The time-course linkage between hemolysis, redox, and metabolic parameters during red blood cell storage with or without uric acid and ascorbic acid supplementation.

Oxidative phenomena are considered to lie at the root of the accelerated senescence observed in red blood cells (RBCs) stored under standard blood bank conditions. It was recently shown that the addition of uric (UA) and/or ascorbic acid (AA) to the preservative medium beneficially impacts the storability features of RBCs related to the handling of pro-oxidant triggers. This study constitutes the next step, aiming to examine the links between hemolysis, redox, and metabolic parameters in control and supplemented RBC units of different storage times. For this purpose, a paired correlation analysis of physiological and metabolism parameters was performed between early, middle, and late storage in each subgroup. Strong and repeated correlations were observed throughout storage in most hemolysis parameters, as well as in reactive oxygen species (ROS) and lipid peroxidation, suggesting that these features constitute donor-signatures, unaffected by the diverse storage solutions. Moreover, during storage, a general "dialogue" was observed between parameters of the same category (e.g., cell fragilities and hemolysis or lipid peroxidation and ROS), highlighting their interdependence. In all groups, extracellular antioxidant capacity, proteasomal activity, and glutathione precursors of preceding time points anticorrelated with oxidative stress lesions of upcoming ones. In the case of supplemented units, factors responsible for glutathione synthesis varied proportionally to the levels of glutathione itself. The current findings support that UA and AA addition reroutes the metabolism to induce glutathione production, and additionally provide mechanistic insight and footing to examine novel storage optimization strategies.

Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring.

Redox imbalance and oxidative stress have emerged as generative causes of the structural and functional degradation of red blood cells (RBC) that happens during their hypothermic storage at blood banks. The aim of the present study was to examine whether the antioxidant enhancement of stored RBC units following uric (UA) and/or ascorbic acid (AA) supplementation can improve their storability as well as post-transfusion phenotypes and recovery by using in vitro and animal models, respectively. For this purpose, 34 leukoreduced CPD/SAGM RBC units were aseptically split in 4 satellite units each. UA, AA or their mixture were added in the three of them, while the fourth was used as control. Hemolysis as well as redox and metabolic parameters were studied in RBC units throughout storage. The addition of antioxidants maintained the quality parameters of stored RBCs, (e.g., hemolysis, calcium homeostasis) and furthermore, shielded them against oxidative defects by boosting extracellular and intracellular (e.g., reduced glutathione; GSH) antioxidant powers. Higher levels of GSH seemed to be obtained through distinct metabolic rewiring in the modified units: methionine-cysteine metabolism in UA samples and glutamine production in the other two groups. Oxidatively-induced hemolysis, reactive oxygen species accumulation and membrane lipid peroxidation were lower in all modifications compared to controls. Moreover, denatured/oxidized Hb binding to the membrane was minor, especially in the AA and mix treatments during middle storage. The treated RBC were able to cope against pro-oxidant triggers when found in a recipient mimicking environment in vitro, and retain control levels of 24h recovery in mice circulation. The currently presented study provides (a) a detailed picture of the effect of UA/AA administration upon stored RBCs and (b) insight into the differential metabolic rewiring when distinct antioxidant "enhancers" are used.

Innate Variability in Physiological and Omics Aspects of the Beta Thalassemia Trait-Specific Donor Variation Effects.

