Mitapivat reprograms the RBC metabolome and improves anemia in a mouse model of hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common, nonimmune, hereditary, chronic hemolytic anemia after hemoglobinopathies. The genetic defects in membrane function causing HS lead to perturbation of the RBC metabolome, with altered glycolysis. In mice genetically lacking protein 4.2 (4.2-/-; Epb42), a murine model of HS, we showed increased expression of pyruvate kinase (PK) isoforms in whole and fractioned RBCs in conjunction with abnormalities in the glycolytic pathway and in the glutathione (GSH) system. Mitapivat, a PK activator, metabolically reprogrammed 4.2-/- mouse RBCs with amelioration of glycolysis and the GSH cycle. This resulted in improved osmotic fragility, reduced phosphatidylserine positivity, amelioration of RBC cation content, reduction of Na/K/Cl cotransport and Na/H-exchange overactivation, and decrease in erythroid vesicles release in vitro. Mitapivat treatment significantly decreased erythrophagocytosis and beneficially affected iron homeostasis. In mild-to-moderate HS, the beneficial effect of splenectomy is still controversial. Here, we showed that splenectomy improves anemia in 4.2-/- mice and that mitapivat is noninferior to splenectomy. An additional benefit of mitapivat treatment was lower expression of markers of inflammatory vasculopathy in 4.2-/- mice with or without splenectomy, indicating a multisystemic action of mitapivat. These findings support the notion that mitapivat treatment should be considered for symptomatic HS.

Gene Expression Profiling as a New Real-Time Assay in Human Biomonitoring of Waste-to-Energy Plant Workers.

Exposure to heavy metals represents one of the most important risk factors for the health of incinerator workers. Indeed, heavy metals can determine increased generation of reactive oxygen species (ROS). In this work, we introduced the use of transcription profiling of detoxifying genes, involved in redox balance and genome integrity, as a highly sensitive assay of heavy metal exposure and subsequent oxidative stress. For this purpose, blood mRNA levels of OGG1, ST13, NQO1 and MT1A genes, as well as urinary concentrations of nine heavy metals and the oxidized base 8-OHdG of 49 subjects (26 controls and 23 employees in the waste-to-energy plant of San Zeno, Arezzo, Italy) were determined. No significant difference between the two populations was observed, thus highlighting, as far as the biomarkers analysed are concerned, the absence of occupational exposure to heavy metals and systemic oxidative stress induction in the workers of the waste-to-energy plant of San Zeno. Correlation analyses underline a close association between heavy metals exposure and changes in expression levels of a number of genes, even at low exposure doses, thus remarking the greater capacity of detection of transcription profiling compared to other biomarkers and the importance of its introduction in future human biomonitoring programs.

Plastic and Placenta: Identification of Polyethylene Glycol (PEG) Compounds in the Human Placenta by HPLC-MS/MS System.

The placenta is a crucial interface between the fetus and the maternal environment. It allows for nutrient absorption, thermal regulation, waste elimination, and gas exchange through the mother's blood supply. Furthermore, the placenta determines important adjustments and epigenetic modifications that can change the phenotypic expression of the individual even long after birth. Polyethylene glycol (PEG) is a polyether compound derived from petroleum with many applications, from medicine to industrial manufacturing. In this study, for the first time, an integration of ultra-high-performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) was used to detect suites of PEG compounds in human placenta samples, collected from 12 placentas, originating from physiological pregnancy. In 10 placentas, we identified fragments of PEG in both chorioamniotic membranes and placental cotyledons, for a total of 36 samples.

Urinary Untargeted Metabolic Profile Differentiates Children with Autism from Their Unaffected Siblings.

