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Positronium is abundantly produced within the molecular voids of a patient's body during positron emission tomography (PET). Its properties dynamically respond to the submolecular architecture of the tissue and the partial pressure of oxygen. Current PET systems record only two annihilation photons and cannot provide information about the positronium lifetime. This study presents the in vivo images of positronium lifetime in a human, for a patient with a glioblastoma brain tumor, by using the dedicated Jagiellonian PET system enabling simultaneous detection of annihilation photons and prompt gamma emitted by a radionuclide. The prompt gamma provides information on the time of positronium formation. The photons from positronium annihilation are used to reconstruct the place and time of its decay. In the presented case study, the determined positron and positronium lifetimes in glioblastoma cells are shorter than those in salivary glands and those in healthy brain tissues, indicating that positronium imaging could be used to diagnose disease in vivo.

Purpose: The aim of this work is to investigate the feasibility of the Jagiellonian Positron Emission Tomography (J-PET) scanner for intra-treatment proton beam range monitoring., Methods: The Monte Carlo simulation studies with GATE and PET image reconstruction with CASToR were performed in order to compare six J-PET scanner geometries. We simulated proton irradiation of a PMMA phantom with a Single Pencil Beam (SPB) and Spread-Out Bragg Peak (SOBP) of various ranges. The sensitivity and precision of each scanner were calculated, and considering the setup's cost-effectiveness, we indicated potentially optimal geometries for the J-PET scanner prototype dedicated to the proton beam range assessment., Results: The investigations indicate that the double-layer cylindrical and triple-layer double-head configurations are the most promising for clinical application. We found that the scanner sensitivity is of the order of 10 -5 coincidences per primary proton, while the precision of the range assessment for both SPB and SOBP irradiation plans was found below 1 mm. Among the scanners with the same number of detector modules, the best results are found for the triple-layer dual-head geometry. The results indicate that the double-layer cylindrical and triple-layer double-head configurations are the most promising for the clinical application, CONCLUSIONS:: We performed simulation studies demonstrating that the feasibility of the J-PET detector for PET-based proton beam therapy range monitoring is possible with reasonable sensitivity and precision enabling its pre-clinical tests in the clinical proton therapy environment. Considering the sensitivity, precision and cost-effectiveness, the double-layer cylindrical and triple-layer dual-head J-PET geometry configurations seem promising for future clinical application., Competing Interests: Declaration of competing interest The Authors declare no conflict of interest., (Copyright © 2024 Associazione Italiana di Fisica Medica e Sanitaria. Published by Elsevier Ltd. All rights reserved.)

Discrete symmetries play an important role in particle physics with violation of CP connected to the matter-antimatter imbalance in the Universe. We report the most precise test of P, T and CP invariance in decays of ortho-positronium, performed with methodology involving polarization of photons from these decays. Positronium, the simplest bound state of an electron and positron, is of recent interest with discrepancies reported between measured hyperfine energy structure and theory at the level of 10 -4 signaling a need for better understanding of the positronium system at this level. We test discrete symmetries using photon polarizations determined via Compton scattering in the dedicated J-PET tomograph on an event-by-event basis and without the need to control the spin of the positronium with an external magnetic field, in contrast to previous experiments. Our result is consistent with QED expectations at the level of 0.0007 and one standard deviation., (© 2024. The Author(s).)

The problem of boron concentration monitoring during the boron neutron capture therapy (BNCT) therapy is one of the main challenges of this type of radiotherapy and is directly related to the nature of the interaction of neutrons with mater. Among the available in vivo methods of boron monitoring positron emission tomography seems to be very promising but it requires a new boron carrier with a β+ emitter, which is not yet clinically available. An alternative solution may be the prompt gamma radiation analysis (PGRA) based on the secondary radiation emitted in the interaction of neutrons with the patient's tissues. This method requires, however, compact gamma radiation detection systems sustaining high counting rates and characterized by very good energy resolution. In this contribution, we present state-of-the-art solutions for monitoring in BNCT based on PGRA. Moreover, we describe a new concept of such a system based on position-sensitive scintillator detectors equipped with an anti-Compton shield and data analysis supported with modern artificial intelligence algorithms., (© The Author(s) 2023. Published by Oxford University Press.)

In positron emission tomography (PET) studies, convolutional neural networks (CNNs) may be applied directly to the reconstructed distribution of radioactive tracers injected into the patient's body, as a pattern recognition tool. Nonetheless, unprocessed PET coincidence data exist in tabular format. This paper develops the transformation of tabular data into n-dimensional matrices, as a preparation stage for classification based on CNNs. This method explicitly introduces a nonlinear transformation at the feature engineering stage and then uses principal component analysis to create the images. We apply the proposed methodology to the classification of simulated PET coincidence events originating from NEMA IEC and anthropomorphic XCAT phantom. Comparative studies of neural network architectures, including multilayer perceptron and convolutional networks, were conducted. The developed method increased the initial number of features from 6 to 209 and gave the best precision results (79.8) for all tested neural network architectures; it also showed the smallest decrease when changing the test data to another phantom.