The broad spectrum of beta-thalassemia (ßThal) mutations may result in mild reduction (ß ++), severe reduction (ß +) or complete absence (ß 0) of beta-globin synthesis. ßThal heterozygotes eligible for blood donation are "good storers" in terms of red blood cell (RBC) fragility, proteostasis and redox parameters of storage lesion. However, it has not been examined if heterogeneity in genetic backgrounds among ßThal-trait donors affects their RBC storability profile. For this purpose, a paired analysis of physiological and omics parameters was performed in freshly drawn blood and CPD/SAGM-stored RBCs donated by eligible volunteers of ß ++ (N = 4), ß + (N = 9) and ß 0 (N = 2) mutation-based phenotypes. Compared to ß +, ß ++ RBCs were characterized by significantly lower RDW and HbA2 but higher hematocrit, MCV and NADPH levels in vivo. Moreover, they had lower levels of reactive oxygen species and markers of oxidative stress, already from baseline. Interestingly, their lower myosin and arginase membrane levels were accompanied by increased cellular fragility and arginine values. Proteostasis markers (proteasomal activity and/or chaperoning-protein membrane-binding) seem to be also diminished in ß ++ as opposed to the other two phenotypic groups. Overall, despite the low number of samples in the sub-cohorts, it seems that the second level of genetic variability among the group of ßThal-trait donors is reflected not only in the physiological features of RBCs in vivo, but almost equally in their storability profiles. Mutations that only slightly affect the globin chain equilibrium direct RBCs towards phenotypes closer to the average control, at least in terms of fragility indices and proteostatic dynamics.

Early and Late-Phase 24 h Responses of Stored Red Blood Cells to Recipient-Mimicking Conditions.

The 24-hour (24 h) post-transfusion survival of donor red blood cells (RBCs) is an important marker of transfusion efficacy. Nonetheless, within that period, donated RBCs may encounter challenges able to evoke rapid stress-responses. The aim of the present study was to assess the effect of exposure to plasma and body temperature upon stored RBCs under recipient-mimicking conditions in vitro from the first hours "post-transfusion" up to 24 h. For this purpose, packed RBCs from seven leukoreduced CPD/SAGM units were reconstituted with plasma of twenty-seven healthy individuals and incubated for 24 h at 37oC. Three units were additionally used to examine stress-responses in 3-hour intervals post mixing with plasma (n = 5) until 24 h. All experiments were performed in shortly-, medium-, and long-stored RBCs. Hemolysis, redox, morphology, membrane protein binding and vesiculation parameters were assessed. Even though spontaneous hemolysis was minimal post-reconstitution, it presented a time-dependent increase. A similar time-course profile was evident for the concentration of procoagulant extracellular vesicles and the osmotic fragility (shortly-stored RBCs). On the contrary, mechanical fragility and reactive oxygen species accumulation were characterized by increases in medium-stored RBCs, evident even from the first hours in the recipient-mimicking environment. Finally, exposure to plasma resulted in rapid improvement of morphology, especially in medium-stored RBCs. Overall, some RBC properties vary significantly during the first 24 h post-mixing, at levels different from both the storage ones and the standard end-of-24 h. Such findings may be useful for understanding the performance of RBCs and their possible clinical effects -especially on susceptible recipients- during the first hours post-transfusion.

Corpuscular Fragility and Metabolic Aspects of Freshly Drawn Beta-Thalassemia Minor RBCs Impact Their Physiology and Performance Post Transfusion: A Triangular Correlation Analysis In Vitro and In Vivo.

The clarification of donor variation effects upon red blood cell (RBC) storage lesion and transfusion efficacy may open new ways for donor-recipient matching optimization. We hereby propose a "triangular" strategy for studying the links comprising the transfusion chain-donor, blood product, recipient-as exemplified in two cohorts of control and beta-thalassemia minor (ßThal+) donors (n = 18 each). It was unraveled that RBC osmotic fragility and caspase-like proteasomal activity can link both donor cohorts to post-storage states. In the case of heterozygotes, the geometry, size and intrinsic low RBC fragility might be lying behind their higher post-storage resistance to lysis and recovery in mice. Moreover, energy-related molecules (e.g., phosphocreatine) and purine metabolism factors (IMP, hypoxanthine) were specifically linked to lower post-storage hemolysis and phosphatidylserine exposure. The latter was also ameliorated by antioxidants, such as urate. Finally, higher proteasomal conservation across the transfusion chain was observed in heterozygotes compared to control donors. The proposed "triangularity model" can be (a) expanded to additional donor/recipient backgrounds, (b) enriched by big data, especially in the post-transfusion state and (c) fuel targeted experiments in order to discover new quality biomarkers and design more personalized transfusion medicine schemes.

