Federica Gevi

Alterations of red blood cell metabolome during cold liquid storage of erythrocyte concentrates in CPD–SAGM ☆

Erythrocyte concentrates for transfusion purposes represent a life-saving therapeutics of primary relevance in the clinical setting. However, efforts have been continuously proposed to improve safety and efficacy of long-term stored red blood cells. By means of liquid chromatography coupled with Q-TOF mass spectrometry, we were able to perform an untargeted metabolomics analysis in order to highlight metabolic species (i.e. low molecular biochemicals including sugars, lipids, nucleotides, aminoacids, etc.), both in red blood cells and supernatants, which showed fluctuations against day 0 controls over storage duration on a weekly basis. We could confirm and expand existing literature about the rapid fall of glycolytic rate and accumulation of glycolysis end products. A shift was observed towards the oxidative phase of pentose phosphate pathway, in response to an exacerbation of oxidative stress (altered glutathione homeostasis and accumulation of peroxidation/inflammatory products in the supernatant). The present study provides the first evidence that over storage duration metabolic fluxes in red blood cells proceed from pentose phosphate pathway towards purine salvage pathway, instead of massively re-entering glycolysis via the nonoxidative phase. This article is part of a Special Issue entitled: Integrated omics.

Proteomics and transcriptomics investigation on longissimus muscles in Large White and Casertana pig breeds.

Consumer complaints against the blandness of modern lean meat and the frequent reference to the more strongly flavored meat that was available years ago have prompted reconsideration of high fat-depositing typical pig breeds. Casertana and Large White pig breeds are characterized by a different tendency toward fat accumulation as they exhibit opposite genetic and physiological traits with respect to the energy metabolism. These physiological differences were investigated in longissimus lumborum muscles through proteomics (2-DE, MS/MS) and microarray approaches. Data were analyzed for pathway and network analyses, as well as GO term enrichment of biological functions. As a result, Casertana showed a greater amount of proteins involved in glycolitic metabolism and mainly rely on fast-mobilizable energy sources. Large White overexpressed cell cycle and skeletal muscle growth related genes. Metabolic behavior and other implications are discussed.

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

SUMMARY 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. HighlightsDendritic cells (DCs) can co‐express Arg1 and IDO1 immunosuppressive enzymesArg1 activity is required for IDO1 induction by TGF‐&bgr; in DCsSpermidine, a downstream Arg1 product, but not arginine starvation, induces IDO1 in DCsArg1+ myeloid derived suppressor cells (MDSCs) can render DCs immunosuppressive via IDO1 &NA; Arginase 1 (Arg1) and indoleamine 2,3‐dioxygenase 1 (IDO1) are immunosuppressive enzymes known to operate in distinct immune cells. Mondanelli and colleagues demonstrate that Arg1 and IDO1 cooperate in conferring long‐term, immunosuppressive effects to dendritic cells.

Cadmium stress responses in Brassica juncea: hints from proteomics and metabolomics.

Among heavy metal stressors, cadmium (Cd) pollution is one leading threat to the environment. In this view, research efforts have been increasingly put forward to promote the individuation of phytoextractor plants that are capable of accumulating and withstanding the toxic metals, including Cd, in the aerial parts. We hereby adopted the hyperaccumulator B. juncea (Indian mustard) as a model to investigate plant responses to Cd stress at low (25 μM) and high (100 μM) doses. Analytical strategies included mass-spectrometry-based determination of Cd and the assessment of its effect on the leaf proteome and metabolome. Results were thus integrated with routine physiological data. Taken together, physiology results highlighted the deregulation of photosynthesis efficiency, ATP synthesis, reduced transpiration, and the impairment of light-independent carbon fixation reactions. These results were supported at the proteomics level by the observed Cd-dependent alteration of photosystem components and the alteration of metabolic enzymes, including ATP synthase subunits, carbonic anhydrase, and enzymes involved in antioxidant responses (especially glutathione and phytochelatin homeostasis) and the Calvin cycle. Metabolomics results confirmed the alterations of energy-generating metabolic pathways, sulfur-compound metabolism (GSH and PCs), and Calvin cycle. Besides, metabolomics results highlighted the up-regulation of phosphoglycolate, a byproduct of the photorespiration metabolism. This was suggestive of the likely increased photorespiration rate as a means to cope with Cd-induced unbalance in stomatal conductance and deregulation of CO2 homeostasis, which would, in turn, promote CO2 depletion and O2 (and thus oxidative stress) accumulation under prolonged photosynthesis in the leaves from plants exposed to high doses of CdCl2. Overall, it emerges that Cd-stressed B. juncea might rely on photorespiration, an adaptation that would prevent the over-reduction of the photosynthetic electron transport chain and photoinhibition.