It was recently demonstrated that newly invented positronium imaging may be used for improving cancer diagnostics by providing additional information about tissue pathology with respect to the standardized uptake value currently available in positron emission tomography (PET). Positronium imaging utilizes the properties of positronium atoms, which are built from the electrons and positrons produced in the body during PET examinations. We hypothesized that positronium imaging would be sensitive to the in vitro discrimination of tumor-like three-dimensional structures (spheroids) built of melanoma cell lines with different cancer activities and biological properties. The lifetime of ortho-positronium (o-Ps) was evaluated in melanoma spheroids from two cell lines (WM266-4 and WM115) differing in the stage of malignancy. Additionally, we considered parameters such as the cell number, spheroid size and melanoma malignancy to evaluate their relationship with the o-Ps lifetime. We demonstrate pilot results for o-Ps lifetime measurement in extracellular matrix-free spheroids. With the statistical significance of two standard deviations, we demonstrated that the higher the degree of malignancy and the rate of proliferation of neoplastic cells, the shorter the lifetime of ortho-positronium. In particular, we observed the following indications encouraging further research: (i) WM266-4 spheroids characterized by a higher proliferation rate and malignancy showed a shorter o-Ps lifetime than WM115 spheroids characterized by a lower growth rate. (ii) Both cell lines showed a decrease in the lifetime of o-Ps after spheroid generation on day 8 compared to day 4 in culture, and the mean o-Ps lifetime was longer for spheroids formed from WM115 cells than for those formed from WM266-4 cells, regardless of spheroid age. The results of this study revealed that positronium is a promising biomarker that may be applied in PET diagnostics for the assessment of the degree of cancer malignancy., (© 2023. The Author(s).)

Extracellular vesicles (EVs) are released by all cells, both in physiological and pathological conditions. Their molecular charge and composition emerge as possible biomarkers, but EVs may also be considered for other clinical applications. This review discusses the role of other features of EVs, such as their lipid components or composition of glycans that form the EV corona and regulate EV biodistribution and uptake by target cells. The importance of EV electric charge has been discussed as a new insight into EV fate and destination.

Background: The Jagiellonian Positron Emission Tomograph is the 3-layer prototype of the first scanner based on plastic scintillators, consisting of 192 half-metre-long strips with readouts at both ends. Compared to crystal-based detectors, plastic scintillators are several times cheaper and could be considered as a more economical alternative to crystal scintillators in future PETs. JPET is also a first multi-photon PET prototype. For the development of multi-photon detection, with photon characterized by the continuous energy spectrum, it is important to estimate the efficiency of J-PET as a function of energy deposition. The aim of this work is to determine the registration efficiency of the J-PET tomograph as a function of energy deposition by incident photons and the intrinsic efficiency of the J-PET scanner in detecting photons of different incident energies. In this study, 3-hit events are investigated, where 2-hits are caused by 511 keV photons emitted in [Formula: see text] annihilations, while the third hit is caused by one of the scattered photons. The scattered photon is used to accurately measure the scattering angle and thus the energy deposition. Two hits by a primary and a scattered photon are sufficient to calculate the scattering angle of a photon, while the third hit ensures the precise labeling of the 511 keV photons., Results: By comparing experimental and simulated energy distribution spectra, the registration efficiency of the J-PET scanner was determined in the energy deposition range of 70-270 keV, where it varies between 20 and 100[Formula: see text]. In addition, the intrinsic efficiency of the J-PET was also determined as a function of the energy of the incident photons., Conclusion: A method for determining registration efficiency as a function of energy deposition and intrinsic efficiency as a function of incident photon energy of the J-PET scanner was demonstrated. This study is crucial for evaluating the performance of the scanner based on plastic scintillators and its applications as a standard and multi-photon PET systems. The method may be also used in the calibration of Compton-cameras developed for the ion-beam therapy monitoring and simultaneous multi-radionuclide imaging in nuclear medicine., (© 2023. The Author(s).)

Background: The aim of this study was to investigate the role of urine-derived extracellular vesicles (uEVs) in diabetic kidney disease (DKD) in patients diagnosed with type 2 diabetes mellitus (T2DM)., Methods: UEVs were characterized by size distribution and microRNA content by next-generation small RNA sequencing and quantitative reverse transcription PCR., Results: A subset of sixteen miRNAs enriched in T2DM patients with DKD, including hsa-miR-514a-5p, hsa-miR‑451a, hsa-miR-126-3p, hsa-miR-214, or hsa-miR‑503 was identified. Eight miRNAs as hsa-miR-21-3p, hsa-miR-4792, hsa-miR‑375, hsa-miR-1268a, hsa-miR-501-5p, or hsa-miR-582 were downregulated. Prediction of potential target genes and pathway enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed possible functions related to cellular processes such as apoptosis, inflammation, and tissue remodeling, that promote diabetic complications, such as DKD. Among them, hsa-miR-375, hsa-miR-503, and hsa-miR-451a make important contribution. Additionally, downregulated hsa-miR-582-5p has not been reported so far in any diabetes-related pathways., Conclusions: This study revealed the most significant miRNAs in uEVs of patients with T2DM. However, as this is a bioinformatic prediction that we performed based on the putative targets of the identified miRNAs. Thus, further in vitro functional studies are needed to confirm our findings. Knowing the fact that EVs are crucial in transferring miRNAs, there is a great need toto discover their involvement in the pathomechanism of T2DM-related kidney disease., (© 2023. The Author(s).)

