Enrichment of hepatic glycogen and plasma glucose from H218 O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice.

Deuterated water (2 H2 O) is a widely used tracer of carbohydrate biosynthesis in both preclinical and clinical settings, but the significant kinetic isotope effects (KIE) of 2 H can distort metabolic information and mediate toxicity. 18 O-water (H2 18 O) has no significant KIE and is incorporated into specific carbohydrate oxygens via well-defined mechanisms, but to date it has not been evaluated in any animal model. Mice were given H2 18 O during overnight feeding and 18 O-enrichments of liver glycogen, triglyceride glycerol (TG), and blood glucose were quantified by 13 C NMR and mass spectrometry (MS). Enrichment of oxygens 5 and 6 relative to body water informed indirect pathway contributions from the Krebs cycle and triose phosphate sources. Compared with mice fed normal chow (NC), mice whose NC was supplemented with a fructose/glucose mix (i.e., a high sugar [HS] diet) had significantly higher indirect pathway contributions from triose phosphate sources, consistent with fructose glycogenesis. Blood glucose and liver TG 18 O-enrichments were quantified by MS. Blood glucose 18 O-enrichment was significantly higher for HS versus NC mice and was consistent with gluconeogenic fructose metabolism. TG 18 O-enrichment was extensive for both NC and HS mice, indicating a high turnover of liver triglyceride, independent of diet. Thus H2 18 O informs hepatic carbohydrate biosynthesis in similar detail to 2 H2 O but without KIE-associated risks.

Proteomic and Metabolic Analysis of Pinus halepensis Mill. Embryonal Masses Induced under Heat Stress.

Understanding the physiological and molecular adjustments occurring during tree stress response is of great importance for forest management and breeding programs. Somatic embryogenesis has been used as a model system to analyze various processes occurring during embryo development, including stress response mechanisms. In addition, "priming" plants with heat stress during somatic embryogenesis seems to favor the acquisition of plant resilience to extreme temperature conditions. In this sense, Pinus halepensis somatic embryogenesis was induced under different heat stress treatments (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) and its effects on the proteome and the relative concentration of soluble sugars, sugar alcohols and amino acids of the embryonal masses obtained were assessed. Heat severely affected the production of proteins, and 27 proteins related to heat stress response were identified; the majority of the proteins with increased amounts in embryonal masses induced at higher temperatures consisted of enzymes involved in the regulation of metabolism (glycolysis, the tricarboxylic acid cycle, amino acid biosynthesis and flavonoids formation), DNA binding, cell division, transcription regulation and the life-cycle of proteins. Finally, significant differences in the concentrations of sucrose and amino acids, such as glutamine, glycine and cysteine, were found.

Peptidomics Unveils Distinct Acetylation Patterns of Histone and Annexin A1 in Differentiated Thyroid Cancer.

Thyroid cancer is a common malignancy of the endocrine system. Nodules are routinely evaluated for malignancy risk by fine needle aspiration biopsy (FNAB), and in cases such as follicular lesions, differential diagnosis between benign and malignant nodules is highly uncertain. Therefore, the discovery of new biomarkers for this disease could be helpful in improving diagnostic accuracy. Thyroid nodule biopsies were subjected to a precipitation step with both the insoluble and supernatant fractions subjected to proteome and peptidome profiling. Proteomic analysis identified annexin A1 as a potential biomarker of thyroid cancer malignancy, with its levels increased in malignant samples. Also upregulated were the acetylated peptides of annexin A1, revealed by the peptidome analysis of the supernatant fraction. In addition, supernatant peptidomic analysis revealed a number of acetylated histone peptides that were significantly elevated in the malignant group, suggesting higher gene transcription activity in malignant tissue. Two of these peptides were found to be robust malignancy predictors, with an area under the receiver operating a characteristic curve (ROC AUC) above 0.95. Thus, this combination of proteomics and peptidomics analyses improved the detection of malignant lesions and also provided new evidence linking thyroid cancer development to heightened transcription activity. This study demonstrates the importance of peptidomic profiling in complementing traditional proteomics approaches.

Posttranslational modifications of proteins are key features in the identification of CSF biomarkers of multiple sclerosis.

