Developments in dairy cow fertility research.

Accurate mass and MS/MS fragmentation data for (a) kynurenine, (b) melatonin, and (c) tryptophan. (TIF 191 kb

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 (Advates, Helixate NexGens and Refactos), 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 NexGens showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refactos showed glutathione S-transferase and b-lactamase, whereas Advates 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.L'artcoo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com

DEOXYNIVALENOL DETOXIFICATION IN TRANSGENIC DURUM WHEAT CONFERS RESISTANCE TO FUSARIUM HEAD BLIGHT AND CROWN ROT DISEASES REDUCING GRAIN MYCOTOXIN CONTAMINATION

Fusarium diseases, including Fusarium head blight (FHB) and crown rot (FCR), represent major agricultural problems worldwide, causing reducti on of grain yield, quality and food safety. Grain contaminati on by Fusarium mycotoxins, mainly deoxynivalenol (DON), is responsible for health problems in humans and animals. DON acts as virulence factor during pathogenesis and its glycosylati on, performed by UDPglucosyltransferases (UGTs) and resulti ng in DON-3-glucoside (D3G) producti on, has been identi fi ed as the main detoxifi cati on strategy in wheat. In this work, we produced Triti cum durum cv. Svevo transgenic lines consti tuti vely expressing the barley HvUGT13248 gene. In them, DON-detoxifi cati on by UGT was found to confer a broad-spectrum resistance against F. graminearum and F. culmorum, aff ecti ng diff erent plant organs and developmental stages during FHB and FCR. When challenged with F. graminearum, the transgenic plants revealed a signifi cant reducti on (up to 30%) of FHB symptoms, mostly evident during early-mid stages of the infecti on progress. Notably, much higher DON-to-D3G conversion ability and considerable decrease of DON and DON+D3G content in wholemeal fl our of transgenic lines vs. nontransgenic control was observed. The higher effi ciency of D3G conversion since early infecti on stages may have reduced fungal progression and, consequently, DON and D3G contaminati on in kernels. Furthermore, we highlighted for the fi rst ti me the possible involvement of the DON-detoxifying mechanism in limiti ng FCR disease caused by F. culmorum. When challenged with the pathogen at the seedling stage, the HvUGT13248-expressing lines showed signifi cant reducti on (~50%) of FCR symptoms throughout the infecti on as compared to non-transgenic plants. Transgenic seedlings revealed also a bett er root tolerance to DON, which could have contributed to a higher seedling vigor during the infecti on. The concomitant effi cacy of the DON-detoxifi cati on strategy against FHB and FCR represents an att racti ng sustainable approach to pursue in breeding programs targeti ng broad-spectrum Fusarium resistance and hence reducti on of mycotoxin contaminati on of durum wheat-derived products

Metabolomic Profile of the Fungus Cryomyces antarctiucus Under Simulated Martian and Space Conditions as Support for Life-Detecion 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 ryptoendolithic 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

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 a-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

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

Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1\ua0(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

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

Proteomica e metabolomica: nuove piattaforme tecnologiche per applicazioni in campo biologico

The word „omics‟, is derived from the Greek suffix, „ome‟ (meaning all, every). In the mid 1990‟s, rapid evolution and growing knowledge concerning genomes led to a quick development of the full range of „omics‟ approaches (genomics, transcriptomics, proteomics, metabolomics). This PhD thesis focuses on the application of new “omics” sciences to characterized samples of biomedical interest such as pharmaceutical products. The first section is focused on the characterization of important pharmaceutical products used by hemophiliac patients, trough proteomics approaches. The first objective of this study was to compare the heterogeneity and the high purity of three rFVIII commercially available rFVIII concentrates before and after thrombin digestion. The second object was to extend the same study to plasma derived factor VIII . The second part of my PhD project was designed mainly to the development and optimization of a new HPLC-MS method for metabolomics. In the last part of my research I used this analytical approach to investigate metabolites in blastocoels fluid that is a routine waste product prior to pre-implantation blastocyst vitrification. The aim of this study was directed to identify a correlation between quali-quantitative profiles of small molecules of metabolic interest and the outcome of embryo transfer through a simple HPLC-MS assay. The last part of my research was dedicated to studing metabolism in red blood cell during storage. Proteome and metabolomic approaches, are widely used for studies complex biological systems and can be used for applications in biological areas.La parola omica deriva dal suffisso greco “ome” che significa: tutto, intero, completo. In particolar modo per quanto riguarda l’area biologica lo scopo delle scienze omiche è quello di avere una visione globale per comprendere la vita e la sua organizzazione. L’evoluzione rapida e la crescita di conoscenza riguardante il genoma hanno portato ad una rapida crescita nello sviluppo di nuove scienze (genomica, trascrittomica, proteomica, metabolomica) La mia tesi di dottorato riguarda le applicazioni delle nuove scienze omiche per caratterizzare campioni di interesse biomedico come ad esempio prodotti farmaceutici. Il primo obiettivo dello studio è stato quello di comparare e caratterizzare fattore VIII ricombinante prodotto da tre ditte farmaceutiche differenti. Questi prodotti attaualmente utilizzati per curare l’emofilia sono stati studiati prima e dopo attivazione attraverso il taglio effettuato dalla trombina. Il secondo obiettivo della mia tesi è stato quello di estendere lo studio proteomico non solo ai fattori VIII ricombinanti ma anche a quelli plasmaderivati. La seconda parte del mio progetto è stata dedicata allo sviluppo e all’ottimizzazione di un nuovo metodo basato sull’utilizzo di HPLC e spettrometro di massa per lo studio della metabolomica. Nell’ultima parte della mia tesi ho utilizzato questo tipo di approcio per studiare la variazione metabolica all’interno del fluido del blastocele. Lo scopo di questo lavoro è stato quello di individuare marker metabolici che possano definire la qualità delle blastocisti da impiantare durante il processo della FIVET. Infine ho utilizzato questo metodo per mettere in evidenza la variazione metabolica nei globuli rossi durante la loro conservazione in sacca.Dottorato di ricerca in Genetica e biologia cellular