Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.

Immune system threat detection hinges on T cells' ability to perceive varying peptide-major histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs.

Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.

Unlabelled: Immune system threat detection hinges on T cells' ability to perceive varying peptide major-histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs.

Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in mice.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress.

Order by chance: origins and benefits of stochasticity in immune cell fate control.

To protect against diverse challenges, the immune system must continuously generate an arsenal of specialized cell types, each of which can mount a myriad of effector responses upon detection of potential threats. To do so, it must generate multiple differentiated cell populations with defined sizes and proportions, often from rare starting precursor cells. Here, we discuss the emerging view that inherently probabilistic mechanisms, involving rare, rate-limiting regulatory events in single cells, control fate decisions and population sizes and fractions during immune development and function.

Selective organ ischaemia/reperfusion identifies liver as the key driver of the post-injury plasma metabolome derangements.

Background: Understanding the molecular mechanisms in perturbation of the metabolome following ischaemia and reperfusion is critical in developing novel therapeutic strategies to prevent the sequelae of post-injury shock. While the metabolic substrates fueling these alterations have been defined, the relative contribution of specific organs to the systemic metabolic reprogramming secondary to ischaemic or haemorrhagic hypoxia remains unclear.

Materials And Methods: A porcine model of selected organ ischaemia was employed to investigate the relative contribution of liver, kidney, spleen and small bowel ischaemia/reperfusion to the plasma metabolic phenotype, as gleaned through ultra-high performance liquid chromatography-mass spectrometry-based metabolomics.

All animals are equal but some animals are more equal than others: Plasma lactate and succinate in hemorrhagic shock-A comparison in rodents, swine, nonhuman primates, and injured patients.

Background: Plasma levels of lactate and succinate are predictors of mortality in critically injured patients in military and civilian settings. In relative terms, these metabolic derangements have been recapitulated in rodent, swine, and nonhuman primate models of severe hemorrhage. However, no direct absolute quantitative comparison has been evaluated across these species.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

Hypoxanthine catabolism is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%).

Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization.

The extracellular matrix (ECM) is readily enriched by decellularizing tissues with nondenaturing detergents to solubilize and deplete the vast majority of cellular components. This approach has been used extensively to generate ECM scaffolds for regenerative medicine technologies and in 3D cell culture to model how the ECM contributes to disease progression. A highly enriched ECM fraction can then be generated using a strong chaotrope buffer that is compatible with downstream bottom-up proteomic analysis or 3D cell culture experiments after extensive dialysis.

Plasma succinate is a predictor of mortality in critically injured patients.

Background: Trauma is the leading cause of mortality under the age of 40 years. Recent observations on metabolic reprogramming during hypoxia and ischemia indicate that hypoxic mitochondrial uncoupling promotes the generation of succinate, which in turn mediates reperfusion injury and inflammatory sequelae upon reoxygenation. Plasma levels of succinate significantly increase in response to trauma and hemorrhage in experimental models and clinical samples, suggesting that succinate may represent a candidate marker of systemic perfusion in trauma.

Hemorrhagic shock and tissue injury drive distinct plasma metabolome derangements in swine.

Background: Tissue injury and hemorrhagic shock induce significant systemic metabolic reprogramming in animal models and critically injured patients. Recent expansions of the classic concepts of metabolomic aberrations in tissue injury and hemorrhage opened the way for novel resuscitative interventions based on the observed abnormal metabolic demands. We hypothesize that metabolic demands and resulting metabolic signatures in pig plasma will vary in response to isolated or combined tissue injury and hemorrhagic shock.

Omics markers of the red cell storage lesion and metabolic linkage.

The introduction of omics technologies in the field of Transfusion Medicine has significantly advanced our understanding of the red cell storage lesion. While the clinical relevance of such a lesion is still a matter of debate, quantitative and redox proteomics approaches, as well quantitative metabolic flux analysis and metabolic tracing experiments promise to revolutionise our understanding of the role of blood processing strategies, inform the design and testing of novel additives or technologies (such as pathogen reduction), and evaluate the clinical relevance of donor and recipient biological variability with respect to red cell storability and transfusion outcomes. By reviewing existing literature in this rapidly expanding research endeavour, we highlight for the first time a correlation between metabolic markers of the red cell storage age and protein markers of haemolysis.

Mass Spectrometry-Based Bottom-Up Proteomics: Sample Preparation, LC-MS/MS Analysis, and Database Query Strategies.

Recent technological advances in mass spectrometry (MS) have made possible the investigation and quantification of complex mixtures of biomolecules. The exceptional sensitivity and resolving power of today's mass spectrometers allow for the detection of proteins and peptides at low femtomole quantities; however, these attributes demand high sample purity to minimize artifacts and achieve the highest degree of biomolecule identification. Tissue preparation for proteomic studies is particularly challenging due to their heterogeneity in cell type, presence of insoluble biomaterials, and wide diversity of biomolecules.

Glutamine metabolism drives succinate accumulation in plasma and the lung during hemorrhagic shock.

Background: Metabolomic investigations have consistently reported succinate accumulation in plasma after critical injury. Succinate receptors have been identified on numerous tissues, and succinate has been directly implicated in postischemic inflammation, organ dysfunction, platelet activation, and the generation of reactive oxygen species, which may potentiate morbidity and mortality risk to patients. Metabolic flux (heavy-isotope labeling) studies demonstrate that glycolysis is not the primary source of increased plasma succinate during protracted shock.

Data on how several physiological parameters of stored red blood cells are similar in glucose 6-phosphate dehydrogenase deficient and sufficient donors.

This article contains data on the variation in several physiological parameters of red blood cells (RBCs) donated by eligible glucose-6-phosphate dehydrogenase (G6PD) deficient donors during storage in standard blood bank conditions compared to control, G6PD sufficient (G6PD(+)) cells. Intracellular reactive oxygen species (ROS) generation, cell fragility and membrane exovesiculation were measured in RBCs throughout the storage period, with or without stimulation by oxidants, supplementation of N-acetylcysteine and energy depletion, following incubation of stored cells for 24 h at 37 °C. Apart from cell characteristics, the total or uric acid-dependent antioxidant capacity of the supernatant in addition to extracellular potassium concentration was determined in RBC units.

Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days.

Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells.

Storage of packed red blood cells (RBCs) is associated with progressive accumulation of lesions, mostly triggered by energy and oxidative stresses, which potentially compromise the effectiveness of the transfusion therapy. Concerns arise as to whether glucose 6-phosphate dehydrogenase deficient subjects (G6PD(-)), ~5% of the population in the Mediterranean area, should be accepted as routine donors in the light of the increased oxidative stress their RBCs suffer from. To address this question, we first performed morphology (scanning electron microscopy), physiology and omics (proteomics and metabolomics) analyses on stored RBCs from healthy or G6PD(-) donors.

Plasma First Resuscitation Reduces Lactate Acidosis, Enhances Redox Homeostasis, Amino Acid and Purine Catabolism in a Rat Model of Profound Hemorrhagic Shock.

The use of aggressive crystalloid resuscitation to treat hypoxemia, hypovolemia, and nutrient deprivation promoted by massive blood loss may lead to the development of the blood vicious cycle of acidosis, hypothermia, and coagulopathy and, utterly, death. Metabolic acidosis is one of the many metabolic derangements triggered by severe trauma/hemorrhagic shock, also including enhanced proteolysis, lipid mobilization, as well as traumatic diabetes. Appreciation of the metabolic benefit of plasma first resuscitation is an important concept.

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen.

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼ 120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications.