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Melanoma is an aggressive disease that arises from mutations in the cells that produce the pigment melanin, melanocytes. Melanoma is characterized by a high mortality rate, due to avoidance of applied therapies and metastasis to other organs. The peculiar features of boron neutron capture therapy (BNCT), particularly its cell-level selectivity, make BNCT a promising modality for melanoma treatment. However, appropriate cellular models should be used to study new therapies or improve the efficacy of existing therapies. Spheroids, which have been used for years for in vitro studies of the efficacy of anti-cancer therapies, have many characteristics shared with tumors through which they can increase the accuracy of the cellular response compared to 2D culture in vitro studies and reduce the use of animals for research in the future. To the best of our knowledge, when we started researching the use of spheroids in BNCT in vitro, there was no publication showing such use. Our study aimed to evaluate the efficacy of a 3D cellular model (spheroids) for testing BNCT on melanoma cells. We assessed boronophenylalanine (10BPA) uptake using inductively coupled plasma mass spectrometry in both spheroids and 2D cultures of melanoma and melanocytes. DNA damage, Ki67 protein expression, and spheroid growth were analyzed. The experimental groups included: (1) IR_B (neutron flux + 50 µg 10B/mL), (2) IR (neutron flux alone), (3) C_B (no irradiation, 50 µg 10B/mL), and (4) C (no irradiation and no treatment with boron). The total absorbed doses were estimated to be 2.1–3.1 Gy for IR_B cells and spheroids as well as 8.3–9.4 Gy for IR_B spheroids, while estimated doses for IR cells were 0.5–1.9 Gy. The results indicated that IR_B spheroids might exhibit a reduced diameter. Melanoma cells in the 3D model showed that their DNA damage levels may be higher than those in the 2D model. Moreover, the Ki67 assay revealed differences in the expression of this marker between irradiated melanoma cell lines. In conclusion, preincubation with 10BPA enhances BNCT efficacy, leading to cell growth inhibition and increased DNA fragmentation. Differences in DNA damage between 2D and 3D models may be due to dissimilarities in cell metabolism caused by a changed cell architecture. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this review article, we present arguments demonstrating that the advent of high sensitivity total-body PET systems and the invention of the method of positronium imaging, open realistic perspectives for the application of positronium as a biomarker for in-vivo assessment of the degree of hypoxia. Hypoxia is a state or condition, in which the availability of oxygen is not sufficient to support physiological processes in tissue and organs. Positronium is a metastable atom formed from electron and positron which is copiously produced in the intramolecular spaces in the living organisms undergoing positron emission tomography (PET). Properties of positronium, such as e.g., lifetime, depend on the size of intramolecular spaces and the concentration in them of oxygen molecules. Therefore, information on the partial pressure of oxygen (pO2) in the tissue may be derived from the positronium lifetime measurement. The partial pressure of oxygen differs between healthy and cancer tissues in the range from 10 to 50 mmHg. Such differences of pO2 result in the change of ortho-positronium lifetime e.g., in water by about 2–7 ps. Thus, the application of positronium as a biomarker of hypoxia requires the determination of the mean positronium lifetime with the resolution in the order of 2 ps. We argue that such resolution is in principle achievable for organ-wise positronium imaging with the total-body PET systems. [ABSTRACT FROM AUTHOR]

Whole gamma imaging: a new concept of PET combined with Compton imaging. Imaging of "invisible" would be not possible without the introduction of the instrument called "scintillation camera" which significantly impacted the role of radionuclide-based imaging techniques and allowed visualize large segments of the body with one data acquisition (tomography). Recently the first total-body PET scanners were taken into operation [[4]], [[5]], [[6]], [[7]], [[8]] changing the imaging paradigm by enabling simultaneous whole-body dynamic and parametric imaging of all tissues in the body simultaneously [[3]]. Also, positronium imaging requiring direct imaging with excellent time resolution may become soon practical, as we presently experience rapid development in improving the PET systems time resolution [[20]], [[21]], [[22]], [[23]]. [Extracted from the article]

Extracellular vesicles (EVs) are nano- and micro-sized double-layered membrane entities derived from most cell types and released into biological fluids. Biological properties (cell-uptake, biocompatibility), and chemical (composition, structure) or physical (size, density) characteristics make EVs a good candidate for drug delivery systems (DDS). Recent advances in the field of EVs (e.g., scaling-up production, purification) and developments of new imaging methods (total-body positron emission tomography [PET]) revealed benefits of radiolabeled EVs in diagnostic and interventional medicine as a potential DDs in theranostics. [ABSTRACT FROM AUTHOR]

Positron emission tomography (PET) imaging is the most quantitative modality for assessing disease activity at the molecular and cellular levels, and therefore, it allows monitoring its course and determining the efficacy of various therapeutic interventions. In this scientific communication, we describe the unparalleled and revolutionary impact of PET imaging on research and day to day practice of medicine. We emphasize the critical importance of the development and synthesis of novel radiotracers (starting from the enormous impact of F-Fluorodeouxyglucose (FDG) introduced by investigators at the University of Pennsylvania (PENN)) and PET instrumentation. These innovations have led to the total-body PET systems enabling dynamic and parametric molecular imaging of all organs in the body simultaneously. We also present our perspectives for future development of molecular imaging by multiphoton PET systems that will enable users to extract substantial information (owing to the evolving role of positronium imaging) about the related molecular and biological bases of various disorders, which are unachievable by the current PET imaging techniques. [ABSTRACT FROM AUTHOR]

In contrast to standard 2D cell cultures, spheroids are three-dimensional (3D) models which can mimic natural conditions of cancer growth and metabolism. Their complex structure can be investigated and analyzed using fluorescence microscopy and micro-tomographic imaging (micro-CT) as a new technique. In this study, we show application of two different melanoma cell lines (WM115 and WM266) with different biological characteristics to form spheroids by a hanging drop method. [ABSTRACT FROM AUTHOR]

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. Urine has shown to be a particularly rich reservoir of extracellular vesicles (EVs) which are potential biomarkers of diabetes and its renal complications. EVs were isolated by Hydrostatic Filtration Dialysis and ultracentrifugation method. Raman spectroscopy was applied to the measurement of spectral signatures of vesicles. Partial Least Squares regression and Cluster Analysis were used for detailed analysis of all chemical differences between study samples. Raman spectroscopy allowed to distinguish diabetic patients (with good and unsatisfactory glycemic control) and healthy subjects. Additionally we showed the potential of Raman spectroscopy technique to be used to differentiate cells cultured in different glycemic conditions. Unlabelled Image [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Lactadherin is a small (53–66 kDa) 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. [ABSTRACT FROM AUTHOR]

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.

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.

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.

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.

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.