Autism Spectrum Disorder (ASD) encompasses a clinical spectrum of neurodevelopmental conditions that display significant heterogeneity in etiology, symptomatology, and severity. We previously compared 30 young children with idiopathic ASD and 30 unrelated typically-developing controls, detecting an imbalance in several compounds belonging mainly to the metabolism of purines, tryptophan and other amino acids, as well as compounds derived from the intestinal flora, and reduced levels of vitamins B6, B12 and folic acid. The present study describes significant urinary metabolomic differences within 14 pairs, including one child with idiopathic ASD and his/her typically-developing sibling, tightly matched by sex and age to minimize confounding factors, allowing a more reliable identification of the metabolic fingerprint related to ASD. By using a highly sensitive, accurate and unbiased approach, suitable for ensuring broad metabolite detection coverage on human urine, and by applying multivariate statistical analysis, we largely replicate our previous results, demonstrating a significant perturbation of the purine and tryptophan pathways, and further highlight abnormalities in the "phenylalanine, tyrosine and tryptophan" pathway, essentially involving increased phenylalanine and decreased tyrosine levels, as well as enhanced concentrations of bacterial degradation products, including phenylpyruvic acid, phenylacetic acid and 4-ethylphenyl-sulfate. The outcome of these within-family contrasts consolidates and extends our previous results obtained from unrelated individuals, adding further evidence that these metabolic imbalances may be linked to ASD rather than to environmental differences between cases and controls. It further underscores the excess of some gut microbiota-derived compounds in ASD, which could have diagnostic value in a network model differentiating the metabolome of autistic and unaffected siblings. Finally, it points toward the existence of a "metabolic autism spectrum" distributed as an endophenotype, with unaffected siblings possibly displaying a metabolic profile intermediate between their autistic siblings and unrelated typically-developing controls.

Metabolomic Profile of the Fungus Cryomyces antarcticus Under Simulated Martian and Space Conditions as Support for Life-Detection Missions on Mars.

The identification of traces of life beyond Earth (e.g., Mars, icy moons) is a challenging task because terrestrial chemical-based molecules may be destroyed by the harsh conditions experienced on extraterrestrial planetary surfaces. For this reason, studying the effects on biomolecules of extremophilic microorganisms through astrobiological ground-based space simulation experiments is significant to support the interpretation of the data that will be gained and collected during the ongoing and future space exploration missions. Here, the stability of the biomolecules of the cryptoendolithic black fungus Cryomyces antarcticus, grown on two Martian regolith analogues and on Antarctic sandstone, were analysed through a metabolomic approach, after its exposure to Science Verification Tests (SVTs) performed in the frame of the European Space Agency (ESA) Biology and Mars Experiment (BIOMEX) project. These tests are building a set of ground-based experiments performed before the space exposure aboard the International Space Station (ISS). The analysis aimed to investigate the effects of different mineral mixtures on fungal colonies and the stability of the biomolecules synthetised by the fungus under simulated Martian and space conditions. The identification of a specific group of molecules showing good stability after the treatments allow the creation of a molecular database that should support the analysis of future data sets that will be collected in the ongoing and next space exploration missions.

Metabolomics of Dry Versus Reanimated Antarctic Lichen-Dominated Endolithic Communities.

Cryptoendolithic communities are almost the sole life form in the ice-free areas of the Antarctic desert, encompassing among the most extreme-tolerant organisms known on Earth that still assure ecosystems functioning, regulating nutrient and biogeochemical cycles under conditions accounted as incompatible with active life. If high-throughput sequencing based studies are unravelling prokaryotic and eukaryotic diversity, they are not yet characterized in terms of stress adaptations and responses, despite their paramount ecological importance. In this study, we compared the responses of Antarctic endolithic communities, with special focus on fungi, both under dry conditions (i.e., when dormant), and after reanimation by wetting, light, and optimal temperature (15 °C). We found that several metabolites were differently expressed in reanimated opposite sun exposed communities, suggesting a critical role in their success. In particular, the saccharopine pathway was up-regulated in the north surface, while the spermine/spermidine pathway was significantly down-regulated in the shaded exposed communities. The carnitine-dependent pathway is up-regulated in south-exposed reanimated samples, indicating the preferential involvement of the B-oxidation for the functioning of TCA cycle. The role of these metabolites in the performance of the communities is discussed herein.

Anti-Inflammatory Potential of Cow, Donkey and Goat Milk Extracellular Vesicles as Revealed by Metabolomic Profile.