Deciphering the Relationship Between Free and Vesicular Hemoglobin in Stored Red Blood Cell Units.

Red blood cells (RBCs) release hemoglobin (Hb)-containing extracellular vesicles (EVs) throughout their lifespan in the circulation, and especially during senescence, by spleen-facilitated vesiculation of their membrane. During ex vivo aging under blood bank conditions, the RBCs lose Hb, both in soluble form and inside EVs that accumulate as a part of storage lesion in the supernatant of the unit. Spontaneous hemolysis and vesiculation are increasingly promoted by the storage duration, but little is known about any physiological linkage between them. In the present study, we measured the levels of total extracellular and EV-enclosed Hb (EV-Hb) in units of whole blood (n = 36) or packed RBCs stored in either CPDA-1 (n = 99) or in CPD-SAGM additive solution (n = 46), in early, middle, and late storage. The spectrophotometry data were subjected to statistical analysis to detect possible correlation(s) between storage hemolysis and EV-Hb, as well as the threshold (if any) that determines the area of this dynamic association. It seems that the percentage of EV-Hb is negatively associated with hemolysis levels from middle storage onward by showing low to moderate correlation profiles in all strategies under investigation. Moreover, 0.17% storage hemolysis was determined as the potential cut-off, above which this inverse correlation is evident in non-leukoreduced CPDA units. Notably, RBC units with hemolysis levels > 0.17% are characterized by higher percentage of nanovesicles (<100 nm) over typical microvesicles (100-400 nm) compared with the lower hemolysis counterparts. Our results suggest an ordered loss of Hb during RBC accelerated aging that might fuel targeted research to elucidate its mechanistic basis.

Beta thalassemia minor is a beneficial determinant of red blood cell storage lesion.

Blood donor genetics and lifestyle affect the quality of red blood cell (RBC) storage. Heterozygotes for beta thalassemia (bThal+) constitute a non-negligible proportion of blood donors in the Mediterranean and other geographical areas. The unique hematological profile of bThal+ could affect the capacity of enduring storage stress, however, the storability of bThal+ RBC is largely unknown. In this study, RBC from 18 bThal+ donors were stored in the cold and profiled for primary (hemolysis) and secondary (phosphatidylserine exposure, potassium leakage, oxidative stress) quality measures, and metabolomics, versus sex- and age-matched controls. The bThal+ units exhibited better levels of storage hemolysis and susceptibility to lysis following osmotic, oxidative and mechanical insults. Moreover, bThal+ RBC had a lower percentage of surface removal signaling, reactive oxygen species and oxidative defects to membrane components at late stages of storage. Lower potassium accumulation and higher uratedependent antioxidant capacity were noted in the bThal+ supernatant. Full metabolomics analyses revealed alterations in purine and arginine pathways at baseline, along with activation of the pentose phosphate pathway and glycolysis upstream to pyruvate kinase in bThal+ RBC. Upon storage, substantial changes were observed in arginine, purine and vitamin B6 metabolism, as well as in the hexosamine pathway. A high degree of glutamate generation in bThal+ RBC was accompanied by low levels of purine oxidation products (IMP, hypoxanthine, allantoin). The bThal mutations impact the metabolism and the susceptibility to hemolysis of stored RBC, suggesting good post-transfusion recovery. However, hemoglobin increment and other clinical outcomes of bThal+ RBC transfusion deserve elucidation by future studies.

Red cell proteasome modulation by storage, redox metabolism and transfusion.

