Metabolic Profiling as an Approach to Differentiate T-Cell Acute Lymphoblastic Leukemia Cell Lines Belonging to the Same Genetic Subgroup.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive tumor mainly affecting children and adolescents. It is driven by multiple genetic mutations that together define the leukemic phenotype. Interestingly, based on genetic alterations and/or deregulated expression, at least six genetic subgroups have been recognized. The TAL/LMO subgroup is one of the most represented genetic subgroups, characterizing 30-45% of pediatric T-ALL cases. The study of lipid and metabolic profiles is increasingly recognized as a valuable tool for comprehending the development and progression of tumors. In this study, metabolic and lipidomic analysis via LC/MS have been carried out on four T-ALL cell lines belonging to the TAL/LMO subgroup (Jurkat, Molt-4, Molt-16, and CCRF-CEM) to identify new potential metabolic biomarkers and to provide a subclassification of T-ALL cell lines belonging to the same subgroup. A total of 343 metabolites were annotated, including 126 polar metabolites and 217 lipid molecules. The statistical analysis, for both metabolic and lipid profiles, shows significant differences and similarities among the four cell lines. The Molt-4 cell line is the most distant cell line and CCRF-CEM shows a high activity in specific pathways when compared to the other cell lines, while Molt-16 and Jurkat show a similar metabolic profile. Additionally, this study highlighted the pathways that differ in each cell line and the possible enzymes involved using bioinformatic tools, capable of predicting the pathways involved by studying the differences in the metabolic profiles. This experiment offers an approach to differentiate T-ALL cell lines and could open the way to verify and confirm the obtained results directly in patients.

An untargeted metabolomics approach to study changes of the medium during human cornea culture.

INTRODUCTION: Two main approaches (organ culture and hypothermia) for the preservation and storage of human donor corneas are globally adopted for corneal preservation before the transplant. Hypothermia is a hypothermic storage which slows down cellular metabolism while organ culture, a corneal culture performed at 28-37 °C, maintains an active corneal metabolism. Researchers, till now, have just studied the impact of organ culture on human cornea after manipulating and disrupting tissues. OBJECTIVES: The aim of the current work was to optimize an analytical procedure which can be useful for discovering biomarkers capable of predicting tissue health status. For the first time, this research proposed a preliminary metabolomics study on medium for organ culture without manipulating and disrupting the valuable human tissues which could be still used for transplantation. METHODS: In particular, the present research proposed a method for investigating changes in the medium, over a storage period of 20 days, in presence and absence of a human donor cornea. An untargeted metabolomics approach using UHPLC-QTOF was developed to deeply investigate the differences on metabolites and metabolic pathways and the influence of the presence of the cornea inside the medium. RESULTS: Differences in the expression of some compounds emerged from this preliminary metabolomics approach, in particular in medium maintained for 10 and 20 days in presence but also in the absence of cornea. A total of 173 metabolites have been annotated and 36 pathways were enriched by pathway analysis. CONCLUSION: The results revealed a valuable untargeted metabolomics approach which can be applied in organ culture metabolomics.

Characterization of Nanovesicles Isolated from Olive Vegetation Water.

Edible plant and fruit-derived nanovesicles (NVs) are membrane-enclosed particles with round-shape morphology and signaling functions, which resemble mammalian cell-derived extracellular vesicles. These NVs can transmit cross-kingdom signals as they contain bioactive molecules and exert biological effects on mammalian cells. Their properties and stability in the gastrointestinal tract suggest NVs as a promising nutraceutical tool. In this study, we have demonstrated for the first time the presence of NVs in olive vegetation water (OVW), a waste by-product generated during olive oil production. Biophysical characterization by scanning electron microscopy, cryo-transmission electron microscopy, and nanoparticle tracking analysis revealed the presence in OVW of NVs having size and morphology similar to that of vesicles isolated from edible plants. Integrated lipidomic, metabolomic, and proteomic analyses showed that OVW-NVs carry a set of lipids, metabolites and proteins which have recognized antioxidant and anti-inflammatory activities. The nature of biomolecules identified in OVW-NVs suggests that these vesicles could exert beneficial effects on mammalian cells and could be used in the nutraceutical and food industries. The successful isolation of OVW-NVs and the characterization of their features strengthen the idea that agricultural waste might represent a source of NVs having features similar to NVs isolated from edible plants/fruits.