Purpose: Cardiac myxoma (CM), the most common cardiac tumor in adults, accounts for 50-75% of benign cardiac tumors. The diagnosis of CM is often elusive, especially in young stroke survivors and transthoracic echocardiography (TTE) is the initial technique for the differential diagnostics of CM. Less invasive cardiac computed tomography (CT) and magnetic resonance imaging (MRI) are not available for the majority of cardiac patients. Here, a robust imaging approach, ortho-Positronium (o-Ps) imaging, is presented to determine cardiac myxoma extracted from patients undergoing urgent cardiac surgery due to unexpected atrial masses. We aimed to assess if the o-Ps atom, produced copiously in intramolecular voids during the PET imaging, serves as a biomarker for CM diagnosing., Methods: Six perioperative CM and normal (adipose) tissue samples from patients, with primary diagnosis confirmed by the histopathology examination, were examined using positron annihilation lifetime spectroscopy (PALS) and micro-CT. Additionally, cell cultures and confocal microscopy techniques were used to picture cell morphology and origin., Results: We observed significant shortening in the mean o-Ps lifetime in tumor with compare to normal tissues: an average value of 1.92(02) ns and 2.72(05) ns for CM and the adipose tissue, respectively. Microscopic differences between tumor samples, confirmed in histopathology examination and micro-CT, did not influenced the major positronium imaging results., Conclusions: Our findings, combined with o-Ps lifetime analysis, revealed the novel emerging positronium imaging marker (o-PS) for cardiovascular imaging. This method opens the new perspective to facilitate the quantitative in vivo assessment of intracardiac masses on a molecular (nanoscale) level., (© 2023. The Author(s).)

Background: Mild therapeutic hypothermia (MTH) is believed to reduce the effectiveness of antiplatelet drugs. Effective dual-antiplatelet therapy after percutaneous coronary intervention (PCI) is mandatory to avoid acute stent thrombosis. The effectiveness of ticagrelor in MTH-treated out-of-hospital cardiac arrest (OHCA) survivors is still a matter of debate. The aim of the study was to evaluate the impact of MTH on the platelet-inhibitory effect of ticagrelor in comatose survivors of OHCA treated with primary PCI., Methods: Eighteen comatose survivors of OHCA with acute coronary syndrome undergoing immediate PCI treated with MTH were compared with 14 patients with uncomplicated primary myocardial infarction after PCI, matched for gender and age, in a prospective, single-center, observational study. Platelet aggregation was evaluated using VerifyNow P₂Y₁₂ point-of-care testing at 3 time points: admission (T₀), during MTH (T₁), and 48-72 h after rewarming (T₂)., Results: Ticagrelor effectively inhibits platelet aggregation in OHCA patients subjected to MTH and in all patients in the control group. The effectiveness of ticagrelor did not differ between the MTH group and the control group (p = 0.581). In 2 cases in the MTH population, the platelet response to ticagrelor was inadequate, and in one of them it remained insufficient during the re-warming phase. There was no stent thrombosis in these patients., Conclusions: The present study confirmed the effectiveness of ticagrelor to inhibit platelets in myocardial infarction patients after OHCA treated with primary PCI undergoing hypothermia. The use of cooling was not associated with an increased risk of stent thrombosis.

This review introduce extracellular vesicles (EVs) to a molecular imaging field. The idea of modern analyses based on the use of omics studies, using high-throughput methods to characterize the molecular content of a single biological system, vesicolomics seems to be the new approach to collect molecular data about EV content, to find novel biomarkers or therapeutic targets. The use of various imaging techniques, including those based on radionuclides as positron emission tomography (PET) or single photon emission computed tomography (SPECT), combining molecular data on EVs , opens up the new space for radiovesicolomics-a new approach to be used in theranostics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stępień, Rząca and Moskal.)