BACKGROUND: Proteasomes are proteolytic complexes with prominent roles in the control of protein homeostasis and cellular viability. However, little is known about the effects of storage and glucose-6-phosphate dehydrogenase deficiency (G6PD-) on the activity and topology of red blood cell (RBC) proteasomes. MATERIALS AND METHODS: We investigated the concentration (by GeLC-MS proteomics analysis and immunoblotting), activity (by using peptide substrates and proteasome inhibitors), and subcellular/extracellular distribution (following cell fractionation and isolation of extracellular vesicles, respectively) of RBC proteasomes in fresh blood and RBCs from control and G6PD- donors following storage in leukoreduced units. RBC proteasome activity was also tested in transfusion-mimicking conditions in vitro. RESULTS: Stored RBCs were characterised by decreased cytosolic proteasome activity compared to fresh RBCs but increased membrane activity and protein concentration levels. Active proteasomes along with other "repair or destroy" proteins are recruited to the membrane during storage. A proportion of them is released in the supernatant in soluble form or inside extracellular vesicles. Significantly increased enzymatic activity and release of proteasomes were observed in G6PD- vs control RBCs. Similar variations were observed in stress protein biomarkers at the G6PD- membrane. The proteasome profile (mainly the caspase-like activity) had significant correlations with the G6PD- metabolome and quality markers of the RBC units. The storage-induced modifications in the proteasome activities were only partly restored in transfusion-mimicking conditions. DISCUSSION: Storage conditions and G6PD deficiency affect (individually and in synergy) the abundance, distribution, activity, and release of RBC proteasomes. The partial irreversibility of these effects in transfusion-mimicking conditions demands further investigation of their clinical impact on transfusion outcomes.

The Post-Storage Performance of RBCs from Beta-Thalassemia Trait Donors Is Related to Their Storability Profile.

Blood donors with beta-thalassemia traits (ßThal+) have proven to be good "storers", since their stored RBCs are resistant to lysis and resilient against oxidative/proteotoxic stress. To examine the performance of these RBCs post-storage, stored ßThal+ and control RBCs were reconstituted in plasma donated from transfusion-dependent beta-thalassemic patients and healthy controls, and incubated for 24 h at body temperature. Several physiological parameters, including hemolysis, were evaluated. Moreover, labeled fresh/stored RBCs from the two groups were transfused in mice to assess 24 h recovery. All hemolysis metrics were better in the group of heterozygotes and distinguished them against controls in the plasma environment. The reconstituted ßThal+ samples also presented higher proteasome activity and fewer procoagulant extracellular vesicles. Transfusion to mice demonstrated that ßThal+ RBCs present a marginal trend for higher recovery, regardless of the recipient's immune background and the RBC storage age. According to correlation analysis, several of these advantageous post-storage characteristics are related to storage phenotypes, like the cytoskeleton composition, low cellular fragility, and enhanced membrane proteostasis that characterize stored ßThal+ RBCs. Overall, it seems that the intrinsic physiology of ßThal+ RBCs benefits them in conditions mimicking a recipient environment, and in the circulation of animal models; findings that warrant validation in clinical trials.

Red Blood Cell Proteasome in Beta-Thalassemia Trait: Topology of Activity and Networking in Blood Bank Conditions.

Proteasomes are multi-catalytic complexes with important roles in protein control. Their activity in stored red blood cells (RBCs) is affected by both storage time and the donor's characteristics. However, apart from their abundancy in the membrane proteome, not much is known about their topology, activity, and networking during the storage of RBCs from beta-thalassemia trait donors (ßThal+). For this purpose, RBC units from fourteen ßThal+ donors were fractionated and studied for proteasome activity distribution and interactome through fluorometric and correlation analyses against units of sex- and aged-matched controls. In all the samples examined, we observed a time-dependent translocation and/or activation of the proteasome in the membrane and a tight connection of activity with the oxidative burden of cells. Proteasomes were more active in the ßThal+ membranes and supernatants, while the early storage networking of 20S core particles and activities showed a higher degree of connectivity with chaperones, calpains, and peroxiredoxins, which were nonetheless present in all interactomes. Moreover, the ßThal+ interactomes were specially enriched in kinases, metabolic enzymes, and proteins differentially expressed in ßThal+ membrane, including arginase-1, piezo-1, and phospholipid scramblase. Overall, it seems that ßThal+ erythrocytes maintain a considerable "proteo-vigilance" during storage, which is closely connected to their distinct antioxidant dynamics and membrane protein profile.

