determined whether EVs secreted by meningioma cells reflect epigenetic, genomic, and proteomic alterations of original tumors. EV concentrations were quantified in patient plasma (n = 46). Short-term meningioma cultures were established (n = 26) and secreted EVs were isolated. Methylation and copy number profiling was performed using 850k arrays, and mutations were identified by targeted gene panel sequencing. Differential quantitative mass spectrometry was employed for proteomic analysis. Levels of circulating EVs were elevated in meningioma patients compared to healthy individuals, and the plasma EV concentration correlated with malignancy grade and extent of peritumoral edema. Postoperatively, EV counts dropped to normal levels, and the magnitude of the postoperative decrease was associated with extent of tumor resection. Methylation profiling of EV-DNA allowed correct tumor classification as meningioma in all investigated cases, and accurate methylation subclass assignment in almost all cases. Copy number variations present in tumors, as well as tumor-specific mutations were faithfully reflected in meningioma EV-DNA. Proteomic EV profiling did not permit original tumor identification but revealed tumor-associated proteins that could potentially be utilized to enrich meningioma EVs from biofluids. Elevated EV levels in meningioma patient plasma could aid in tumor diagnosis and assessment of treatment response. Meningioma EV-DNA mirrors genetic and epigenetic tumor alterations and facilitates molecular tumor classification.

Activated polymorphonuclear derived extracellular vesicles are potential biomarkers of periprosthetic joint infection.

Extracellular vesicles (EVs) are considered as crucial players in a wide variety of biological processes. Although their importance in joint diseases or infections has been shown by numerous studies, much less is known about their function in periprosthetic joint infection (PJI). Our aim was to investigate activated polymorphonuclear (PMN)-derived synovial EVs in patients with PJI. (1) Is there a difference in the number and size of extracellular vesicles between periprosthetic joint aspirates of patients with PJI and aseptic loosening? (2) Are these vesicles morphologically different in the two groups? (3) Are there activated PMN-derived EVs in septic samples evaluated by flow cytometry after CD177 labelling? (4) Is there a difference in the protein composition carried by septic and aseptic vesicles? Thirty-four patients (n = 34) were enrolled into our investigation, 17 with PJI and 17 with aseptic prosthesis loosening. Periprosthetic joint fluid was aspirated and EVs were separated. Samples were analysed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) and flow cytometry (after Annexin V and CD177 labelling). The protein content of the EVs was studied by mass spectrometry (MS). NTA showed particle size distribution in both groups between 150 nm and 450 nm. The concentration of EVs was significantly higher in the septic samples (p = 0.0105) and showed a different size pattern as compared to the aseptic ones. The vesicular nature of the particles was confirmed by TEM and differential detergent lysis. In the septic group, FC analysis showed a significantly increased event number both after single and double labelling with fluorochrome conjugated Annexin V (p = 0.046) and Annexin V and anti-CD177 (p = 0.0105), respectively. MS detected a significant difference in the abundance of lactotransferrin (p = 0.00646), myeloperoxidase (p = 0.01061), lysozyme C (p = 0.04687), annexin A6 (p = 0.03921) and alpha-2-HS-glycoprotein (p = 0.03146) between the studied groups. An increased number of activated PMN derived EVs were detected in the synovial fluid of PJI patients with a characteristic size distribution and a specific protein composition. The activated PMNs-derived extracellular vesicles can be potential biomarkers of PJI.