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) with the Bi 3 + liquid metal ion gun was used to investigate the content of lipids and amino acids (AAs) in extracellular vesicles (EVs). We induced metabolic changes in human pancreatic β-cells by stimulation with high glucose concentrations (35 mM) and tested the hypothesis of hyperglycemia (HG) has a detrimental effect on lipids and AAs in released EV subpopulations: ectosomes and exosomes. As a result of HG treatment, selected fatty acids (FAs) such as arachidonic, myristic and palmitic acids, changed their abundance in ectosomes and exosomes. Also, intensities of the characteristic peaks for cholesterol (m/z 95.09; 147.07; 161.11; 369.45) along with the molecular ion m/z 386.37 [C 27 H 46 O + ] under HG conditions, both for ectosomes and exosomes, have changed significantly. Comparative analysis of HG EVs and normoglycemic (NG) ones showed statistically significant differences in the signal intensities of four AAs: valine (m/z 72.08 and 83.05), isoleucine (m/z 86.10), phenylalanine (m/z 120.08 and 132.05) and tyrosine (m/z 107.05 and 136.09). We confirmed that ToF-SIMS is a useful technique to study selected AAs and lipid profiles in various EV subpopulations. Our study is the first demonstration of changes in FAs and AAs in exosomes and ectosomes derived from β-cells under the influence of HG., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Extracellular vesicles, especially the larger fraction (LEVs - large extracellular vesicles), are believed to be an important means of intercellular communication. Earlier studies on LEVs have shown their healing properties, especially in the vascular cells of diabetic patients. Uptake of LEVs by endothelial cells and internalization of their cargo have also been demonstrated. Endothelial cells change their properties under hyperglycemic conditions (HGC), which reduces their activity and is the cause of endothelial dysfunction. The aim of our study was to investigate how human umbilical vein endothelial cells (HUVECs) change their biological properties: shape, mobility, cell surface stiffness, as well as describe the activation of metabolic pathways after exposure to the harmful effects of HGC and the administration of LEVs released by endothelial cells. We obtained LEVs from HUVEC cultures in HGC and normoglycemia (NGC) using the filtration and ultracentrifugation methods. We assessed the size of LEVs and the presence of biomarkers such as phosphatidylserine, CD63, beta-actin and HSP70. We analyzed the LEVs uptake efficiency by HUVECs, HUVEC shape, actin cytoskeleton remodeling, surface stiffness and finally gene expression by mRNA analysis. Under HGC conditions, HUVECs were larger and had a stiffened surface and a strengthened actin cortex compared to cells under NGC condition. HGC also altered the activation of metabolic pathways, especially those related to intracellular transport, metabolism, and organization of cellular components. The most interesting observation in our study is that LEVs did not restore cell motility disturbed by HGC. Although, LEVs were not able to reverse this deleterious effect of HGC, they activated transcription of genes involved in protein synthesis and vesicle trafficking in HUVECs., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

In this study, we verified the hypothesis that Raman signature of urinary extracellular vesicles (UEVs) can be used to stratify patients with diabetes at various stages of chronic kidney disease (CKD). Patients with type 2 diabetes diagnosed with different stages of CKD and healthy subjects were enrolled in the study. UEVs were isolated using low-vacuum filtration followed by ultracentrifugation. Correlation analysis, multiple linear regression and principal component analysis were used to find differences between spectral fingerprints of UEVs derived from both groups of patients. Electron microscopy and nanoparticle tracking analysis were applied to characterize the size and morphology of UEVs. We observed significant correlations between selected Raman bands measured for UEVs and clinical parameters. We found significant differences in the area under the specific bands originating mainly from proteins and lipids between the study groups. Based on the tryptophan and amide III bands, we were able to predict the estimated glomerular filtration rate (eGFR). Principal component analysis, partial least squares regression (PLSR) and correlation analysis of the UEV Raman spectra supported the results obtained from the direct analysis of Raman spectra. Our analysis revealed that PLSR and a regression model including tryptophan and amide III bands allows to estimate the value of eGFR., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In vivo assessment of cancer and precise location of altered tissues at initial stages of molecular disorders are important diagnostic challenges. Positronium is copiously formed in the free molecular spaces in the patient’s body during positron emission tomography (PET). The positronium properties vary according to the size of inter- and intramolecular voids and the concentration of molecules in them such as, e.g., molecular oxygen, O 2 ; therefore, positronium imaging may provide information about disease progression during the initial stages of molecular alterations. Current PET systems do not allow acquisition of positronium images. This study presents a new method that enables positronium imaging by simultaneous registration of annihilation photons and deexcitation photons from pharmaceuticals labeled with radionuclides. The first positronium imaging of a phantom built from cardiac myxoma and adipose tissue is demonstrated. It is anticipated that positronium imaging will substantially enhance the specificity of PET diagnostics.

Charged lepton system symmetry under combined charge, parity, and time-reversal transformation (CPT) remains scarcely tested. Despite stringent quantum-electrodynamic limits, discrepancies in predictions for the electron-positron bound state (positronium atom) motivate further investigation, including fundamental symmetry tests. While CPT noninvariance effects could be manifested in non-vanishing angular correlations between final-state photons and spin of annihilating positronium, measurements were previously limited by knowledge of the latter. Here, we demonstrate tomographic reconstruction techniques applied to three-photon annihilations of ortho-positronium atoms to estimate their spin polarisation without magnetic field or polarised positronium source. We use a plastic-scintillator-based positron-emission-tomography scanner to record ortho-positronium (o-Ps) annihilations with single-event estimation of o-Ps spin and determine the complete spectrum of an angular correlation operator sensitive to CPT-violating effects. We find no violation at the precision level of 10 -4 , with an over threefold improvement on the previous measurement., (© 2021. The Author(s).)

For this study, we tested and optimized silicon surface functionalization procedures for capturing urinary extracellular vesicles (uEVs). The influence of the silane type (APTES or GOPS) and protein concentration on the efficiency of uEVs binding was investigated. Human lactadherin protein (LACT) was used to capture uEVs. We applied surface characterization techniques, including ellipsometry, atomic force microscopy, and time-of-flight secondary ion mass spectrometry, to observe changes in the biosensor surface after each functionalization step. uEVs were purified by a low-vacuum filtration method and concentrated by ultracentrifugation. The physical parameters of uEVs after the isolation procedure, such as morphology and size distribution, were determined using transmission electron microscopy and tunable resistive pulse sensing methods. We observed a gradual growth of the molecular layer after subsequent stages of modification of the silicon surface. The ToF-SIMS results showed no changes in the mean intensities for the characteristic peaks of amino acids and lipids in positive and negative polarization, in terms of the surface-modifying silane (APTES or GOPS) used. The most optimal concentration of LACT for the tested system was 25 µg/mL.