Osmotic hemolysis is a donor-specific feature of red blood cells under various storage conditions and genetic backgrounds.

BACKGROUND: Transfusion research has recently focused on the discovery of red blood cell (RBC) storage capacity biomarkers and the elucidation of donor variation effects. This shift of focus can further strengthen personalization of transfusion therapy, by revealing probable links between donor biology, RBC storage lesion profile, and posttransfusion performance. STUDY DESIGN AND METHODS: We performed a paired correlation analysis of osmotic fragility in freshly drawn RBCs and during cold storage in different preservative solutions at weekly intervals until unit's expiration date (n = 231), or following 24 h reconstitution in allogeneic plasma (n = 32) from healthy controls or transfusion-dependent beta-thalassemia patients. RESULTS: We observed exceptional correlation profiles (r > 0.700, p < 10-5 in most cases) of RBC osmotic fragility in the ensemble of samples, as well as in subgroups characterized by distinct genetic backgrounds (sex, beta-thalassemia traits, glucose-6-phosphate dehydrogenase deficiency) and storage strategies (additive solutions, whole blood, RBC concentrates). The mean corpuscular fragility (MCF) of fresh and stored RBCs at each storage time significantly correlated with the MCF of stored RBCs measured at all subsequent time points of the storage period (e.g., MCF values of storage day 21 correlated with those of storage days 28, 35 and 42). A similar correlation profile was also observed between the osmotic hemolysis of fresh/stored RBCs before and following in vitro reconstitution in plasma from healthy controls or beta-thalassemia patients. CONCLUSION: Our findings highlighted the potential of osmotic fragility to serve as a donor-signature on RBCs at every step of any individual transfusion chain (donor, blood product, and probably, recipient).

Leukoreduction makes a difference: A pair proteomics study of extracellular vesicles in red blood cell units.

Prestorage filtration of blood to remove contaminating donor leukocytes and platelets has substantially increased the safety level of transfusion therapy. We have previously shown that leukoreduction has a mitigating effect on the storage lesion profile by lowering the extent of hemolysis and of RBC aging and removal phenotypes, including surface signaling and microvesiculation. Even though protein composition may determine the fate of EVs in the recipient, the probable effect of leukoreduction on the EV proteome has been scarcely investigated. In the present paired study, we characterized the proteome of EVs released in prestorage leukoreduced (L) and nonleukoreduced (N) RBC units prepared from the same donors, by immunoblotting and qualitative proteomics analyses at two storage intervals. Apart from common proteofrms typically associated with the established EV biogenesis mechanisms, the comparative proteomics analyses revealed that both leukoreduction and storage duration affect the complexity of the EV proteome. Membrane and cytoskeleton-related proteins and regulators, metabolic enzymes and plasma proteins exhibited storage duration dependent variation in L- and N-EVs. Specific proteoforms prevailed in each EV group, such as transferrin in L-units or platelet glycoproteins, leukocyte surface molecules, MHC HLA, histones and tetraspanin CD9 in N-units. Of note, several unique proteins have been associated with immunomodulatory, vasoregulatory, coagulatory and anti-bacterial activities or cell adhesion events. The substantial differences between EV composition under the two RBC preparation methods shed light in the underlying EV biogenesis mechanisms and stimuli and may lead to different EV interactions and effects to target cells post transfusion.

From Proteomic Mapping to Invasion-Metastasis-Cascade Systemic Biomarkering and Targeted Drugging of Mutant BRAF-Dependent Human Cutaneous Melanomagenesis.