In recent years, extracellular vesicles (EVs), cell-derived micro and nano-sized structures enclosed in a double-layer membrane, have been in the spotlight for their high potential in diagnostic and therapeutic applications. Indeed, they act as signal mediators between cells and/or tissues through different mechanisms involving their complex cargo and exert a number of biological effects depending upon EVs subtype and cell source. Being produced by almost all cell types, they are found in every biological fluid including milk. Milk EVs (MEVs) can enter the intestinal cells by endocytosis and protect their labile cargos against harsh conditions in the intestinal tract. In this study, we performed a metabolomic analysis of MEVs, from three different species (i.e., bovine, goat and donkey) by mass spectroscopy (MS) coupled with Ultrahigh-performance liquid chromatography (UHPLC). Metabolites, both common or specific of a species, were identified and enriched metabolic pathways were investigated, with the final aim to evaluate their anti-inflammatory and immunomodulatory properties in view of prospective applications as a nutraceutical in inflammatory conditions. In particular, metabolites transported by MEVs are involved in common pathways among the three species. These metabolites, such as arginine, asparagine, glutathione and lysine, show immunomodulating effects. Moreover, MEVs in goat milk showed a greater number of enriched metabolic pathways as compared to the other kinds of milk.

Urine Metabolome during Parturition.

In recent years, some studies have described metabolic changes during human childbirth labor. Metabolomics today is recognized as a powerful approach in a prenatal research context, since it can provide detailed information during pregnancy and it may enable the identification of biomarkers with potential diagnostic or predictive. This is an observational, longitudinal, prospective cohort study of a total of 51 serial urine samples from 15 healthy pregnant women, aged 29-40 years, which were collected before the onset of labor (out of labor, OL). In the same women, during labor (in labor or dilating phase, IL-DP). Samples were analyzed by hydrophilic interaction ultra-performance liquid chromatography coupled with mass spectrometry (HILIC-UPLC-MS), a highly sensitive, accurate, and unbiased approach. Metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathway. This method was used to identify the potential biomarkers. The top 20 most discriminative metabolites contributing to the complete separation of OL and IL-DP were identified. Urinary metabolites displaying the largest differences between OL and IL-DP belonged to steroid hormone, particularly conjugated estrogens and amino acids much of this difference is determined by the fetal contribution. In addition, our results highlighted the efficacy of using urine samples instead of more invasive techniques to evaluate the difference in metabolic analysis between OL and IL-DP.

A metabolomics approach to investigate urine levels of neurotransmitters and related metabolites in autistic children.

Since recently metabolic abnormalities in autistic children have been associated with ASD disturbs, the aim of this study is to determine the neurotransmitter levels in urine samples of autistic children and to analyse the altered metabolic pathway involved in their production. Thus, ASD-specific urinary metabolomic patterns were explored in 40 ASD children and 40 matched controls using untargeted metabolomics through UHPLC-mass spectrometry (Q-exactive analyser), and by using XCMS Metlin software for data interpretation. Through this new advanced technique, a more considerable number of urinary altered metabolites were recorded in autistic children, than in the previous investigations, which allowed us to collect metabolites involved in neurotransmitter production. In these subjects, a high amount of dopamine was revealed and an increased amount of homovanillic acid, to the detriment of noradrenaline and adrenaline production, as well as MHPG and vanillylmandelic acid, which were found lower. This indicates that the accumulation of dopamine is not due to its greater production, but its lesser biotransformation into noradrenaline, due to the blockage of the dopamine ß-hydroxylase enzyme by 4-cresol and vitamin C, both found in high quantities in autistic subjects. Finally, a decreased amount of the active form of vitamin B6, pyridoxal phosphate (P5P), implicated in biotransformation of glutamate into γ-aminobutyric acid (GABA), was also detected, justifying the lower levels of latter. All of these alterations are correlated with a peculiar intestinal microbiome in autistic subjects, supporting the idea of a microbiota-gut-brain axis, then altered levels of neurotransmitters and altered neuronal transmission exist.

Specific adaptations are selected in opposite sun exposed Antarctic cryptoendolithic communities as revealed by untargeted metabolomics.