Chronic Rapamycin administration via drinking water mitigates the pathological phenotype in a Krabbe disease mouse model through autophagy activation.

Krabbe disease (KD) is a rare disorder arising from the deficiency of the lysosomal enzyme galactosylceramidase (GALC), leading to the accumulation of the cytotoxic metabolite psychosine (PSY) in the nervous system. This accumulation triggers demyelination and neurodegeneration, and despite ongoing research, the underlying pathogenic mechanisms remain incompletely understood, with no cure currently available. Previous studies from our lab revealed the involvement of autophagy dysfunctions in KD pathogenesis, showcasing p62-tagged protein aggregates in the brains of KD mice and heightened p62 levels in the KD sciatic nerve. We also demonstrated that the autophagy inducer Rapamycin (RAPA) can partially reinstate the wild type (WT) phenotype in KD primary cells by decreasing the number of p62 aggregates. In this study, we tested RAPA in the Twitcher (TWI) mouse, a spontaneous KD mouse model. We administered the drug ad libitum via drinking water (15 mg/L) starting from post-natal day (PND) 21-23. We longitudinally monitored the mouse motor performance through grip strength and rotarod tests, and a set of biochemical parameters related to the KD pathogenesis (i.e. autophagy markers expression, PSY accumulation, astrogliosis and myelination). Our findings demonstrate that RAPA significantly enhances motor functions at specific treatment time points and reduces astrogliosis in TWI brain, spinal cord, and sciatic nerves. Utilizing western blot and immunohistochemistry, we observed a decrease in p62 aggregates in TWI nervous tissues, corroborating our earlier in-vitro results. Moreover, RAPA treatment partially removes PSY in the spinal cord. In conclusion, our results advocate for considering RAPA as a supportive therapy for KD. Notably, as RAPA is already available in pharmaceutical formulations for clinical use, its potential for KD treatment can be rapidly evaluated in clinical trials.

Untargeted Lipidomic Approach for Studying Different Nervous System Tissues of the Murine Model of Krabbe Disease.

Krabbe disease is a rare neurodegenerative disease with an autosomal recessive character caused by a mutation in the GALC gene. The mutation leads to an accumulation of psychosine and a subsequent degeneration of oligodendrocytes and Schwann cells. Psychosine is the main biomarker of the disease. The Twitcher mouse is the most commonly used animal model to study Krabbe disease. Although there are many references to this model in the literature, the lipidomic study of nervous system tissues in the Twitcher model has received little attention. This study focuses on the comparison of the lipid profiles of four nervous system tissues (brain, cerebellum, spinal cord, and sciatic nerve) in the Twitcher mouse compared to the wild-type mouse. Altogether, approximately 230 molecular species belonging to 19 lipid classes were annotated and quantified. A comparison at the levels of class, molecular species, and lipid building blocks showed significant differences between the two groups, particularly in the sciatic nerve. The in-depth study of the lipid phenotype made it possible to hypothesize the genes and enzymes involved in the changes. The integration of metabolic data with genetic data may be useful from a systems biology perspective to gain a better understanding of the molecular basis of the disease.

Circulating extracellular particles from severe COVID-19 patients show altered profiling and innate lymphoid cell-modulating ability.

