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.

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.

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.

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.

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.

Drug-Induced Lysosomal Impairment Is Associated with the Release of Extracellular Vesicles Carrying Autophagy Markers.

Amiodarone is a cationic amphiphilic drug used as an antiarrhythmic agent. It induces phospholipidosis, i.e., the accumulation of phospholipids within organelles of the endosomal-lysosomal system. Extracellular vesicles (EVs) are membrane-enclosed structures released by any type of cell and retrieved in every fluid of the body. EVs have been initially identified as a system to dispose cell waste, but they are also considered to be an additional manner to transmit intercellular signals. To understand the role of EVs in drug-induced phospholipidosis, we investigated EVs release in amiodarone-treated HEK-293 cells engineered to produce fluorescently labelled EVs. We observed that amiodarone induces the release of a higher number of EVs, mostly of a large/medium size. EVs released upon amiodarone treatment do not display significant morphological changes or altered size distribution, but they show a dose-dependent increase in autophagy associated markers, indicating a higher release of EVs with an autophagosome-like phenotype. Large/medium EVs also show a higher content of phospholipids. Drugs inducing lysosomal impairment such as chloroquine and bafilomycin A1 similarly prompt a higher release of EVs enriched in autophagy markers. This result suggests a mechanism associated with amiodarone-induced lysosomal impairment more than a connection with the accumulation of specific undigested substrates. Moreover, the implementation of the lysosomal function by overexpressing TFEB, a master gene regulator of lysosomal biogenesis, prevents the amiodarone-induced release of EVs, suggesting that this could be a feasible target to attenuate drug-induced abnormalities.

Lipostar, a Comprehensive Platform-Neutral Cheminformatics Tool for Lipidomics.

To date, the main limitations for LC-MS-based untargeted lipidomics reside in the lack of adequate computational and cheminformatics tools that are able to support the analysis of several thousands of species from biological samples, enabling data mining and automating lipid identification and external prediction processes. To address these issues, we developed Lipostar, novel vendor-neutral high-throughput software that effectively supports both targeted and untargeted LC-MS lipidomics, implementing data acquisition, user-friendly multivariate analysis (to be used for model generation and new sample predictions), and advanced lipid identification protocols that can work with or without the support of preformed lipid databases. Moreover, Lipostar integrates the lipidomic processes with a full metabolite identification (MetID) procedure, essential in drug safety applications and in translational studies. Case studies demonstrating a number of Lipostar features are also presented.

LC/MS lipid profiling from human serum: a new method for global lipid extraction.

Over the last decade, technological advances have improved the sensitivity and selectivity of LC/MS analyzers, providing very efficient tools for lipidomics research. In particular, the nine lipid classes that constitute 99 % of the human serum lipidome (sterols, cholesteryl esters, phosphocholines, phosphoethanolamines, sphingomyelins, triacylglycerols, fatty acids, lysophosphocholines, and diacylglycerols) can be easily detected. However, until today there has not been a unique technique for sample preparation that provides a satisfactory recovery for all of these nine classes together. In this work, we have developed and validated a new one-phase extraction (OPE) method that overcomes this limitation. This method was also compared with the gold standard lipid extraction methods such as Folch, Bligh & Dyer, and recently developed methods with methanol and methyl-tert-butyl ether. Results demonstrate that the mixture of methanol/chloroform/MTBE (MMC) provides a recovery very close to 100 % for all nine lipid classes of the human serum investigated. For this extraction method, 100 µL of human serum is incubated with 2 mL of the solvents mixture, then vortexed and centrifuged. For its simplicity of execution, rapidity, reproducibility, and the reduced volume of sample required, this method opens the door to the use of human serum lipid profiling for large-scale applications in scientific research and clinical trials.

Metabolism of JWH-015, JWH-098, JWH-251, and JWH-307 in silico and in vitro: a pilot study for the detection of unknown synthetic cannabinoids metabolites.