Antarctic cryptoendolithic communities are self-supporting borderline ecosystems spreading across the extreme conditions of the Antarctic desert and represent the predominant life-form in the ice-free areas of McMurdo Dry Valleys, accounted as the closest terrestrial Martian analogue. Components of these communities are highly adapted extremophiles and extreme-tolerant microorganisms, among the most resistant known to date. Recently, studies investigated biodiversity and community composition in these ecosystems but the metabolic activity of the metacommunity has never been investigated. Using an untargeted metabolomics, we explored stress-response of communities spreading in two sites of the same location, subjected to increasing environmental pressure due to opposite sun exposure, accounted as main factor influencing the diversity and composition of these ecosystems. Overall, 331 altered metabolites (206 and 125 unique for north and south, respectively), distinguished the two differently exposed communities. We also selected 10 metabolites and performed two-stage Receiver Operating Characteristic (ROC) analysis to test them as potential biomarkers. We further focused on melanin and allantoin as protective substances; their concentration was highly different in the community in the shadow or in the sun. These results clearly indicate that opposite insolation selected organisms in the communities with different adaptation strategies in terms of key metabolites produced.

Multidisciplinary characterization of melanin pigments from the black fungus Cryomyces antarcticus.

Melanin is a natural pigment present in almost all biological groups, and is composed of indolic polymers and characterized by black-brown colorization. Furthermore, it is one of the pigments produced by extremophiles including those living in the Antarctic desert, and is mainly involved in their protection from high UV radiation, desiccation, salinity and oxidation. Previous studies have shown that melanized species have an increased capability to survive high level of radiation compared with the non-melanized counterpart. Understanding the molecular composition of fungal melanin could help to understand this peculiar capability. Here, we aimed to characterize the melanin pigment extracted from the Antarctic black fungus Cryomyces antarcticus, which is a good test model for radioprotection researches, by studying its chemical properties and spectral data. Our results demonstrated that, in spite of having a specific type of melanin as the majority of fungi, the fungus possesses the ability to produce both 1,8-dihydroxynaphthalene (DHN) and L 3-4 dihydroxyphenylalanine (L-DOPA) melanins, opening interesting scenarios for the protection role against radiation. Researches on fungal melanin have a huge application in different fields, including radioprotection, bioremediation, and biomedical applications. KEY POINTS: • Isolation and characterization by multidisciplinary approaches of fungal melanins. • Discovery that pathways for producing DOPA and DHN are both active even in its extreme habitat. • Hypothesis supporting the possibility of using melanin pigment for radioprotection.

Equipping Durum Wheat-Thinopyrum ponticum Recombinant Lines With a Thinopyrum elongatum Major QTL for Resistance to Fusarium Diseases Through a Cytogenetic Strategy.

Prompted by recent changes in climate trends, cropping areas, and management practices, Fusarium head blight (FHB), a threatening disease of cereals worldwide, is also spreading in unusual environments, where bread wheat (BW) and durum wheat (DW) are largely cultivated. The scarcity of efficient resistance sources within adapted germplasm is particularly alarming for DW, mainly utilized for human consumption, which is therefore at high risk of kernel contamination by health-dangerous mycotoxins (e.g., deoxynivalenol = DON). To cope with this scenario, we looked outside the wheat primary gene pool and recently transferred an exceptionally effective FHB resistance QTL (Fhb-7EL) from Thinopyrum elongatum 7EL chromosome arm onto a Thinopyrum ponticum 7el1L arm segment, containing additional valuable genes (including Lr19 for leaf rust resistance and Yp for yellow pigment content), distally inserted onto 7DL of BW lines. Two such lines were crossed with two previously developed DW-Th. ponticum recombinants, having 7el1L distal portions on 7AL arms. Genomic in situ hybridization (GISH) analysis showed homologous pairing, which is enabled by 7el1L segments common to the BW and DW recombinant chromosomes, to occur with 42-78% frequency, depending on the shared 7el1L amount. Aided by 7EL/7el1L-linked markers, 7EL+7el1L tetraploid recombinant types were isolated in BC1 progenies to DW of all cross combinations. Homozygous 7EL+7el1L recombinant plants and null segregates selected in BC2F2 progenies were challenged by Fusarium graminearum spike inoculation to verify the Fhb-7EL efficacy in DW. Infection outcomes confirmed previous observations in BW, with >90% reduction of disease severity associated with Fhb-7EL presence vs. its absence. The same differential effect was detected on seed set and weight of inoculated spikes, with genotypes lacking Fhb-7EL having ∼80% reduction compared with unaffected values of Fhb-7EL carriers. In parallel, DON content in flour extracts of resistant recombinants averaged 0.67 ppm, a value >800 times lower than that of susceptible controls. Furthermore, as observed in BW, the same Fhb-7EL also provided the novel DW recombinants with resistance to Fusarium crown rot (∼60% symptom reduction) as from seedling infection with Fusarium culmorum. Through alien segment stacking, we succeeded in equipping DW with a very effective barrier against different Fusarium diseases and other positive attributes for crop security and safety.