BACKGROUND: Multiple sclerosis is an inflammatory and degenerative disease of the central nervous system (CNS) characterized by demyelination and concomitant axonal loss. The lack of a single specific test, and the similarity to other inflammatory diseases of the central nervous system, makes it difficult to have a clear diagnosis of multiple sclerosis. Therefore, laboratory tests that allows a clear and definite diagnosis, as well as to predict the different clinical courses of the disease are of utmost importance. Herein, we compared the cerebrospinal fluid (CSF) proteome of patients with multiple sclerosis (in the relapse-remitting phase of the disease) and other diseases of the CNS (inflammatory and non-inflammatory) aiming at identifying reliable biomarkers of multiple sclerosis. METHODS: CSF samples from the discovery group were resolved by 2D-gel electrophoresis followed by identification of the protein spots by mass spectrometry. The results were analyzed using univariate (Student's t test) and multivariate (Hierarchical Cluster Analysis, Principal Component Analysis, Linear Discriminant Analysis) statistical and numerical techniques, to identify a set of protein spots that were differentially expressed in CSF samples from patients with multiple sclerosis when compared with other two groups. Validation of the results was performed in samples from a different set of patients using quantitative (e.g., ELISA) and semi-quantitative (e.g., Western Blot) experimental approaches. RESULTS: Analysis of the 2D-gels showed 13 protein spots that were differentially expressed in the three groups of patients: Alpha-1-antichymotrypsin, Prostaglandin-H2-isomerase, Retinol binding protein 4, Transthyretin (TTR), Apolipoprotein E, Gelsolin, Angiotensinogen, Agrin, Serum albumin, Myosin-15, Apolipoprotein B-100 and EF-hand calcium-binding domain-containing protein. ELISA experiments allowed validating part of the results obtained in the proteomics analysis and showed that some of the alterations in the CSF proteome are also mirrored in serum samples from multiple sclerosis patients. CSF of multiple sclerosis patients was characterized by TTR oligomerization, thus highlighting the importance of analyzing posttranslational modifications of the proteome in the identification of novel biomarkers of the disease. CONCLUSIONS: The model built based on the results obtained upon analysis of the 2D-gels and in the validation phase attained an accuracy of about 80% in distinguishing multiple sclerosis patients and the other two groups.

Specific Antiproliferative Properties of Proteinaceous Toxin Secretions from the Marine Annelid Eulalia sp. onto Ovarian Cancer Cells.

As Yondelis joins the ranks of approved anti-cancer drugs, the benefit from exploring the oceans' biodiversity becomes clear. From marine toxins, relevant bioproducts can be obtained due to their potential to interfere with specific pathways. We explored the cytotoxicity of toxin-bearing secretions of the polychaete Eulalia onto a battery of normal and cancer human cell lines and discovered that the cocktail of proteins is more toxic towards an ovarian cancer cell line (A2780). The secretions' main proteins were identified by proteomics and transcriptomics: 14-3-3 protein, Hsp70, Rab3, Arylsulfatase B and serine protease, the latter two being known toxins. This mixture of toxins induces cell-cycle arrest at G2/M phase after 3h exposure in A2780 cells and extrinsic programmed cell death. These findings indicate that partial re-activation of the G2/M checkpoint, which is inactivated in many cancer cells, can be partly reversed by the toxic mixture. Protein-protein interaction networks partake in two cytotoxic effects: cell-cycle arrest with a link to RAB3C and RAF1; and lytic activity of arylsulfatases. The discovery of both mechanisms indicates that venomous mixtures may affect proliferating cells in a specific manner, highlighting the cocktails' potential in the fine-tuning of anti-cancer therapeutics targeting cell cycle and protein homeostasis.

Comparative Proteomic Analysis of Nodulated and Non-Nodulated Casuarina glauca Sieb. ex Spreng. Grown under Salinity Conditions Using Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS).

Casuarina glauca displays high levels of salt tolerance, but very little is known about how this tree adapts to saline conditions. To understand the molecular basis of C. glauca response to salt stress, we have analyzed the proteome from branchlets of plants nodulated by nitrogen-fixing Frankia Thr bacteria (NOD+) and non-nodulated plants supplied with KNO3 (KNO3+), exposed to 0, 200, 400, and 600 mM NaCl. Proteins were identified by Short Gel, Long Gradient Liquid Chromatography coupled to Tandem Mass Spectrometry and quantified by Sequential Window Acquisition of All Theoretical Mass Spectra -Mass Spectrometry. 600 proteins were identified and 357 quantified. Differentially Expressed Proteins (DEPs) were multifunctional and mainly involved in Carbohydrate Metabolism, Cellular Processes, and Environmental Information Processing. The number of DEPs increased gradually with stress severity: (i) from 7 (200 mM NaCl) to 40 (600 mM NaCl) in KNO3+; and (ii) from 6 (200 mM NaCl) to 23 (600 mM NaCl) in NOD+. Protein-protein interaction analysis identified different interacting proteins involved in general metabolic pathways as well as in the biosynthesis of secondary metabolites with different response networks related to salt stress. Salt tolerance in C. glauca is related to a moderate impact on the photosynthetic machinery (one of the first and most important stress targets) as well as to an enhancement of the antioxidant status that maintains cellular homeostasis.