This pilot study was performed to study the main metabolic reactions of four synthetic cannabinoids: JWH-015, JWH-098, JWH-251, and JWH-307 in order to setup a screening method for the detection of main metabolites in biological fluids. In silico prediction of main metabolic reactions was performed using MetaSite(™) software. To evaluate the agreement between software prediction and experimental reactions, we performed in vitro experiments on the same JWHs using rat liver slices. The obtained samples were analyzed by liquid chromatography-quadrupole time-of-flight and the identification of metabolites was executed using Mass-MetaSite(™) software that automatically assigned the metabolite structures to the peaks detected based on their accurate masses and fragmentation. A comparison between the experimental findings and the in silico metabolism prediction using MetaSite(™) software showed a good accordance between experimental and in silico data. Thus, the use of in silico metabolism prediction might represent a useful tool for the forensic and clinical toxicologist to identify possible main biomarkers for synthetic cannabinoids in biological fluids, especially urine, following their administration.

Evidence of Lysosomal ß-Hexosaminidase Enzymatic Activity Associated with Extracellular Vesicles: Potential Applications for the Correction of Sandhoff Disease.

Extracellular vesicles (EVs) can be isolated from biological fluids and cell culture medium. Their nanometric dimension, relative stability, and biocompatibility have raised considerable interest for their therapeutic use as delivery vehicles of macromolecules, namely nucleic acids and proteins. Deficiency in lysosomal enzymes and associated proteins is at the basis of a group of genetic diseases known as lysosomal storage disorders (LSDs), characterized by the accumulation of undigested substrates into lysosomes. Among them, GM2 gangliosidoses are due to a deficiency in the activity of lysosomal enzyme ß-hexosaminidase, leading to the accumulation of the GM2 ganglioside and severe neurological symptoms. Current therapeutic approaches, including enzyme replacement therapy (ERT), have proven unable to significantly treat these conditions. Here, we provide evidence that the lysosomal ß-hexosaminidase enzyme is associated with EVs released by HEK cells and that the EV-associated activity can be increased by overexpressing the α-subunit of ß-hexosaminidase. The delivery of EVs to ß-hexosaminidase-deficient fibroblasts results in a partial cross-correction of the enzymatic defect. Overall findings indicate that EVs could be a source of ß-hexosaminidase that is potentially exploitable for developing therapeutic approaches for currently untreatable LSDs.

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.

Increased Vasoactive Intestinal Peptide (VIP) in polycystic ovary syndrome patients undergoing IVF.

Introduction: Polycystic ovary syndrome (PCOS) is a common multifactorial and polygenic disorder of the endocrine system, affecting up to 20% of women in reproductive age with a still unknown etiology. Follicular fluid (FF) represents an environment for the normal development of follicles rich in metabolites, hormones and neurotransmitters, but in some instances of PCOS the composition can be different. Vasoactive intestinal peptide (VIP) is an endogenous autonomic neuropeptide involved in follicular atresia, granulosa cell physiology and steroidogenesis. Methods: ELISA assays were performed to measure VIP and estradiol levels in human follicular fluids, while AMH, FSH, LH, estradiol and progesterone in the plasma were quantified by chemiluminescence. UHPLC/QTOF was used to perform the untargeted metabolomic analysis. Results: Our ELISA and metabolomic results show: i) an increased concentration of VIP in follicular fluid of PCOS patients (n=9) of about 30% with respect to control group (n=10) (132 ± 28 pg/ml versus 103 ± 26 pg/ml, p=0,03) in women undergoing in vitro fertilization (IVF), ii) a linear positive correlation (p=0.05, r=0.45) between VIP concentration and serum Anti-Müllerian Hormone (AMH) concentration and iii) a linear negative correlation between VIP and noradrenaline metabolism. No correlation between VIP and estradiol (E2) concentration in follicular fluid was found. A negative correlation was found between VIP and noradrenaline metabolite 3,4-dihydroxyphenylglycolaldehyde (DOPGAL) in follicular fluids. Conclusion: VIP concentration in follicular fluids was increased in PCOS patients and a correlation was found with noradrenaline metabolism indicating a possible dysregulation of the sympathetic reflex in the ovarian follicles. The functional role of VIP as noradrenergic modulator in ovarian physiology and PCOS pathophysiology was discussed.