The concomitant lower concentrations of vitamins B6, B9 and B12 may cause methylation deficiency in autistic children.

Autism spectrum disorder (ASD) is characterized by severe and persistent difficulties in social communication and social interaction at multiple levels. Recently, metabolic disorders have been associated with most cases of patients with ASD. The aim of this study was to investigate, through a new and more sophisticated mass technique, such as UHPLC-mass spectrometry (Q-exactive analyzer), alteration in metabolisms analyzing ASD children urine samples from children showing simultaneous vitamin B6, B9 and B12 deficiencies. This in order to study how these concurrent deficiencies may influence some phenotypic aspects of autistic disorder. Thus, urinary metabolic patterns specific to ASD were explored at an early age in 60 children with ASD, showing lower three vitamins levels, and 60 corresponding controls (age group 3-8, M: F=42:18). The results showed significant block of cystathionine formation with consequent accumulation of homocysteine. A lower glutathione levels (GSH), with reduction of essential intracellular reducing environment required for normal immune function, detoxification capacity and redox-sensitive enzyme activity. Increased concentration of 5-methyltetrahydrofolate, which leads to a lower availability of methyl group and significant decrease in urinary methionine and S-adenosyl-L-methionine (SAM) concentrations, the major methyl donor. The latter justify the well-known reduction in protein and DNA methylation reported in autistic children. As a final consideration, the concomitant deficiencies of all three B vitamins, recorded in a significant number of autistic children, suggests that intestinal dysbiosis in these patients may be the main cause of a reduction in their absorption, in addition to the genetic mutation of a specific gene.

Untargeted Metabolomics of Plant Leaf Tissues.

Untargeted metabolomics is a useful approach for the simultaneous analysis of a vast array of compounds from a single extract. Metabolomic profiling is the relative multi-parallel quantification of a mixture of low molecular weight compounds, or classes of compounds, and it is most often performed by using ultra performance liquid chromatography (UPLC) coupled with mass spectrometry (MS). Being an extension of the classical targeted methods, this approach allows a broader view of the main biochemical events within a particular sample. This chapter exemplifies and provides experimental details on the basic steps to perform a non-targeted metabolomic analysis on plant leaf tissues: sample collection and homogenization, extraction of metabolites, raw data acquisition, and processing into formats for data mining and informatics. In particular, the approach was applied to two spring wheat varieties with different level of drought tolerance (Kavir, drought-resistant; Bahar, drought-sensitive) developed by the CIMMYT (International Center for the Improvement of Corn and Wheat).

Deoxynivalenol Detoxification in Transgenic Wheat Confers Resistance to Fusarium Head Blight and Crown Rot Diseases.

Fusarium diseases, including Fusarium head blight (FHB) and Fusarium crown rot (FCR), reduce crop yield and grain quality and are major agricultural problems worldwide. These diseases also affect food safety through fungal production of hazardous mycotoxins. Among these, deoxynivalenol (DON) acts as a virulence factor during pathogenesis on wheat. The principal mechanism underlying plant tolerance to DON is glycosylation by specific uridine diphosphate-dependent glucosyltransferases (UGTs), through which DON-3-ß-d-glucoside (D3G) is produced. In this work, we tested whether DON detoxification by UGT could confer to wheat a broad-spectrum resistance against Fusarium graminearum and F. culmorum. These widespread Fusarium species affect different plant organs and developmental stages in the course of FHB and FCR. To assess DON-detoxification potential, we produced transgenic durum wheat plants constitutively expressing the barley HvUGT13248 and bread wheat plants expressing the same transgene in flower tissues. When challenged with F. graminearum, FHB symptoms were reduced in both types of transgenic plants, particularly during early to mid-infection stages of the infection progress. The transgenic durum wheat displayed much greater DON-to-D3G conversion ability and a considerable decrease of total DON+D3G content in flour extracts. The transgenic bread wheat exhibited a UGT dose-dependent efficacy of DON detoxification. In addition, we showed, for the first time, that DON detoxification limits FCR caused by F. culmorum. FCR symptoms were reduced throughout the experiment by nearly 50% in seedlings of transgenic plants constitutively expressing HvUGT13248. Our results demonstrate that limiting the effect of the virulence factor DON via in planta glycosylation restrains FHB and FCR development. Therefore, ability for DON detoxification can be a trait of interest for wheat breeding targeting FHB and FCR resistance.