Introduction: Extracellular vesicles (EVs) and particles (EPs) represent reliable biomarkers for disease detection. Their role in the inflammatory microenvironment of severe COVID-19 patients is not well determined. Here, we characterized the immunophenotype, the lipidomic cargo and the functional activity of circulating EPs from severe COVID-19 patients (Co-19-EPs) and healthy controls (HC-EPs) correlating the data with the clinical parameters including the partial pressure of oxygen to fraction of inspired oxygen ratio (PaO2/FiO2) and the sequential organ failure assessment (SOFA) score. Methods: Peripheral blood (PB) was collected from COVID-19 patients (n=10) and HC (n=10). EPs were purified from platelet-poor plasma by size exclusion chromatography (SEC) and ultrafiltration. Plasma cytokines and EPs were characterized by multiplex bead-based assay. Quantitative lipidomic profiling of EPs was performed by liquid chromatography/mass spectrometry combined with quadrupole time-of-flight (LC/MS Q-TOF). Innate lymphoid cells (ILC) were characterized by flow cytometry after co-cultures with HC-EPs or Co-19-EPs. Results: We observed that EPs from severe COVID-19 patients: 1) display an altered surface signature as assessed by multiplex protein analysis; 2) are characterized by distinct lipidomic profiling; 3) show correlations between lipidomic profiling and disease aggressiveness scores; 4) fail to dampen type 2 innate lymphoid cells (ILC2) cytokine secretion. As a consequence, ILC2 from severe COVID-19 patients show a more activated phenotype due to the presence of Co-19-EPs. Discussion: In summary, these data highlight that abnormal circulating EPs promote ILC2-driven inflammatory signals in severe COVID-19 patients and support further exploration to unravel the role of EPs (and EVs) in COVID-19 pathogenesis.

Mechanotransduction Impairment in Primary Fibroblast Model of Krabbe Disease.

Krabbe disease (KD) is a genetic disorder caused by the absence of the galactosylceramidase (GALC) functional enzyme. No cure is currently available. Here, we investigate the mechanotransduction process in primary fibroblasts collected from the twitcher mouse, a natural KD murine model. Thanks to mechanotransduction, cells can sense their environment and convert external mechanical stimuli into biochemical signals that result in intracellular changes. In GALC-deficient fibroblasts, we show that focal adhesions (FAs), the protein clusters necessary to adhere and migrate, are increased, and that single-cell migration and wound healing are impaired. We also investigate the involvement of the autophagic process in this framework. We show a dysregulation in the FA turnover: here, the treatment with the autophagy activator rapamycin boosts cell migration and improves the clearance of FAs in GALC-deficient fibroblasts. We propose mechanosensing impairment as a novel potential pathological mechanism in twitcher fibroblasts, and more in general in Krabbe disease.

Comparison between Sickle Cell Disease Patients and Healthy Donors: Untargeted Lipidomic Study of Erythrocytes.

Sickle cell disease (SCD) is one of the most common severe monogenic disorders in the world caused by a mutation on HBB gene and characterized by hemoglobin polymerization, erythrocyte rigidity, vaso-occlusion, chronic anemia, hemolysis, and vasculopathy. Recently, the scientific community has focused on the multiple genetic and clinical profiles of SCD. However, the lipid composition of sickle cells has received little attention in the literature. According to recent studies, changes in the lipid profile are strongly linked to several disorders. Therefore, the aim of this study is to dig deeper into lipidomic analysis of erythrocytes in order to highlight any variations between healthy and patient subjects. 241 lipid molecular species divided into 17 classes have been annotated and quantified. Lipidomic profiling of SCD patients showed that over 24% of total lipids were altered most of which are phospholipids. In-depth study of significant changes in lipid metabolism can give an indication of the enzymes and genes involved. In a systems biology scenario, these variations can be useful to improve the understanding of the biochemical basis of SCD and to try to make a score system that could be predictive for the severity of clinical manifestations.

Accurate Analysis of Lipid Building Blocks Using the Tool LipidOne.

LC/MS-based analysis techniques combined with specialized lipid platforms allow the qualitative and quantitative determination of thousands of lipid molecules. Each individual molecule can be considered as an assembly of smaller parts, often called building blocks that are the result of a myriad of biochemical synthesis and transformation processes. LipidOne is a new lipidomic tool that automatically highlights all qualitative and quantitative changes in lipid building blocks both among all detected lipid classes and between experimental groups. Thanks to LipidOne, the discovered differences among lipid building blocks can be easily linked to the activity of specific enzymes.

LC/MS Q-TOF Metabolomic Investigation of Amino Acids and Dipeptides in Pleurotus ostreatus Grown on Different Substrates.