Validation of an LC-MS/MS Method for the Quantification of Caffeine and Theobromine Using Non-Matched Matrix Calibration Curve.

Caffeine is one of the most widely consumed psycho-stimulants. The study of the beneficial effects of caffeine consumption to decrease the risk of developing several neuropsychiatric pathologies is receiving increasing attention. Thus, accurate and sensitive methods have been developed, mainly by LC-MS/MS, in order to quantify caffeine and its metabolites. These quantifications of caffeine and its metabolites by LC-MS/MS require a considerable effort to select or find a surrogate matrix, without the compounds of interest, to be used in the calibration curves. Thus, we evaluated the possibility of using calibration curves prepared in solvent instead of calibration curves prepared in human plasma. Results show that the calibration curves prepared in solvent and in human plasma were similar by comparing their slopes and interceptions, and the accuracy and precision were within the limits of acceptance for both calibration curves. This work demonstrates that, by using internal standards, it is possible to use a calibration curve in solvent instead of a calibration curve in plasma to perform an accurate and precise quantification of caffeine and theobromine.

Ser¹¹9 phosphorylation modulates the activity and conformation of PRRXL1, a homeodomain transcription factor.

PRRXL1 [paired related homeobox-like 1; also known as DRG11 (dorsal root ganglia 11)] is a paired-like homeodomain transcription factor expressed in DRG and dSC (dorsal spinal cord) nociceptive neurons. PRRXL1 is crucial for the establishment and maintenance of nociceptive circuitry, as Prrxl1(-/-) mice present neuronal loss, reduced pain sensitivity and failure to thrive. In the present study, we show that PRRXL1 is highly phosphorylated in vivo, and that its multiple band pattern on electrophoretic analysis is the result of different phosphorylation states. PRRXL1 phosphorylation appears to be differentially regulated along the dSC and DRG development and it is mapped to two functional domains. One region comprises amino acids 107-143, whereas the other one encompasses amino acids 227-263 and displays repressor activity. Using an immunoprecipitation-MS approach, two phosphorylation sites were identified, Ser¹¹9 and Ser²³8. Phosphorylation at Ser¹¹9 is shown to be determinant for PRRXL1 conformation and transcriptional activity. Ser¹¹9 phosphorylation is thus proposed as a mechanism for regulating PRRXL1 function and conformation during nociceptive system development.

A Comparative Analysis of the Venom System between Two Morphotypes of the Sea Anemone Actinia equina.

The current study investigates the venom-delivery system of green and red morphotypes of the sea anemone Actinia equina to disclose its potential as a source of bioactive compounds. We compared the two morphotypes using electron and optical microscopy, proteomics, and toxicity assessment on zebrafish embryos. Specialized venom-injecting cells (nematocysts) are equally distributed and found in the tentacles of both varieties. Proteomics revealed proteins of interest in both red and green Actinia, yielding the three most abundant Gene Ontology (GO) terms related to the biological processes "proteolysis", "hemolysis in another organism" and "lipid catabolic process". Neurotoxins and cytolytic toxins similar to known cnidarian toxins like PsTX-60A and AvTX-60A, for instance, were identified in both types. Extracts from green and red anemones were toxic to zebrafish embryos, with green anemone venom appearing to be more potent. The findings highlight the presence of proteinaceous toxins in A. equina and the potential for different varieties to possess distinct bioactive compounds. Notably, pore-forming toxins are suggested for molecular probes and immunotoxins, making them valuable assets for potential biotechnological and biomedical purposes.

The secretome of macrophages has a differential impact on spinal cord injury recovery according to the polarization protocol.

