Cristina Marrocco

Meat quality of the longissimus lumborum muscle of Casertana and Large White pigs: Metabolomics and proteomics intertwined

Longissimus lumborum muscles from high fat-deposing Casertana and lean meat Large White pigs were assayed for meat quality parameters, including early and ultimate post mortem pH, water holding capacity and Minolta L*a*b*values. These parameters were correlated to results from differential proteomic and targeted metabolomic analyses. Higher levels of glycolytic enzymes and lactate accumulation were related to slow pH drop in Casertana pigs, albeit not to rapid pH lowering in LW counterparts. On the other hand, the individuation of pyruvate kinaseM1 and tropomyosin levels in LW were related to water holding capacity and Minolta values at 24h after slaughter. Bioinformatic analyses strengthened the correlation between over-expression of structural proteins in LW and more accentuated growth aptitude in this breed. Conversely, enzymes taking part into branching glycolytic reactions, such as glycerol 3-phosphate and creatine kinase M, were related to accentuated lipogenesis and slower albeit prolonged glycolytic rate in Casertana, respectively. Breed-specific differences at the protein level were not only related to growth performances and fat accumulation tendency in vivo, but they also affected post mortem performances through a direct influence on the forcedly anaerobic behavior of pig muscles after slaughter.

Love me tender: an Omics window on the bovine meat tenderness network.

Meat tenderness prediction is a challenging task, especially in Maremmana, an Italian autochtonous and highly appreciated beef breed. In the present study we reported an integrated proteomics, phosphoproteomics and metabolomics overview of meat tenderness in longissimus dorsi from 15 male Maremmana individuals, through the discrimination of tender and tough groups via standard meat tenderness indicators (WBS, MFI(4 h), MFI(10 days), sarcomere length) and their correlation with results from Omics analyses. Results revealed that the tender meat group was characterized by higher levels of glycolytic enzymes, which were less phosphorylated and overall more active (lactate accumulation was higher in the tender group), than in tough counterparts. Additionally, we could observe a higher level of oxidative stress in the tender group. No proteomics nor phosphoproteomics result hinted at the widely accepted role of calpains and cathepsins, except for the indication of calcium homeostasis dysregulation. Nevertheless, myofibrillar degradation was monitored and related to structural protein fragmentations. Fragmentation of structural proteins and activities of glycolytic enzymes were inversely related to their phosphorylation levels, suggesting that PTMs might add further levels of complexity in the frame of meat tenderness.

Chianina beef tenderness investigated through integrated Omics.

In the present study we performed an integrated proteomics, interactomics and metabolomics analysis of Longissimus dorsi tender and tough meat samples from Chianina beef cattle. Results were statistically handled as to obtain Pearsons correlation coefficients of the results from Omics investigation in relation to canonical tenderness-related parameters, including Warner Bratzler shear force, myofibrillar degradation (at 48 h and 10 days after slaughter), sarcomere length and total collagen content. As a result, we could observe that the tender meat group was characterized by higher levels of glycolytic enzymes, which were over-phosphorylated and produced accumulation of glycolytic intermediates. Oxidative stress promoted meat tenderness and elicited heat shock protein responses, which in turn triggered apoptosis-like cascades along with PARP fragmentation. Phosphorylation was found to be a key process in post mortem muscle conversion to meat, as it was shown not only to modulate glycolytic enzyme activities, but also mediate the stability of structural proteins at the Z-disk. On the other hand, phosphorylation of HSPs has been supposed to alter their functions through changing their affinity for target interactors. Analogies and breed-specific differences are highlighted throughout the text via a direct comparison of the present results against the ones obtained in a parallel study on Maremmana Longissimus dorsi. It emerges that, while the main cornerstones and the final outcome are maintained, post mortem metabolism in tender and tough meat yielding individuals is subtly modulated via specific higher levels of enzymes and amino acidic residue phosphorylation in a breed-specific fashion, and whether calcium homeostasis dysregulation was a key factor in Maremmana, higher early post mortem phosphocreatine levels in the Chianina tender group could favor a slower and prolonged glycolytic rate, prolonging the extent of the minimum hanging period necessary to obtain tender meat from this breed by a few days.