The well-established correlation between diet and health arouses great interest in seeking new health-promoting functional foods that may contribute to improving health and well-being. Herein, the metabolomic investigation of Pleurotus ostreatus samples grown on two different substrates (black poplar wood logs, WS, and lignocellulosic byproducts, LcS) revealed the high potential of such a mushroom as a source of bioactive species. The liquid chromatography/mass spectrometry combined with quadrupole time-of-flight (LC/MS Q-TOF) analysis allowed the identification of essential and nonessential amino acids along with the outstanding presence of dipeptides. Multivariate statistical models highlighted important differences in the expression of both classes of compounds arising from the growth of P. ostreatus strains on WS and LcS. The former, in particular, was correlated to an increased expression of carnitine-based amino acid derivatives and proline-based dipeptides. This finding may represent a potential strategy to drive the expression of bioactive compounds of interest to obtain enriched mushrooms or useful functional ingredients from them.

Chronic lithium administration in a mouse model for Krabbe disease.

Krabbe disease (KD; or globoid cell leukodystrophy) is an autosomal recessive lysosomal storage disorder caused by deficiency of the galactosylceramidase (GALC) enzyme. No cure is currently available for KD. Clinical applied treatments are supportive only. Recently, we demonstrated that two differently acting autophagy inducers (lithium and rapamycin) can improve some KD hallmarks in-vitro, laying the foundation for their in-vivo pre-clinical testing. Here, we test lithium carbonate in-vivo, in the spontaneous mouse model for KD, the Twitcher (TWI) mouse. The drug is administered ad libitum via drinking water (600 mg/L) starting from post natal day 20. We longitudinally monitor the mouse motor performance through the grip strength, the hanging wire and the rotarod tests, and a set of biochemical parameters related to the KD pathogenesis [i.e., GALC enzymatic activity, psychosine (PSY) accumulation and astrogliosis]. Additionally, we investigate the expression of some crucial markers related to the two pathways that could be altered by lithium: the autophagy and the ß-catenin-dependent pathways. Results demonstrate that lithium has not a significant rescue effect on the TWI phenotype, although it can slightly and transiently improves muscle strength. We also show that lithium, with this administration protocol, is unable to stimulate autophagy in the TWI mice central nervous system, whereas results suggest that it can restore the ß-catenin activation status in the TWI sciatic nerve. Overall, these data provide intriguing inputs for further evaluations of lithium treatment in TWI mice.

LipidOne: user-friendly lipidomic data analysis tool for a deeper interpretation in a systems biology scenario.

SUMMARY: LC/MS-based analysis techniques combined with specialized lipid tool allow for the qualitative and quantitative determination of thousands of lipid molecules. Some recent bioinformatics tools have been developed to study changes in the lipid profile in case-control experiments and correlate these changes to different enzyme activity or gene expression. However, the existing tools have the limitation to treat only the assembled lipid molecules. In reality, each individual molecule can be considered as an assembly of smaller parts, often called building blocks. These are the result of a myriad of biochemical synthesis and transformation processes that, from a systems biology perspective, should not be ignored. Here, we present LipidOne, a new lipidomic tool which highlights all qualitative and quantitative changes in lipid building blocks both among all detected lipid classes and among experimental groups. Thanks to LipidOne, even differences in lipid building blocks can now be linked to the activity of specific classes of enzymes, transcripts and genes. AVAILABILITY AND IMPLEMENTATION: LipidOne software is freely available at www.dcbb.unipg.it/LipidOne and https://github.com/matteogiulietti/LipidOne. CONTACT: roberto.pellegrino@unipg.it. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

α-Linolenic acid and product octadecanoids in Styrian pumpkin seeds and oils: How processing impacts lipidomes of fatty acid, triacylglycerol and oxylipin molecular structures.

Styrian pumpkin seed oil is a conditioned green-colored oil renowned for nutty smell and taste. Due to α-linolenic acid (ALA) contents below 1% of total fatty acids and the prospect of nutritional health claims based on its potential oxidation products, we investigated the fate of ALA and product oxylipins in the course of down-stream processing of seeds and in oils. Lipidomic analyses with Lipid Data Analyzer 2.8.1 revealed: Processing did not change (1) main fatty acid composition in the oils, (2) amounts of triacylglycerol species, (3) structures of triacylglycerol molecular species containing ALA. (4) Minor precursor ALA in fresh Styrian and normal pumpkins produced 6 product phytoprostanes in either cultivar, quantitatively more in the latter. (5) In oil samples 7 phytoprostanes and 2 phytofurans were detected. The latter two are specific for their presence in pumpkin seed oils, of note, quantitatively more in conditioned oils than in cold-pressed native oils.