Melanoma is classified among the most notoriously aggressive human cancers. Despite the recent progress, due to its propensity for metastasis and resistance to therapy, novel biomarkers and oncogenic molecular drivers need to be promptly identified for metastatic melanoma. Hence, by employing nano liquid chromatography-tandem mass spectrometry deep proteomics technology, advanced bioinformatics algorithms, immunofluorescence, western blotting, wound healing protocols, molecular modeling programs, and MTT assays, we comparatively examined the respective proteomic contents of WM115 primary (n = 3955 proteins) and WM266-4 metastatic (n = 6681 proteins) melanoma cells. It proved that WM115 and WM266-4 cells have engaged hybrid epithelial-to-mesenchymal transition/mesenchymal-to-epithelial transition states, with TGF-ß controlling their motility in vitro. They are characterized by different signatures of SOX-dependent neural crest-like stemness and distinct architectures of the cytoskeleton network. Multiple signaling pathways have already been activated from the primary melanoma stage, whereas HIF1α, the major hypoxia-inducible factor, can be exclusively observed in metastatic melanoma cells. Invasion-metastasis cascade-specific sub-routines of activated Caspase-3-triggered apoptosis and LC3B-II-dependent constitutive autophagy were also unveiled. Importantly, WM115 and WM266-4 cells exhibited diverse drug response profiles, with epirubicin holding considerable promise as a beneficial drug for metastatic melanoma clinical management. It is the proteome navigation that enables systemic biomarkering and targeted drugging to open new therapeutic windows for advanced disease.

Proteome of Stored RBC Membrane and Vesicles from Heterozygous Beta Thalassemia Donors.

Genetic characteristics of blood donors may impact the storability of blood products. Despite higher basal stress, red blood cells (RBCs) from eligible donors that are heterozygous for beta-thalassemia traits (ßThal+) possess a differential nitrogen-related metabolism, and cope better with storage stress compared to the control. Nevertheless, not much is known about how storage impacts the proteome of membrane and extracellular vesicles (EVs) in ßThal+. For this purpose, RBC units from twelve ßThal+ donors were studied through proteomics, immunoblotting, electron microscopy, and functional ELISA assays, versus units from sex- and aged-matched controls. ßThal+ RBCs exhibited less irreversible shape modifications. Their membrane proteome was characterized by different levels of structural, lipid raft, transport, chaperoning, redox, and enzyme components. The most prominent findings include the upregulation of myosin proteoforms, arginase-1, heat shock proteins, and protein kinases, but the downregulation of nitrogen-related transporters. The unique membrane proteome was also mirrored, in part, to that of ßThal+ EVs. Network analysis revealed interesting connections of membrane vesiculation with storage and stress hemolysis, along with proteome control modulators of the RBC membrane. Our findings, which are in line with the mild but consistent oxidative stress these cells experience in vivo, provide insight into the physiology and aging of stored ßThal+ RBCs.

Sex-related aspects of the red blood cell storage lesion.

BACKGROUND: Several factors contribute to the manifestation of red blood cell (RBC) storage lesions, with one of the most interesting being the "donor variation effect". Since many haematological characteristics of blood donors are sex-dependent, sex hormones and their age-dependent variation may affect the storage profile of RBCs. MATERIALS AND METHODS: Fresh blood from 200 healthy male and female donors underwent haematological, biochemical and physiological analysis. Three selected groups of donors (men, n=8; pre-menopausal women, n=8; and post-menopausal women, n=4) exhibiting as similar as possible baseline values were recruited for blood donation in leukoreduced CPD/SAGM units. RBC indices, haemolysis and propensity for haemolysis, reactive oxygen species (ROS) and plasma antioxidant capacity were measured bi-weekly. RESULTS: Female blood was characterised by lower plasma antioxidant capacity and free haemoglobin (Hb) levels in vivo, in spite of the higher RBC osmotic fragility, compared to male blood. Comparatively low Hb concentration was also measured in stored RBCs from female donors, as in vivo. Mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), and plasma antioxidant capacity were also lower in female donors throughout storage, even though baseline levels were equal to those of the male group. There was no difference in propensity of stored RBCs for haemolysis between male and female units but intracellular ROS levels were significantly lower in female RBCs. Increased end-of-storage extracellular potassium and recruitment of protein stress markers (clusterin, Hb) to the RBC membrane were observed in the units of post- vs pre-menopausal female donors at mid-storage onwards. DISCUSSION: Donor's sex has an impact on Hb concentration and redox parameters of stored RBCs. In addition, menopause seems to promote RBC membrane remodelling, at least during prolonged storage. Our pilot study provides new insights on the different effects on RBC storage lesion according to sex.