Proteomic analysis of platelets treated with gamma irradiation versus a commercial photochemical pathogen reduction technology.

Several strategies are currently being tested to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion. Within the framework of the Italian Platelet Technology Assessment Study, we investigated the variations of the protein profiles (proteomics) of apheresis PLT concentrates (PCs) upon treatment with riboflavin and ultraviolet (UV) light (Mirasol; 6.24 J/mL; 280‐400 nm).

Red blood cell subpopulations in freshly drawn blood: application of proteomics and metabolomics to a decades-long biological issue.

BACKGROUND It has long been known that red blood cells comprise various subpopulations, which can be separated through Percoll density gradients. MATERIALS AND METHODS In this study, we performed integrated flow cytometry, proteomic and metabolomic analyses on five distinct red blood cell subpopulations obtained by Percoll density gradient separation of freshly drawn leucocyte-depleted erythrocyte concentrates. The relation of density gradient fractions to cell age was confirmed through band 4.1a/4.1b assays. RESULTS We observed a decrease in size and increase in cell rugosity in older (denser) populations. Metabolomic analysis of fraction 5 (the oldest population) showed a decrease of glycolytic metabolism and of anti-oxidant defence-related mechanisms, resulting in decreased activation of the pentose phosphate pathway, less accumulation of NADPH and reduced glutathione and increased levels of oxidized glutathione. These observations strengthen conclusions about the role of oxidative stress in erythrocyte ageing in vivo, in analogy with results of recent in vitro studies. On the other hand, no substantial proteomic differences were observed among fractions. This result was partly explained by intrinsic technical limitations of the two-dimensional gel electrophoresis approach and the probable clearance from the bloodstream of erythrocytes with membrane protein alterations. Since this clearance effect is not present in vitro (in blood bank conditions), proteomic studies have shown substantial membrane lesions in ageing red blood cells in vitro. CONCLUSION This analysis shows that the three main red blood cell subpopulations, accounting for over 92% of the total RBC, are rather homogeneous soon after withdrawal. Major age-related alterations in vivo probably affect enzyme activities through post-translational mechanisms rather than through changes in the overall proteomic profile of RBC.

Thiol-based regulation of glyceraldehyde-3-phosphate dehydrogenase in blood bank–stored red blood cells: a strategy to counteract oxidative stress

Red blood cell (RBC) glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is a glycolytic enzyme normally inhibited upon binding to the membrane‐spanning protein Band 3, but active when free in the cytosol. Accumulating evidence in other cells indicates that oxidative thiol modifications in cytosolic GAPDH drive this molecule into functional avenues that deviate from glycolysis. This study aimed to investigate the role of GAPDH in oxidative stress–dependent metabolic modulations occurring in SAGM‐stored RBCs, to increase the knowledge of the molecular mechanisms affecting RBC survival and viability under blood banking conditions.

Proteomic analysis of plasma derived from platelet buffy coats during storage at room temperature. An application of ProteoMiner™ technology.

The present study was aimed at revealing new insights into the analysis of storage-related processes occurring in the supernatants of platelet concentrates (PCs) derived from pooled buffy coats suspended in whole plasma. To reduce the dynamic range of plasma protein concentrations and access low-abundance proteins, we made use of a solid-phase combinatorial peptide ligand library, known under the trade name of ProteoMiner™. Afterwards, two-dimensional electrophoresis (2-DE) was coupled with mass spectrometry (MS) to reveal changes in proteomic profiles. Several storage-induced protein alterations were identified including changes to major plasma proteins. In particular, a precursor of the secretory form of clusterin was shown to accumulate during storage of PC supernatants, together with platelet-derived tropomyosin, suggesting a progressive loss of platelet integrity. Platelet-released proteins following activation have also been detected (alpha-1-B-glycoprotein, kininogen-1, and serpin proteinase inhibitor 8). Moreover, specific protein fragments (vitronectin, plakoglobin, hornerin, and apolipoprotein A-IV) were found to be modulated upon storage, possibly indicating a time-dependent buffy-coat PC deterioration. Globally, our findings provided the disclosure of unique proteins in PC supernatants with respect to previous studies conducted in similar experimental conditions, suggesting ProteoMiner enrichment technology to be a possible complementary tool in the identification of diagnostically relevant proteins as age/quality biomarkers of therapeutic products.