Metabolomic Profiling and Biological Activities of Pleurotus columbinus Quél. Cultivated on Different Agri-Food Byproducts.

The genus Pleurotus (Fr.) P. Kumm (Pleurotaceae, Basidiomycota) comprises a cosmopolitan group of mushrooms highly appreciated for their nutritional value and health-promoting benefits. Despite there being many studies about the phytochemical composition of Pleurotus spp., there are very few reports dealing with the phytochemistry, antioxidant and antimicrobial activities of P. columbinus Quél. In this study, a mass spectrometry ultra-performance liquid chromatography mass spectrometry (UHPLC)-QTOF method, coupled with principal component analysis (PCA), was applied to the P. columbinus metabolome in order to investigate the influence of different agri-food residues as growth substrates for P. columbinus cultivation, on the bioactive chemical profile of fruiting bodies and evaluated their potential as antioxidants and antimicrobials. Additionally, a quantitative HPLC-DAD-MS analysis was conducted on phenolic and flavonoid compounds, that could explain, albeit partially, the observed biological effects of P. columbinus extracts. The qualitative metabolic profile identified 97 metabolites, whereas the quantitative HPLC-DAD-MS analysis confirmed the presence of phenolic and flavonoids, in the mushroom extracts, which also showed intrinsic scavenging/reducing and antimicrobial effects. The antibacterial effects were particularly evident against Escherichia coli, whereas Tricophyton and Aspergillus were the dermatophytes more sensitive to the mushroom extracts. The present study supports more in-depth investigations, aimed at evaluating the influence of growth substrate on P. columbinus antimicrobial and antioxidant properties. The extracts from P. columbinus revealed valuable sources of primary and secondary metabolites, thus suggesting potential applications in the formulation of food supplements with biological properties, above all in terms of antioxidant and antimicrobial properties.

Kinetics of hydrocortisone sodium phosphate penetration into the human aqueous humor after topical application.

AIM OF THE STUDY: Hydrocortisone is a soft steroid with low anti-inflammatory properties and a short duration of action, used to manage several ocular conditions. The clinical benefits and side effects associated with hydrocortisone are well documented, but its basic pharmacokinetic in the eye is yet to be fully elucidated. The purpose of this study is to investigate the anterior chamber penetration capabilities of hydrocortisone when used in different concentrations as eye drops treatment. MATERIALS AND METHODS: This is a double-blind, single-centre, randomised clinical trial performed at the Department of Medicine and Surgery of the University of Perugia (Italy) on consecutive patients who undergone phacoemulsification with intraocular lens implantation. Patients were randomly assigned on the morning of surgery to receive a single instillation of 0.33% (group A) or 0.001% (group B) hydrocortisone sodium phosphate solution. Group of patients C did not receive any treatment and was used to measure the hydrocortisone endogenous levels. Before surgery, one aliquot of aqueous humor for each patient was aspirated. The time of collection for each sample was recorded. Hydrocortisone concentrations were then stratified into six interval classes of 30 minutes each. RESULTS: The mean concentration of hydrocortisone was significantly higher in group A (25.2 ± 12.4 ng/mL) compared with group B (7.11 ± 1.51 ng/mL) and compared with the mean hydrocortisone endogenous levels (3.92 ± 1.18 ng/mL) (P < .0001). No statistically significant differences of hydrocortisone mean concentrations between group B and the mean endogenous levels were found. CONCLUSIONS: Considering the frequent need for prolonged topical steroid therapies and the possible consequent undesirable side effects, ophthalmologists should consider the lowest clinically effective dose of hydrocortisone useful to obtain the desired therapeutic effect and in an adequate time, to minimise the amount of steroids into the anterior chamber and to avoid side effects like intra-ocular pressure increase or cataract development.