The Multi-Faced Extracellular Vesicles in the Plasma of Chronic Kidney Disease Patients.

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles released by most cells in body fluids and extracellular matrix. They function as signal transducers in intercellular communication, contributing to the maintenance of cell and tissue integrity. EVs biogenesis is deregulated in various pathologies, in structural and functional connection to the pathophysiology of donor cells. Consequently, EVs are considered diagnostic and monitoring factors in many diseases. Despite consensus as to their activity in promoting coagulation and inflammation, there is evidence suggesting protective roles for EVs in stress states. Chronic kidney disease (CKD) patients are at high risk of developing cardiovascular defects. The pathophysiology, comorbidities, and treatment of CKD may individually and in synergy affect extracellular vesiculation in the kidney, endothelium, and blood cells. Oxidative and mechanical stresses, chronic inflammation, and deregulation of calcium and phosphate homeostasis are established stressors of EV release. EVs may affect the clinical severity of CKD by transferring biological response modifiers between renal, vascular, blood, and inflammatory cells. In this Review, we focus on EVs circulating in the plasma of CKD patients. We highlight some recent advances in the understanding of their biogenesis, the effects of dialysis, and pharmacological treatments on them and their potential impact on thrombosis and vascular defects. The strong interest of the scientific community to this exciting field of research may reveal hidden pieces in the pathophysiology of CKD and thus, innovative ways to treat it. Overcoming gaps in EV biology and technical difficulties related to their size and heterogeneity will define the success of the project.

Human Melanoma-Cell Metabolic Profiling: Identification of Novel Biomarkers Indicating Metastasis.

Melanoma is the most aggressive type of skin cancer, leading to metabolic rewiring and enhancement of metastatic transformation. Efforts to improve its early and accurate diagnosis are largely based on preclinical models and especially cell lines. Hence, we herein present a combinational Nuclear Magnetic Resonance (NMR)- and Ultra High Performance Liquid Chromatography-High-Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS)-mediated untargeted metabolomic profiling of melanoma cells, to landscape metabolic alterations likely controlling metastasis. The cell lines WM115 and WM2664, which belong to the same patient, were examined, with WM115 being derived from a primary, pre-metastatic, tumor and WM2664 clonally expanded from lymph-node metastases. Metabolite samples were analyzed using NMR and UHPLC-HRMS. Multivariate statistical analysis of high resolution NMR and MS (positive and negative ionization) results was performed by Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), while metastasis-related biomarkers were determined on the basis of VIP lists, S-plots and Student's t-tests. Receiver Operating Characteristic (ROC) curves of NMR and MS data revealed significantly differentiated metabolite profiles for each cell line, with WM115 being mainly characterized by upregulated levels of phosphocholine, choline, guanosine and inosine. Interestingly, WM2664 showed notably increased contents of hypoxanthine, myo-inositol, glutamic acid, organic acids, purines, pyrimidines, AMP, ADP, ATP and UDP(s), thus indicating the critical roles of purine, pyrimidine and amino acid metabolism during human melanoma metastasis.

Malignancy Grade-Dependent Mapping of Metabolic Landscapes in Human Urothelial Bladder Cancer: Identification of Novel, Diagnostic, and Druggable Biomarkers.