Analysis of TAp73-dependent signaling via omics technologies.

Transactivation-proficient (TA) p73 is a transcription factor belonging to the p53 family, which regulates a variety of biological processes, including neurogenesis, differentiation, apoptosis, and DNA damage checkpoint response. In the present study, we adopted multiple Omics approaches, based upon the simultaneous application of metabolomics, lipidomics, and proteomics, in order to dissect the intracellular pathways activated by p73. As cellular model, we utilized a clone of the human osteosarcoma SAOS-2 cell line that allows the expression of TAp73α in an inducible manner. We found that TAp73α promoted mitochondrial activity (accumulation of metabolic intermediates and up-regulation of proteins related to the Krebs cycle), boosted glutathione homeostasis, increased arginine-citrulline-NO metabolism, altered purine synthesis, and promoted the pentose phosphate pathway toward NADPH accumulation for reducing and biosynthetic purposes. Indeed, lipid metabolism was driven toward the accumulation and oxidation of long-chain fatty acids with pro-apoptotic potential. In parallel, the expression of TAp73α was accompanied by the dephosphorylation of key proteins of the mitotic spindle assembly checkpoint. In conclusion, the obtained results confirm existing evidence from transcriptomics analyses and suggest a role for TAp73α in the regulation of cellular metabolism, cell survival, and cell growth.

Red blood cell populations and membrane levels of peroxiredoxin 2 as candidate biomarkers to reveal blood doping

BACKGROUND Blood doping represents one main trend in doping strategies. Blood doping refers to the practice of boosting the number of red blood cells (RBCs) in the bloodstream in order to enhance athletic performance, by means of blood transfusions, administration of erythropoiesis-stimulating substances, blood substitutes, natural or artificial altitude facilities, and innovative gene therapies. While detection of recombinant EPO and homologous transfusion is already feasible through electrophoretic, mass spectrometry or flow cytometry-based approaches, no method is currently available to tackle doping strategies relying on autologous transfusions. MATERIALS AND METHODS We exploited an in vitro model of autologous transfusion through a 1:10 dilution of concentrated RBCs after 30 days of storage upon appropriate dilution in freshly withdrawn RBCs from the same donor. Western blot towards membrane Prdx2 and Percoll density gradients were exploited to assess their suitability as biomarkers of transfusion. RESULTS Membrane Prdx2 was visible in day 30 samples albeit not in day 0, while it was still visible in the 1:10 dilution of day 30 in day 0 RBCs. Cell gradients also highlighted changes in the profile of the RBC subpopulations upon dilution of stored RBCs in the fresh ones. DISCUSSION From this preliminary in vitro investigation it emerges that Prdx2 and RBC populations might be further tested as candidate biomarkers of blood doping through autologous transfusion, though it is yet to be assessed whether the kinetics in vivo of Prdx2 exposure in the membrane of transfused RBCs will endow a sufficient time-window to allow reliable anti-doping testing.

Label-free quantitation of phosphopeptide changes in erythrocyte membranes: towards molecular mechanisms underlying deformability alterations in stored red blood cells

While the probability of troublesome adverse effects related to the transfusion of older red blood cell (RBC) units is still a matter of debate and of clinical investigation, what is now known for certain is that blood storage affects the biochemical and biological properties of RBCs. The