Metabolic patterns in insulin-resistant male hypogonadism.

Male hypogonadism associated with insulin resistance (IR) very often leads to metabolic syndrome, at variance with hypogonadism in its first stadium of insulin sensitivity (IS). A plasma metabolomic investigation of these patients can provide useful information in comparison with the values of IS patients. To this aim plasma from insulin-resistant males with hypogonadism were analysed by using ultra high-performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (HRMS). Thus, metabolites were compared to the controls through multivariate statistical analysis and grouped by metabolic pathways. Metabolite database searches and pathway analyses identified imbalances in 18-20 metabolic pathways. Glucose metabolism (e.g., glycolysis and the Krebs cycle) is fuelled by amino acids degradation, in particular of branched amino acids, in individuals with lean body mass. Gluconeogenesis is strongly activated. Some crucial pathways such as glycerol are skewed. Mitochondrial electron transport is affected with a reduction in ATP production. Beta-oxidation of short and medium chain fatty acids did not represent an energy source in hypogonadism, at variance with long and branched fatty acids, justifying the increase in fat mass. Carnosine and ß-alanine are strongly reduced resulting in increased fatigue and mental confusion. A comparison of IR with IS male hypogonadism will contribute to a better understanding of how these two hormones work in synergy or antagonise each other in humans. It could also help to select patients who will respond to hormone treatment, and provide accurate biomarkers to measure the response to treatment eventually leading to better strategies in preventing systemic complications in patients not fit for hormone replacement therapy.

Metabolic patterns in insulin-sensitive male hypogonadism.

Male hypogonadism is a disorder characterised by low levels of the hormone testosterone. At beginning subjects with low levels of testosterone do not show insulin resistance (insulin-sensitive patients), which develops over time (insulin-resistance patients). To analyse the metabolic alterations mainly related to decreased testosterone, we performed metabolomics investigations on the plasma of males with hypogonadism who showed normal insulin levels. Plasma from patients with low testosterone (<8 nmol/l) and homeostatic model assessment for insulin-resistance-index (HOMAi) < 2.5, as well as matched controls, was analysed by UHPLC and mass spectrometry. Then metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathways. Glycolysis was not altered, as expected for the presence of insulin activity, but imbalances in several other pathways were found, such as the pentose phosphate pathway (PPP), glycerol shuttle, malate shuttle, Krebs cycle (TCA) and lipid metabolism. The PPP was significantly upregulated. Moreover, while the first steps of the Krebs cycle were downregulated, 2-oxoglutarate was replenished via glutaminolysis. Since glutaminolysis leads to an activation of the malate aspartate cycle, greater amounts of NADH and ATP with respect to the control were recorded. The activation of the glycerol shuttle was also recorded, with consequent lower triglyceride production and downregulation of beta-oxidation. This explained the moderately increased dyslipidaemia, as well as the mild increase in body mass index (BMI) observed in insulin-sensitive hypogonadism. Finally, a significant decrease in carnosine was recorded, explaining the muscle weakness commonly observed.

Redox Status, Procoagulant Activity, and Metabolome of Fresh Frozen Plasma in Glucose 6-Phosphate Dehydrogenase Deficiency.