Introduction: The inflammatory response after spinal cord injury (SCI) is an important contributor to secondary damage. Infiltrating macrophages can acquire a spectrum of activation states, however, the microenvironment at the SCI site favors macrophage polarization into a pro-inflammatory phenotype, which is one of the reasons why macrophage transplantation has failed. Methods: In this study, we investigated the therapeutic potential of the macrophage secretome for SCI recovery. We investigated the effect of the secretome in vitro using peripheral and CNS-derived neurons and human neural stem cells. Moreover, we perform a pre-clinical trial using a SCI compression mice model and analyzed the recovery of motor, sensory and autonomic functions. Instead of transplanting the cells, we injected the paracrine factors and extracellular vesicles that they secrete, avoiding the loss of the phenotype of the transplanted cells due to local environmental cues. Results: We demonstrated that different macrophage phenotypes have a distinct effect on neuronal growth and survival, namely, the alternative activation with IL-10 and TGF-ß1 (M(IL-10+TGF-ß1)) promotes significant axonal regeneration. We also observed that systemic injection of soluble factors and extracellular vesicles derived from M(IL-10+TGF-ß1) macrophages promotes significant functional recovery after compressive SCI and leads to higher survival of spinal cord neurons. Additionally, the M(IL-10+TGF-ß1) secretome supported the recovery of bladder function and decreased microglial activation, astrogliosis and fibrotic scar in the spinal cord. Proteomic analysis of the M(IL-10+TGF-ß1)-derived secretome identified clusters of proteins involved in axon extension, dendritic spine maintenance, cell polarity establishment, and regulation of astrocytic activation. Discussion: Overall, our results demonstrated that macrophages-derived soluble factors and extracellular vesicles might be a promising therapy for SCI with possible clinical applications.

An Exploration of Novel Bioactives from the Venomous Marine Annelid Glycera alba.

The immense biodiversity of marine invertebrates makes them high-value targets for the prospecting of novel bioactives. The present study investigated proteinaceous toxins secreted by the skin and proboscis of Glycera alba (Annelida: Polychaeta), whose congenerics G. tridactyla and G. dibranchiata are known to be venomous. Proteomics and bioinformatics enabled the detection of bioactive proteins that hold potential for biotechnological applications, including toxins like glycerotoxins (GLTx), which can interfere with neuromuscular calcium channels and therefore have value for the development of painkillers, for instance. We also identified proteins involved in the biosynthesis of toxins. Other proteins of interest include venom and toxin-related bioactives like cysteine-rich venom proteins, many of which are known to interfere with the nervous system. Ex vivo toxicity assays with mussel gills exposed to fractionated protein extracts from the skin and proboscis revealed that fractions potentially containing higher-molecular-mass venom proteins can exert negative effects on invertebrate prey. Histopathology, DNA damage and caspase-3 activity suggest significant cytotoxic effects that can be coadjuvated by permeabilizing enzymes such as venom metalloproteinases M12B. Altogether, these encouraging findings show that venomous annelids are important sources of novel bioactives, albeit illustrating the challenges of surveying organisms whose genomes and metabolisms are poorly understood.

Self-recycling and partially conservative replication of mycobacterial methylmannose polysaccharides.

The steep increase in nontuberculous mycobacteria (NTM) infections makes understanding their unique physiology an urgent health priority. NTM synthesize two polysaccharides proposed to modulate fatty acid metabolism: the ubiquitous 6-O-methylglucose lipopolysaccharide, and the 3-O-methylmannose polysaccharide (MMP) so far detected in rapidly growing mycobacteria. The recent identification of a unique MMP methyltransferase implicated the adjacent genes in MMP biosynthesis. We report a wide distribution of this gene cluster in NTM, including slowly growing mycobacteria such as Mycobacterium avium, which we reveal to produce MMP. Using a combination of MMP purification and chemoenzymatic syntheses of intermediates, we identified the biosynthetic mechanism of MMP, relying on two enzymes that we characterized biochemically and structurally: a previously undescribed α-endomannosidase that hydrolyses MMP into defined-sized mannoligosaccharides that prime the elongation of new daughter MMP chains by a rare α-(1→4)-mannosyltransferase. Therefore, MMP biogenesis occurs through a partially conservative replication mechanism, whose disruption affected mycobacterial growth rate at low temperature.

Chronic treatment with D2-antagonist haloperidol leads to inhibitory/excitatory imbalance in striatal D1-neurons.