BACKGROUND: Urothelial bladder cancer (UBC) is one of the cancers with the highest mortality rate and prevalence worldwide; however, the clinical management of the disease remains challenging. Metabolomics has emerged as a powerful tool with beneficial applications in cancer biology and thus can provide new insights on the underlying mechanisms of UBC progression and/or reveal novel diagnostic and therapeutic schemes. METHODS: A collection of four human UBC cell lines that critically reflect the different malignancy grades of UBC was employed; RT4 (grade I), RT112 (grade II), T24 (grade III), and TCCSUP (grade IV). They were examined using Nuclear Magnetic Resonance, Mass Spectrometry, and advanced statistical approaches, with the goal of creating new metabolic profiles that are mechanistically associated with UBC progression toward metastasis. RESULTS: Distinct metabolic profiles were observed for each cell line group, with T24 (grade III) cells exhibiting the most abundant metabolite contents. AMP and creatine phosphate were highly increased in the T24 cell line compared to the RT4 (grade I) cell line, indicating the major energetic transformation to which UBC cells are being subjected during metastasis. Thymosin ß4 and ß10 were also profiled with grade-specific patterns of expression, strongly suggesting the importance of actin-cytoskeleton dynamics for UBC advancement to metastatic and drug-tolerant forms. CONCLUSIONS: The present study unveils a novel and putatively druggable metabolic signature that holds strong promise for early diagnosis and the successful chemotherapy of UBC disease.

Proteomic mapping of Drosophila transgenic elav.L-GAL4/+ brain as a tool to illuminate neuropathology mechanisms.

Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.

Exploitation of Drosophila Choriogenesis Process as a Model Cellular System for Assessment of Compound Toxicity: the Phloroglucinol Paradigm.

Phloroglucinol (1,3,5 tri-hydroxy-benzene) (PGL), a natural phenolic substance, is a peroxidase inhibitor and has anti-oxidant, anti-diabetic, anti-inflammatory, anti-thrombotic, radio-protective, spasmolytic and anti-cancer activities. PGL, as a medicine, is administered to patients to control the symptoms of irritable bowel syndrome and acute renal colic, in clinical trials. PGL, as a phenolic substance, can cause cytotoxic effects. Administration of PGL up to 300 mg/kg (bw) is well tolerated by animals, while in cell lines its toxicity is developed at concentrations above the dose of 10 µg/ml. Furthermore, it seems that tumor or immortalized cells are more susceptible to the toxic power of PGL, than normal cells. However, studies of its cytotoxic potency, at the cellular level, in complex, differentiated and meta-mitotic biological systems, are still missing. In the present work, we have investigated the toxic activity of PGL in somatic epithelial cells, constituting the follicular compartment of a developing egg-chamber (or, follicle), which directs the choriogenesis (i.e. chorion assembly) process, during late oogenesis of Drosophila melanogaster. Our results reveal that treatment of in vitro growing Drosophila follicles with PGL, at a concentration of 0.2 mM (or, 25.2 µg/ml), does not lead to follicle-cell toxicity, since the protein-synthesis program and developmental pattern of choriogenesis are normally completed. Likewise, the 1 mM dose of PGL was also characterized by lack of toxicity, since the chorionic proteins were physiologically synthesized and the chorion structure appeared unaffected, except for a short developmental delay, being observed. In contrast, concentrations of 10, 20 or 40 mM of PGL unveiled a dose-dependent, increasing, toxic effect, being initiated by interruption of protein synthesis and disassembly of cell-secretory machinery, and, next, followed by fragmentation of the granular endoplasmic reticulum (ER) into vesicles, and formation of autophagic vacuoles. Follicle cells enter into an apoptotic process, with autophagosomes and large vacuoles being formed in the cytoplasm, and nucleus showing protrusions, granular nucleolus and condensed chromatin. PGL, also, proved able to induce disruption of nuclear envelope, activation of nucleus autophagy (nucleophagy) and formation of a syncytium-like pattern being produced by fusion of plasma membranes of two or more individual follicle cells. Altogether, follicle cell-dependent choriogenesis in Drosophila has been herein presented as an excellent, powerful and reliable multi-cellular, differentiated, model biological (animal) system for drug-cytotoxicity assessment, with the versatile compound PGL serving as a characteristic paradigm. In conclusion, PGL is a substance that may act beneficially for a variety of pathological conditions and can be safely used for differentiated somatic -epithelial- cells at clinically low concentrations. At relatively high doses, it could potentially induce apoptotic and autophagic cell death, thus being likely exploited as a therapeutic agent against a number of pathologies, including human malignancies.