OBJECTIVE: Transfusion of fresh frozen plasma (FFP) helps in maintaining the coagulation parameters in patients with acquired multiple coagulation factor deficiencies and severe bleeding. However, along with coagulation factors and procoagulant extracellular vesicles (EVs), numerous bioactive and probably donor-related factors (metabolites, oxidized components, etc.) are also carried to the recipient. The X-linked glucose 6-phosphate dehydrogenase deficiency (G6PD-), the most common human enzyme genetic defect, mainly affects males. By undermining the redox metabolism, the G6PD- cells are susceptible to the deleterious effects of oxidants. Considering the preferential transfusion of FFP from male donors, this study aimed at the assessment of FFP units derived from G6PD- males compared with control, to show whether they are comparable at physiological, metabolic and redox homeostasis levels. METHODS: The quality of n = 12 G6PD- and control FFP units was tested after 12 months of storage, by using hemolysis, redox, and procoagulant activity-targeted biochemical assays, flow cytometry for EV enumeration and phenotyping, untargeted metabolomics, in addition to statistical and bioinformatics tools. RESULTS: Higher procoagulant activity, phosphatidylserine positive EVs, RBC-vesiculation, and antioxidant capacity but lower oxidative modifications in lipids and proteins were detected in G6PD- FFP compared with controls. The FFP EVs varied in number, cell origin, and lipid/protein composition. Pathway analysis highlighted the riboflavin, purine, and glycerolipid/glycerophospholipid metabolisms as the most altered pathways with high impact in G6PD-. Multivariate and univariate analysis of FFP metabolomes showed excess of diacylglycerols, glycerophosphoinositol, aconitate, and ornithine but a deficiency in riboflavin, flavin mononucleotide, adenine, and arginine, among others, levels in G6PD- FFPs compared with control. CONCLUSION: Our results point toward a different redox, lipid metabolism, and EV profile in the G6PD- FFP units. Certain FFP-needed patients may be at greatest benefit of receiving FFP intrinsically endowed by both procoagulant and antioxidant activities. However, the clinical outcome of G6PD- FFP transfusion would likely be affected by various other factors, including the signaling potential of the differentially expressed metabolites and EVs, the degree of G6PD-, the redox status in the recipient, the amount of FFP units transfused, and probably, the storage interval of the FFP, which deserve further investigation by future studies.

A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells.

Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings.

The cardioprotective effect of sildenafil is mediated by the activation of malate dehydrogenase and an increase in the malate-aspartate shuttle in cardiomyocytes.

Recent evidence has shown the cardioprotective effect of PDE5 inhibition in myocardial ischemia/reperfusion injury, heart failure and cardiac hypertrophy. To investigate the biochemical changes that occur during PDE5 inhibition in cardiac cells, this study assessed the metabolic profile of the HL1 cell line, a murine atrial cell line with adult cardiomyocyte properties. After one hour of treatment with sildenafil, glycolysis was moderately but selectively stimulated, unlike the pentose phosphate pathway and the Krebs cycle. Moreover, malate and a-Ketoglutarate accumulated, paralleled by a decrease in aspartate and glutamate. Interestingly, increased activity of malate dehydrogenase (MDH) was also detected in these cells after sildenafil treatment. Thus, we hypothesized that sildenafil stimulates the malate-aspartate shuttle (MAS) with the final effect of transferring electrons and protons from glycolysis-derived cytosolic NADH into the matrix for use by the electron transport chain, using malate as an electron carrier. Through this metabolic modification, sildenafil may counteract what is often observed in ischemia, i.e. reduced MAS flux as well as a dramatic acceleration of glycolysis, which switches to lactate production. Additionally, the results observed in HL1 cells were also found in isolated mouse hearts. The documented metabolic alteration in cardiomyocytes upon treatment with sildenafil occurred by stimulating cGMP production, which did not activate PKG (cGMP-PKG signaling), since the addition of DT-2, a PKG inhibitor, did not block malate accumulation and increased MDH activity. Conversely, the addition of chelerythrine, a PKC inhibitor, counteracted both malate accumulation and MAS activation, supporting previous evidence that, upon the addition of sildenafil, some PKC isoforms may be implicated in cardioprotection (cGMP-PKC signaling). Interestingly, an increase in cGMP, driven by sildenafil, another cGMP stimulator such as nitroprusside (SNP), or a C-type natriuretic peptide (CNP) which does not inhibit PDE5, led to MAS stimulation and increased MDH activity.