Striatal dysfunction has been implicated in the pathophysiology of schizophrenia, a disorder characterized by positive symptoms such as hallucinations and delusions. Haloperidol is a typical antipsychotic medication used in the treatment of schizophrenia that is known to antagonize dopamine D2 receptors, which are abundantly expressed in the striatum. However, haloperidol's delayed therapeutic effect also suggests a mechanism of action that may go beyond the acute blocking of D2 receptors. Here, we performed proteomic analysis of striatum brain tissue and found more than 400 proteins significantly altered after 30 days of chronic haloperidol treatment in mice, namely proteins involved in glutamatergic and GABAergic synaptic transmission. Cell-type specific electrophysiological recordings further revealed that haloperidol not only reduces the excitability of striatal medium spiny neurons expressing dopamine D2 receptors (D2-MSNs) but also affects D1-MSNs by increasing the ratio of inhibitory/excitatory synaptic transmission (I/E ratio) specifically onto D1-MSNs but not D2-MSNs. Therefore, we propose the slow remodeling of D1-MSNs as a mechanism mediating the delayed therapeutic effect of haloperidol over striatum circuits. Understanding how haloperidol exactly contributes to treating schizophrenia symptoms may help to improve therapeutic outcomes and elucidate the molecular underpinnings of this disorder.

Effects of microplastics alone and with adsorbed benzo(a)pyrene on the gills proteome of Scrobicularia plana.

Microplastics (MPs) are globally present in the marine environment, but the biological effects on marine organisms at the molecular and cellular levels remain scarce. Due to their lipophilic nature, MPs can adsorb other contaminants present in the marine environment, which may increase their detrimental effects once ingested by organisms. This study investigates the effects of low-density polyethylene (PE) MPs with and without adsorbed benzo[a]pyrene (BaP) in the gills proteome of the peppery furrow shell clam, Scrobicularia plana. Clams were exposed to PE MPs (11-13 µm; 1 mg L-1) for 14 days. BaP was analyzed in whole clams' soft tissues, and a proteomic approach was applied in the gills using SWATH/DIA analysis. Proteomic responses suggest that virgin MPs cause disturbance by altering cytoskeleton and cell structure, energy metabolism, conformational changes, oxidative stress, fatty acids, DNA binding and, neurotransmission highlighting the potential risk of this type of MPs for the clam health. Conversely, when clam gills were exposed to MPs adsorbed with BaP a higher differentiation of protein expression was observed that besides changes in cytoskeleton and cell structure, oxidative stress, energy metabolism and DNA binding also induce changes in glucose metabolism, RNA binding and apoptosis. These results indicate that the presence of both stressors (MPs and BaP) have a higher toxicological risk to the health of S. plana.

Thermopriming-associated proteome and sugar content responses in Pinus radiata embryogenic tissue.

Improving the capacity of plants to face adverse environmental conditions requires a deep understanding of the molecular mechanisms governing stress response and adaptation. Proteomics, combined with metabolic analyses, offers a wide resource of information to be used in plant breeding programs. Previous studies have shown that somatic embryogenesis in Pinus spp. is a suitable tool not only to investigate stress response processes but also to modulate the behaviour of somatic plants. Based on this, the objective of this study was to analyse the protein and soluble sugar profiles of Pinus radiata embryonal masses after the application of high temperatures to unravel the mechanisms involved in thermopriming and memory acquisition at early stages of the somatic embryogenesis process. Results confirmed that heat provokes deep readjustments in the life cycle of proteins, together with a significant reduction in the carbon-flux of central-metabolism pathways. Heat-priming also promotes the accumulation of proteins involved in oxidative stress defence, in the synthesis of specific amino acids such as isoleucine, influences cell division, the organization of the cytoskeleton and cell-walls, and modifies the levels of free soluble sugars like glucose or fructose. All this seems to be regulated by proteins linked with epigenetic, transcriptional and post-transcriptional mechanisms.

COVID-19 Salivary Protein Profile: Unravelling Molecular Aspects of SARS-CoV-2 Infection.

COVID-19 is the most impacting global pandemic of all time, with over 600 million infected and 6.5 million deaths worldwide, in addition to an unprecedented economic impact. Despite the many advances in scientific knowledge about the disease, much remains to be clarified about the molecular alterations induced by SARS-CoV-2 infection. In this work, we present a hybrid proteomics and in silico interactomics strategy to establish a COVID-19 salivary protein profile. Data are available via ProteomeXchange with identifier PXD036571. The differential proteome was narrowed down by the Partial Least-Squares Discriminant Analysis and enrichment analysis was performed with FunRich. In parallel, OralInt was used to determine interspecies Protein-Protein Interactions between humans and SARS-CoV-2. Five dysregulated biological processes were identified in the COVID-19 proteome profile: Apoptosis, Energy Pathways, Immune Response, Protein Metabolism and Transport. We identified 10 proteins (KLK 11, IMPA2, ANXA7, PLP2, IGLV2-11, IGHV3-43D, IGKV2-24, TMEM165, VSIG10 and PHB2) that had never been associated with SARS-CoV-2 infection, representing new evidence of the impact of COVID-19. Interactomics analysis showed viral influence on the host immune response, mainly through interaction with the degranulation of neutrophils. The virus alters the host's energy metabolism and interferes with apoptosis mechanisms.