Background: Melanoma cells release extracellular vesicles (EVs) subpopulations which differ in size, phenotype and molecular content. Melanoma derived EVs play a role in the development and progression of cancer by delivering surface receptors and bioactive (proteins, lipids, nucleic acids) or signaling molecules to target cells., Methods: We applied Fourier Transform Infrared Spectroscopy (FTIR) to compare infrared spectra of absorption for different subpopulations of EVs originating from normal human melanocytes, primary cutaneous melanoma (WM115) and metastatic cutaneous melanoma (WM266-4)., Results: FTIR results showed that exosome and ectosome populations differ in content of protein and lipid components. We obtained higher lipid to protein ratio for ectosomes in comparison with exosomes what confirms that exosomes are very densely packed with protein cargo. We identified the lowest value of saturated fatty acids/unsaturated fatty acids parameter in the metastatic WM266-4 cell line and ectosomes derived from WM266-4 cell line in comparison with normal melanocytes and the primary WM115 cell line. We identified the alterations in the content of secondary structures of proteins present in EV subpopulations originating from melanocytes and melanoma cells in different malignancy., Conclusions: Obtained results revealed differences in the molecular composition of melanoma derived EVs subtypes, including protein secondary structure, and showed progressive structural changes during cancer development., Competing Interests: The authors declare that they have no conflicts of interest., (© 2020 The Authors.)

Microvesicles (MVs) are found in several types of body fluids and are promising disease biomarkers and therapeutic targets. This study aimed to develop a novel biofunctionalized surface for binding plasma microvesicles (PMVs) based on a lab-on-a-chip (LOC) approach. A new lactadherin (LACT)-functionalized surface was prepared and examined for monitoring PMVs. Moreover, two different strategies of LACT immobilization on a silicon surface were applied to compare different LACT orientations. A higher PMV to LACT binding efficiency was observed for LACT bonded to an αvβ3 integrin-functionalized surface compared with that for LACT directly bonded to a glutaraldehyde-modified surface. Effective binding of PMVs and its components for both LACT immobilization strategies was confirmed using spectral ellipsometry and time-of-flight secondary ion mass spectrometry methods. The proposed PMV capturing system can be used as a foundation to design novel point-of-care (POC) diagnostic devices to detect and characterize PMVs in clinical samples. Graphical Abstract.

Total-body PET opens a new diagnostic paradigm with prospects for personalized disease treatment, yet the high cost of the current crystal-based PET technology limits dissemination of total-body PET in hospitals and even in research clinics. The J-PET tomography system is based on axially arranged low-cost plastic scintillator strips. It constitutes a realistic cost-effective solution of a total-body PET for broad clinical applications. High sensitivity of total-body J-PET and triggerless data acquisition enable multiphoton imaging, opening possibilities for multitracer and positronium imaging, thus promising quantitative enhancement of specificity in cancer and inflammatory disease assessment., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent years have brought great focus on the development of drug delivery systems based on extracellular vesicles (EVs). Considering the possible applications of EVs as drug carriers, the isolation process is a crucial step. To solve the problems involved in EV isolation, we developed and validated a new EV isolation method-low-vacuum filtration (LVF)-and compared it with two commonly applied procedures-differential centrifugation (DC) and ultracentrifugation (UC). EVs isolated from endothelial cell culture media were characterized by (a) Transmission Electron Microscopy (TEM), (b) Nanoparticle Tracking Analysis (NTA), (c) Western blot and (d) Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Additionally, the membrane surface was imaged with Environmental Scanning Electron Microscopy (ESEM). We found that LVF was a reproducible and efficient method for EV isolation from conditioned media. Additionally, we observed a correlation between ATR-FTIR spectra quality and EV and protein concentration. ESEM imaging confirmed that the actual pore diameter was close to the values calculated theoretically. LVF is an easy, fast and inexpensive EV isolation method that allows for the isolation of both ectosomes and exosomes from high-volume sources with good repeatability. We believe that it could be an efficient alternative to commonly applied methods.