Protective effects of the neuropeptides PACAP, substance P and the somatostatin analogue octreotide in retinal ischemia: a metabolomic analysis

Ischemia is a primary cause of neuronal death in retinal diseases and the somatostatin subtype receptor 2 agonist octreotide (OCT) is known to decrease ischemia-induced retinal cell death. Using a recently optimized ex vivo mouse model of retinal ischemia, we tested the anti-ischemic potential of two additional neuropeptides, pituitary adenylate cyclase activating peptide (PACAP) and substance P (SP), and monitored the major changes occurring at the metabolic level. Metabolomics analyses were performed via fast HPLC online using a microTOF-Q MS instrument, a workflow that is increasingly becoming the gold standard in the field of metabolomics. The metabolomic approach allowed detection of the most significant alterations induced in the retina by ischemia and of the significance of the protective effects exerted by OCT, PACAP or SP. All treatments were shown to reduce ischemia-induced cell death, vascular endothelial growth factor over-expression and glutamate release. The metabolomic analysis showed that OCT and, to a lesser extent, also PACAP or SP, were able to counteract the ischemia-induced oxidative stress and to promote, with various efficacies, (i) decreased accumulation of glutamate and normalization of glutathione homeostasis; (ii) reduced build-up of α-ketoglutarate, which might serve as a substrate for the enhanced biosynthesis of glutamate in response to ischemia; (iii) reduced accumulation of peroxidized lipids and inflammatory mediators; (iv) the normalization of glycolytic fluxes and thus preventing the over-accumulation of lactate or either promoting the down-regulation of the glyoxalate anti-oxidant system; (v) a reduced metabolic shift from glycolysis towards the PPP or either a blockade at the non-oxidative phase of the PPP; and (vi) tuning down of purine metabolism. In addition, OCT seemed to stimulate nitric oxide production. None of the treatments was able to restore ATP production, although ATP reservoirs were partly replenished by OCT, PACAP or SP. These data indicate that, in addition to that of somatostatin, peptidergic systems such as those of PACAP and SP deserve attention in view of peptide-based therapies to treat ischemic retinal disorders.

Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation

Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full‐length cDNA sequence of human factor VIII (FLrFVIII) or B‐domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advate®, Helixate NexGen® and Refacto®), either FLrFVIII or BDDrFVIII, were investigated by 1‐ and 2‐DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2‐D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B‐domain‐truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGen® showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refacto® showed glutathione S‐transferase and β‐lactamase, whereas Advate® apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2‐DE is mandatory for revealing the presence of contaminants.

Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism

BackgroundAutism spectrum disorder (ASD) is still diagnosed through behavioral observation, due to a lack of laboratory biomarkers, which could greatly aid clinicians in providing earlier and more reliable diagnoses. Metabolomics on human biofluids provides a sensitive tool to identify metabolite profiles potentially usable as biomarkers for ASD. Initial metabolomic studies, analyzing urines and plasma of ASD and control individuals, suggested that autistic patients may share some metabolic abnormalities, despite several inconsistencies stemming from differences in technology, ethnicity, age range, and definition of “control” status.MethodsASD-specific urinary metabolomic patterns were explored at an early age in 30 ASD children and 30 matched controls (age range 2–7, M:F = 22:8) using hydrophilic interaction chromatography (HILIC)-UHPLC and mass spectrometry, a highly sensitive, accurate, and unbiased approach. Metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathway.ResultsUrinary metabolites displaying the largest differences between young ASD and control children belonged to the tryptophan and purine metabolic pathways. Also, vitamin B6, riboflavin, phenylalanine-tyrosine-tryptophan biosynthesis, pantothenate and CoA, and pyrimidine metabolism differed significantly. ASD children preferentially transform tryptophan into xanthurenic acid and quinolinic acid (two catabolites of the kynurenine pathway), at the expense of kynurenic acid and especially of melatonin. Also, the gut microbiome contributes to altered tryptophan metabolism, yielding increased levels of indolyl 3-acetic acid and indolyl lactate.ConclusionsThe metabolic pathways most distinctive of young Italian autistic children largely overlap with those found in rodent models of ASD following maternal immune activation or genetic manipulations. These results are consistent with the proposal of a purine-driven cell danger response, accompanied by overproduction of epileptogenic and excitotoxic quinolinic acid, large reductions in melatonin synthesis, and gut dysbiosis. These metabolic abnormalities could underlie several comorbidities frequently associated to ASD, such as seizures, sleep disorders, and gastrointestinal symptoms, and could contribute to autism severity. Their diagnostic sensitivity, disease-specificity, and interethnic variability will merit further investigation.

Red Blood Cell Homeostasis: Pharmacological Interventions to Explore Biochemical, Morphological and Mechanical Properties.

During their passage through the circulation, red blood cells (RBCs) encounter severe physiological conditions consisting of mechanical stress, oxidative damage and fast changes in ionic and osmotic conditions. In order to survive for 120 days, RBCs adapt to their surroundings by subtle regulation of membrane organization and metabolism. RBC homeostasis depends on interactions between the integral membrane protein band 3 with other membrane and cytoskeletal proteins, and with key enzymes of various metabolic pathways. These interactions are regulated by the binding of deoxyhemoglobin to band 3, and by a signaling network revolving around Lyn kinase and Src family kinase-mediated phosphorylation of band 3. Here we show that manipulation of the interaction between the lipid bilayer and the cytoskeleton, using various pharmacological agents that interfere with protein-protein interactions and membrane lipid organization, has various effects on: (1) morphology, as shown by high resolution microscopy and quantitative image analysis; (2) organization of membrane proteins, as indicated by immunofluorescence confocal microscopy and quantitative as well as qualitative analysis of vesicle generation; (3) membrane lipid organization, as indicated by flow cytometric analysis of phosphatidylserine exposure; (4) deformability, as assessed in capillary-mimicking circumstances using a microfluidics system; (5) deformability as determined using a spleen-mimicking device; (6) metabolic activity as indicated by metabolomics. Our data show that there is a complex relationship between red cell morphology, membrane organization and deformability. Also, our data show that red blood cells have a relatively high resistance to disturbance of membrane organization in vitro, which may reflect their capacity to withstand mechanical, oxidative and osmotic stress in vivo.

Targeted Mass Spectrometry-Based Metabolomic Profiling Through Multiple Reaction Monitoring of Liver and Other Biological Matrices

In a systemic viewpoint, relevant biological information on living systems can be grasped from the study of small, albeit pivotal molecules which constitute the fundamental bricks of metabolic pathways. This holds true for liver which plays, among its unique functions, a key role in metabolism. The nonbiased analysis of all this small-molecule complement in its entirety is known as metabolomics. However, no practical approach currently exists to investigate all metabolic species simultaneously without including a technical bias towards acidic or basic compounds, especially when performing mass spectrometry-based investigations. Technical aspects of rapid resolution reversed phase HPLC online with mass spectrometry are hereby described. Such an approach allows to discriminate and quantify a wide array of metabolites with extreme specificity and sensitivity, thus enabling to perform complex investigations even on extremely low quantities of biological material. The advantages also include the possibility to perform targeted investigations on a single (or a handful of) metabolite(s) simoultaneously through single (multiple) reaction monitoring, which further improves the dynamic range of concentrations to be monitored.Such an approach has already proven to represent a valid tool in the direct (on the liver) or indirect (on human red blood cell metabolism which is hereby presented as a representative model, but also on blood plasma or other biological fluids) assessment of metabolic poise modulation and pharmacokinetics for drug development.

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

Graphical abstract Figure. No Caption available. ABSTRACT 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.