Ocular penetration of topical antibiotics: study on the penetration of chloramphenicol, tobramycin and netilmicin into the anterior chamber after topical administration.

BACKGROUND: To compare penetration in the aqueous humour of topically applied antibiotics. DESIGN: Randomized prospective study, Department of Ophthalmology, University of Perugia, Italy PARTICIPANTS: Patients undergoing cataract surgery. METHODS: One hundred twenty-two patients were included: 14 received one drop of chloramphenicol suspension; 12 one application of chloramphenicol gel; 11 one drop of netilmicin suspension; 13 one drop of tobramycin suspension; 37 repeated instillations of chloramphenicol suspension every 10 min for a total of four drops; and 35 repeated instillations of chloramphenicol gel every 10 min for a total of four drops. Samples were taken immediately before surgery from the anterior chamber in order to determine the antibiotic by means of high-performance liquid chromatography. Samples were taken 45-190 min after the eye drops were instilled. MAIN OUTCOME MEASURES: Intraocular penetration of chloramphenicol, netilmicin and tobramicyn. RESULTS: After a single administration, netilmicin and tobramycin were undetectable, whereas the chloramphenicol suspension reached a mean concentration of 0.23 ± 0.21 µg/mL, and the chloramphenicol gel a mean concentration of 0.13 ± 0.14 µg/mL. After repeated administrations, the mean concentrations of the chloramphenicol suspension and gel were 0.60 ± 0.26 µg/mL and 0.58 ± 0.18 µg/mL, respectively. CONCLUSIONS: Tobramycin and netilmicin do not reach detectable concentrations, whereas chloramphenicol, after multiple administrations, reaches concentrations that are effective against Haemophilus influenzae and Haemophilus parainfluenzae, Legionella pneumophila, Moraxella catarrhalis, Neisseria meningitidis, Pasteurella multocida and Streptococcus pneumoniae. This means that chloramphenicol can be rationally used in the prophylaxis and treatment of infections supported by sensitive germs.

Lipidomics of coconut, almond and soybean milks - Comprehensive characterization of triacylglycerol class and comparison with bovine milk.

Nowadays, plant-based milk consumption, as part of a healthy diet, is continuously increasing. In this paper, for the first time a lipidomic analysis on molecular species of triacylglycerol (TG) fraction of plant-based beverages (almond, soy, coconut) was performed by liquid chromatography quadrupole time-of-flight mass spectrometry. A total of 557 TG molecular species was measured, showing significantly different profiles between milk alternatives, compared with bovine milk. The most abundant TG molecular species were TG 18:1_18:1_18:1 and 18:1_18:1_18:2 for almond, TG 18:2_18:2_18:2 and 16:0_18:2_18:2 for soy, TG 12:0_10:0_12:0 and 12:0_12:0_14:0 for coconut. Unconventional fatty acids were detected in almond and soy. The main TG with ethereal linkage were TG-O 56:2, TG-O 56:4, and TG-O 56:5, while the main oxygenated TG was TG 54:5;1O. A total of 30 molecular species were identified as biomarkers for milk differentiation by principal component analysis, providing an interesting support for milk authentication and detection of adulteration on a larger sampling.

Biogenic Amine Content Analysis of Three Chicken-Based Dry Pet Food Formulations.