Purpose: In living organisms, the positron-electron annihilation (occurring during the PET imaging) proceeds in about 30% via creation of a metastable ortho-positronium atom. In the tissue, due to the pick-off and conversion processes, over 98% of ortho-positronia annihilate into two 511 keV photons. In this article, we assess the feasibility for reconstruction of the mean ortho-positronium lifetime image based on annihilations into two photons. The main objectives of this work include the (i) estimation of the sensitivity of the total-body PET scanners for the ortho-positronium mean lifetime imaging using 2γ annihilations and (ii) estimation of the spatial and time resolution of the ortho-positronium image as a function of the coincidence resolving time (CRT) of the scanner., Methods: Simulations are conducted assuming that radiopharmaceutical is labeled with 44 Sc isotope emitting one positron and one prompt gamma. The image is reconstructed on the basis of triple coincidence events. The ortho-positronium lifetime spectrum is determined for each voxel of the image. Calculations were performed for cases of total-body detectors build of (i) LYSO scintillators as used in the EXPLORER PET and (ii) plastic scintillators as anticipated for the cost-effective total-body J-PET scanner. To assess the spatial and time resolution, the four cases were considered assuming that CRT is equal to 500 ps, 140 ps, 50 ps, and 10 ps., Results: The estimated total-body PET sensitivity for the registration and selection of image forming triple coincidences (2γ+γ prompt ) is larger by a factor of 13.5 (for LYSO PET) and by factor of 5.2 (for plastic PET) with respect to the sensitivity for the standard 2γ imaging by LYSO PET scanners with AFOV = 20 cm. The spatial resolution of the ortho-positronium image is comparable with the resolution achievable when using TOF-FBP algorithms already for CRT = 50 ps. For the 20-min scan, the resolution better than 20 ps is expected for the mean ortho-positronium lifetime image determination., Conclusions: Ortho-positronium mean lifetime imaging based on the annihilations into two photons and prompt gamma is shown to be feasible with the advent of the high sensitivity total-body PET systems and time resolution of the order of tens of picoseconds.

Purpose: The time-over-threshold (TOT) technique is being used widely due to itsimplications in developing the multi-channel readouts, mainly when fast signal processing is required. Using the TOT technique, as a measure of energy loss instead of charge integration methods, significantly reduces the signal readout costs by combining the time and energy information. Therefore, this approach can potentially be utilized in J-PET tomograph which is built from plastic scintillators characterized by fast light signals. The drawback in adopting this technique lies in the non-linear correlation between input energy loss and TOT of the signal. The main motivation behind this work is to develop the relationship between TOT and energy loss and validate it by the J-PET tomograph setup., Methods: The experiment was performed using a 22 Na beta emitter source placed in the center of the J-PET tomograph. This isotope produces photons of two different energies: 511 keV photons from the positron annihilation (direct annihilation or through the formation of a para-positronium atom or pick-off process of ortho-positronium atoms) and a 1275 keV prompt photon. This allows the study of the correlation between TOT values and energy loss for energy ranges up to 1000 keV. Since the photon interacts predominantly via Compton scattering inside the plastic scintillator, there is no direct information of the energy deposition. However, using the J-PET geometry, one can measure the scattering angle of the interacting photon. Since the 22 Na source emits photons of two different energies, it is necessary to know unambiguously the energy of incident photons and their corresponding scattering angles in order to estimate energy deposition. In summary, this work presents a dedicated algorithm developed to tag photons of different energies and studying their scattering angles to calculate the energy deposition by the interacting photons., Results: A new method was elaborated to measure the energy loss by photons interacting with plastic scintillators used in the J-PET tomograph. We find the relationship between the energy loss and TOT is non-linear and can be described by the functions TOT = A0 + A1 * ln(E dep + A2) + A3 * (ln(E dep + A2)) 2 and TOT = A0 - A1 * A2[Formula: see text]. In addition, we also introduced a theoretical model to calculate the TOT as a function of energy loss in plastic scintillators., Conclusions: A relationship between TOT and energy loss by photons interacting inside the plastic scintillators used in J-PET scanner is established for a deposited energy range of 100-1000 keV.

Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers; however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC-MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial-mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential., Competing Interests: The authors declare no conflicts of interest.

Introduction: Diabetes mellitus (DM) is the most common metabolic disease. A WHO report from 2016 indicates that 422 million people worldwide suffer from DM or hyperglycaemia because of impaired glucose metabolism. Chronic hyperglycaemia leads to micro- and macrovessel damage, which may result in life-threatening complications. The Wnt pathway regulates cell proliferation and survival by modulating the expression of genes that control cell differentiation. Three linked Wnt pathways have been discovered thus far: a β-catenin-dependent pathway and two pathways independent of β-catenin - the planar cell polarity pathway and calcium-dependent pathway. The Wnt pathway regulates genes associated with inflammation, cell cycle, angiogenesis, fibrinolysis and other molecular processes., Areas Covered: This review presents the current state of knowledge regarding the contribution of the Wnt pathway to endothelial ageing under hyperglycaemic conditions and provides new insights into the molecular basis of diabetic endothelial dysfunction., Conclusion: The β-catenin-dependent pathway is a potential target in the prophylaxis and treatment of early-stage diabetes-related vascular complications. However, the underlying molecular mechanisms remain largely undetermined and require further investigation.

Raman spectroscopy was applied to the measurement of urinary and in vitro endothelium-derived extracellular vesicles (EVs) isolated by hydrostatic filtration dialysis (HFD) method. Raman spectra obtained for urinary EVs (UEVs) showed distinct differences in the fingerprint region. In contrast, average Raman spectra of endothelium-derived EVs samples were almost identical. Cluster Analysis of UEVs significantly discriminated diabetic samples from control, moreover endothelium-derived EVs revealed stronger similarity between long hyperglycemia and normoglycemia samples compared to short hyperglycemia. Results obtained from Partial Least Squares analysis corresponded well with integral intensities of selected bands. Our proof-of-concept approach demonstrates the potential for Raman spectroscopy to be used both for identification of EVs molecular signatures in urine samples from patients with type 2 diabetes mellitus and good glycemic control and unsatisfactory glycemic control as well as for in vitro hyperglycemic model. This noninvasive technique may be useful in identifying new biomarkers of diabetes and renal complications., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background : Platelet-derived microvesicles (PMVs), shed from platelet surface membranes, constitute the majority of circulating microvesicles and have been implicated in procoagulant, pro-inflammatory and pro-atherosclerotic effects. Our aim was to compare plasma PMVs numbers in relation to platelet reactivity during dual antiplatelet therapy (DAPT) with various P2Y 12 adenosine diphosphate (ADP) receptor antagonists. Methods: In pre-discharge men treated with DAPT for an acute coronary syndrome, plasma PMVs were quantified by flow cytometry on the basis of CD62P (P-selectin) and CD42 (glycoprotein Ib) positivity, putative indices of PMVs release from activated and all platelets, respectively. ADP-induced platelet aggregation was measured by multiple-electrode aggregometry. Results: Clinical characteristics were similar in patients on clopidogrel (n=16), prasugrel (n=10) and ticagrelor (n=12). Platelet reactivity was comparably reduced on ticagrelor or prasugrel versus clopidogrel (p<0.01). Compared to clopidogrel-treated patients, CD42 + /CD62P + PMVs counts were 3-4-fold lower in subjects receiving ticagrelor (p=0.001) or prasugrel (p<0.05), while CD42 + PMVs were significantly reduced on ticagrelor (by about 6-fold, p<0.001), but not prasugrel (p=0.3). CD42 + /CD62P + PMVs numbers correlated positively to the ADP-induced aggregation on clopidogrel (p<0.01) or prasugrel (p<0.05), which was absent in ticagrelor users (p=0.8). CD42 + PMVs counts were unrelated to platelet reactivity (p>0.5). Conclusions: Higher antiplatelet potency of prasugrel and ticagrelor versus clopidogrel is associated with decreased plasma CD42 + /CD62P + PMVs numbers. However, in contrast to thienopyridines, the association of reduced CD42 + /CD62P + PMVs counts with ticagrelor use appears independent of its anti-aggregatory effect. Despite similar platelet-inhibitory activity of ticagrelor and prasugrel, only the treatment with ticagrelor seems associated with lower total PMVs release. Our preliminary findings may suggest a novel pleiotropic effect of ticagrelor extending beyond pure anti-aggregatory properties of the drug., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Introduction: RANTES regulates leukocyte recruitment to areas affected by the inflammatory process. Microvesicles (MVs) belong to a subpopulation of extracellular vesicles and show proangiogenic potential by transferring bioactive molecules to target cells., Objectives: the aim of this study was to determine the relationship between circulating proangiogenic factors (MVs and RANTES) and diabetes complications in patients with different severities of diabetic retinopathy (DR). CCR5 (CD195) receptors transported by annexin V-labeled MVs were also investigated., Patients and Methods: Diabetic patients (n = 61), among whom 35 had confirmed DR classified according to guidelines, and controls (n = 25) were included. MVs were isolated by centrifugation and analyzed using flow cytometry, RANTES was assessed by ELISA., Results: the study group differed from the control group with respect to BMI, age, heart rate and systolic blood pressure. Additionally, glucose and creatinine concentrations were significantly increased: 5.30 [5.09-5.62] vs. 9.38 [7.48-11.55] (p <0.0001) mmol/l and 74.59 [64-84] vs. 89.00 [77.11-105.44] μmol/l (p = 0.0005), respectively. RANTES concentrations were significantly increased in diabetic patients compared to those of controls (15.5 (9.7-18.1) vs. 8.9 (0.9-14.6) μg/ml (p = 0.011)), and RANTES concentration significantly increased with respect to nonproliferative DR progression. Moreover, the number of CCR5-positive MVs was significantly increased in patients with heavy nonproliferative diabetic retinopathy (HNPDR) compared to those with so nonproliferative DR (SNPDR): 1178 [836-2254] vs. 394 [275-799] counts/μl., Conclusions: Correlation of RANTES concentrations with the stage of nonproliferative DR and the statistically significant dependence of CCR5-positive MVs with disease progression suggest that MVs and RANTES can be considered new biomarkers.