The pet food market is constantly expanding, and more and more attention is paid to the feeding of pets. Dry foods stand out and are often preferred due to their long shelf life, ease of administration, and low cost. In this context, dry foods are formulated from fresh meats, meat meals, or a mix of the two. These raw materials are often meat not fit for human consumption; they might be subject to contamination and proliferation of microorganisms which, by degrading the organic component, can lead to the formation of undesirable by-products such as biogenic amines. These nitrogenous compounds obtained by decarboxylation of amino acids can therefore be found in high-protein foods, and their ingestion in large quantities can cause intoxication and be harmful. This study aims at analyzing the possible presence of biogenic amines in three different formulations of chicken-based kibbles for pets: one obtained from fresh meat, one from meat meal, and one from a mix of the two. This study is also focused on the presence of free amino acids as they represent the key substrate for decarboxylating enzymes. Mass spectrometry (Q-TOF LC/MS) was used to analyze the presence of biogenic amines and free amino acids. The results show that fresh-meat-based products have a lower content of biogenic amines, and at the same time a higher quantity of free amino acids; on the contrary, meat-meal- and mix-based products have a greater quantity of biogenic amines and a lower concentration of free amino acids, suggesting that there has been a higher microbial proliferation as proved by the total aerobic mesophilic bacteria counts. It is therefore clear that fresh-meat-based kibbles are to be preferred when they are used for preparing dry pet food due to the lowest concentration of biogenic amines.

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.

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.

Bioactivity and Metabolomic Profile of Extracts Derived from Mycelial Solid Cultures of Hypsizygus marmoreus.

The beech mushroom (Hypsizygus marmoreus) is a highly nutritious, edible medicinal mushroom native to East Asia. The present research investigated the impact of different substrates on the metabolite compositions of H. marmoreus mycelia cultivated in vitro. The substrates tested included malt extract agar, malt extract agar enriched with barley malt, and malt extract agar enriched with grape pomace. The study also assessed antimicrobial and antiradical activities of the extracts against gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), gram-negative bacteria (Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa), yeasts (Candida albicans, C. tropicalis, and C. parapsilosis), and dermatophytes (Trichophyton mentagrophytes, T. tonsurans, T. rubrum, Arthroderma quadrifidum, A. gypseum, A. curreyi, and A. insingulare). The results revealed that the H. marmoreus mycelia extracts demonstrated antibacterial and antifungal activities against the tested microorganisms. Extracts obtained from the cultivation in substrates enriched with either barley malt or grape pomace exhibited the highest antibacterial activity among all the tested bacterial strains except for P. aeruginosa. The same extracts showed the highest inhibitory effect against C. albicans and C. parapsilosis. Noteworthy, the extract from the mushroom cultivated in the substrate enriched with grape pomace also exhibited remarkable efficacy against T. mentagrophytes and T. tonsurans. Terpenoid and carbapenem compounds could be related to the antimicrobial properties of the extracts from mushrooms cultivated in substrates enriched with grape pomace. In comparison, the higher antiradical properties could be related to the content of indole compounds. In conclusion, growth substrate selection affects the nutritional and medicinal properties of H. marmoreus, making it a valuable contribution to the understanding of the cultivation of this mushroom.

Diversity of Pleurotus spp. (Agaricomycetes) and Their Metabolites of Nutraceutical and Therapeutic Importance.

Pleurotus spp. have been gaining popularity as a source for the creation of functional foods, nutraceuticals and novel pharmaceuticals. Despite Pleurotus is a specious genus including 208 legitimate species, only a few of them such as P. ostreatus are commercially accessible. The genetic and metabolic diversity of Pleurotus both at specific and subspecific level is therefore of main concern for many researchers. In addition to the conventional morphological approach, molecular and biochemical markers have been greatly contributing to investigate these issues. In this study, samples from six Pleurotus species (P. eryngii is represented by three varieties) were molecularly identified and the phylogeny was inferred to assess the relationships between the various taxa. Strains in pure culture obtained from 6 out of 7 species were cultivated as mycelium in vitro to investigate the metabolites by untargeted LC-MS/MS-based metabolomics. The results pointed out species-specific metabolite patterns and highlighted a clear difference between the P. eryngii group and P. ostreatus, although the latter appears more versatile depending on the strain. This is the first study pointing out and comparing different metabolite patterns in Italian samples of Pleurotus species, including P. eryngii varieties.