Ectosomes ( Ects ) are a subpopulation of extracellular vesicles formed by the process of plasma membrane shedding. In the present study, we profiled ectosome-specific microRNAs (miRNAs) in patients with type 2 diabetes mellitus (T2DM) and analyzed their pro- and anti-angiogenic potential. Methods: We used different approaches for detecting and enumerating Ects , including atomic force microscopy, cryogenic transmission electron microscopy, and nanoparticle tracking analysis. Furthermore, we used bioinformatics tools to analyze functional data obtained from specific miRNA enrichment signatures during angiogenesis and vasculature development. Results: Levels of miR-193b-3p, miR-199a-3p, miR-20a-3p, miR-26b-5p, miR-30b-5p, miR-30c-5p, miR-374a-5p, miR-409-3p, and miR-95-3p were significantly different between Ects obtained from patients with T2DM and those obtained from healthy controls. Conclusion : Our results showed differences in the abundance of pro- and anti-angiogenic miRNAs in Ects of patients with T2DM, and are suggestive of mechanisms underlying the development of vascular complications due to impaired angiogenesis in such patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Micro-RNAs (miRNAs) are small non-coding RNAs that act as negative regulators of the genomic output. Their intrinsic importance within cell biology and human disease is well known. Their mechanism of action based on the base pairing binding to their cognate targets have helped the development not only of many computer applications for the prediction of miRNA target recognition but also of specific applications for functional assessment and analysis. Learning about miRNA function requires practical training in the use of specific computer and web-based applications that are complementary to wet-lab studies. In order to guide the learning process about miRNAs, we have created miRNAtools (http://mirnatools.eu), a web repository of miRNA tools and tutorials. This article compiles tools with which miRNAs and their regulatory action can be analyzed and that function to collect and organize information dispersed on the web. The miRNAtools website contains a collection of tutorials that can be used by students and tutors engaged in advanced training courses. The tutorials engage in analyses of the functions of selected miRNAs, starting with their nomenclature and genomic localization and finishing with their involvement in specific cellular functions., Competing Interests: The authors declare no conflict of interest.

Lactadherin is a small (53-66kDa) multifunctional glycoprotein belonging to the secreted extracellular matrix protein family. It has a multi-domain structure and is involved in many biological and physiological processes, including phagocytosis, angiogenesis, atherosclerosis, tissue remodeling, and haemostasis regulation. Lactadherin binds phosphatidylserine (PS)-enriched cell surfaces in a receptor-independent manner. Interaction between lactadherin and PS is crucial for regulation of blood coagulation processes. This review summarizes recent knowledge on the possible role of lactadherin in haemostasis control, emphasizing the great significance of the interaction between lactadherin and PS expressed on activated platelets and extracellular vesicles. The possible role of lactadherin as a therapeutic target and biomarker is also discussed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of this study was to check the relationship between the density of urinary EVs, their size distribution, and the progress of early renal damage in type 2 diabetic patients (DMt2). Patients were enrolled to this study, and glycated hemoglobin (HbA1c) below 7% was a threshold for properly controlled diabetic patients (CD) and poorly controlled diabetic patients (UD). Patients were further divided into two groups: diabetic patients without renal failure (NRF) and with renal failure (RF) according to the Glomerular Filtration Rate. Density and diameter of EVs were determined by Tunable Resistive Pulse Sensing. Additionally, EVs were visualized by means of Transmission and Environmental Scanning Electron Microscopy. Nano-liquid chromatography coupled offline with mass spectrometry (MALDI-TOF-MS/MS) was applied for proteomic analysis. RF had reduced density of EVs compared to NRF. The size distribution study showed that CD had larger EVs (mode) than UD (115 versus 109 nm; p < 0.05); nevertheless the mean EVs diameter was smaller in controls than in the CD group (123 versus 134 nm; p < 0.05). It was demonstrated that EVs are abundant in urine. Albumin, uromodulin, and number of unique proteins related to cell stress and secretion were detected in the EVs fraction. Density and size of urinary EVs reflect deteriorated renal function and can be considered as potential renal damage biomarkers., Competing Interests: The authors declare that they have no competing interests.

Background: Vitamin D is a major regulator of mineral bone metabolism. The lower vitamin D levels in patients with acute myocardial infarction (AMI) and the seasonal variation of vitamin D levels are proposed., Aim: The evaluation of the seasonal relationship of 25(OH)D levels in patients with AMI and analysis of confounding factors (gender or diabetes mellitus) affecting the levels of vitamin D in AMI patients., Methods: Fifty-nine consecutive patients with mean age 58 ± 9.4 years were admitted to the Department of Invasive Cardiology. Subjects had diagnosed uncomplicated myocardial infarction. Blood samples for analysis were collected on patient admission to the cardiac unit after heparin treatment. Samples for routine laboratory tests were immediately processed. For 25(OH)D, the 25-hydroxycholecalciferol test, which measures total vitamin D levels in serum (DRG Instruments GmbH, Marburg, Germany), was applied., Results: Median serum 25(OH)D concentration in AMI patients was below the recommended optimal values 7.1 (2.3-13.3) ng/mL. Fifty-three (89.8%) patients had vitamin D deficiency (VDD) below 20 ng/mL, six (10.2%) patients had suboptimal 25(OH)D levels (between 20 ng/mL and 30 ng/mL), and no one had the recommended reference range. The seasonal effect of 25(OH)D variations among AMI patients was observed with the lowest levels in the beginning of the year (January-March) and the highest levels at the end of the year (September-December) (p = 0.007). Patients with normoglycaemia had significantly higher (9.2 [2.3-16.8] ng/mL) vitamin D levels compared to patients with impaired glucose tolerance (2.3 [2.3-3.9] ng/mL) or diabetes mellitus (8.5 [2.5-13.3] ng/mL) (p = 0.01)., Conclusions: A high prevalence of VDD in AMI patients has been confirmed. Supplementation of vitamin D in AMI patients with hyperglycaemia can bring greater benefits.