Increased Intake of Both Caffeine and Non-Caffeine Coffee Components Is Associated with Reduced NAFLD Severity in Subjects with Type 2 Diabetes.

Coffee may protect against non-alcoholic fatty liver disease (NAFLD), but the roles of the caffeine and non-caffeine components are unclear. Coffee intake by 156 overweight subjects (87% with Type-2-Diabetes, T2D) was assessed via a questionnaire, with 98 subjects (all T2D) also providing a 24 h urine sample for quantification of coffee metabolites by LC-MS/MS. NAFLD was characterized by the fatty liver index (FLI) and by Fibroscan® assessment of fibrosis. No associations were found between self-reported coffee intake and NAFLD parameters; however, total urine caffeine metabolites, defined as Σcaffeine (caffeine + paraxanthine + theophylline), and adjusted for fat-free body mass, were significantly higher for subjects with no liver fibrosis than for those with fibrosis. Total non-caffeine metabolites, defined as Σncm (trigonelline + caffeic acid + p-coumaric acid), showed a significant negative association with the FLI. Multiple regression analyses for overweight/obese T2D subjects (n = 89) showed that both Σcaffeine and Σncm were negatively associated with the FLI, after adjusting for age, sex, HbA1c, ethanol intake and glomerular filtration rate. The theophylline fraction of Σcaffeine was significantly increased with both fibrosis and the FLI, possibly reflecting elevated CYP2E1 activity-a hallmark of NAFLD worsening. Thus, for overweight/obese T2D patients, higher intake of both caffeine and non-caffeine coffee components is associated with less severe NAFLD. Caffeine metabolites represent novel markers of NAFLD progression.

Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure.

The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcification. The reproducibility of proteome studies is key in clinical translation of the findings. Thus, we aimed to optimize a protocol for aortic valve homogenization and protein extraction and to develop a standard operating procedure (SOP), which researchers can use to maximize protein yield while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenization with a Potter apparatus with a more advanced bead-beating system. Once we confirmed the superiority of the latter, we further optimized it by testing the effect of beads size, the number of homogenization cycles, tube capacity, lysis buffer/tissue mass ratio, and two different lysis buffers. Optimal protein extraction was achieved with 2.8 mm zirconium dioxide beads, in two homogenization cycles, in the presence of 20 µL RIPA buffer/mg tissue, using 2 mL O-ring cryotubes. As a proof of concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterized, resulting in the quantification of proteins across six orders of magnitude and uncovering some putative proteins dysregulated by sex.

Analysis of glucose metabolism in farmed European sea bass (Dicentrarchus labrax L.) using deuterated water.

Glucose metabolism in free-swimming fasted and fed seabass was studied using deuterated water ((2)H(2)O). After transfer to seawater enriched with 4.9% (2)H(2)O for 6-h or for 72-h, positional and mole percent enrichment (MPE) of plasma glucose and water were quantified by (2)H NMR and ESI-MS/MS. Plasma water (2)H-enrichment reached that of seawater within 6h. In both fasted and fed fish, plasma glucose MPE increased asymptotically attaining ~55% of plasma water enrichment by 72 h. The distribution of (2)H-enrichment between the different glucose positions was relatively uniform. The gluconeogenic contribution to glucose that was synthesized during (2)H(2)O administration was estimated from the ratio of position 5 and 2 glucose enrichments. For both fed and fasted fish, gluconeogenesis accounted for 98±1% of the glucose that was produced during the 72-h (2)H(2)O administration period. For fasted fish, gluconeogenic contributions measured after 6h were identical to 72-h values (94±3%). For fed fish, the apparent gluconeogenic contribution at 6-h was significantly lower compared to 72-h (79±5% versus 98±1%, p<0.05). This may reflect a brief augmentation of gluconeogenic flux by glycogenolysis after feeding and/or selective enrichment of plasma glucose position 2 via futile glucose-glucose-6-phosphate cycling.

Proteome-Wide Analysis of Heat-Stress in Pinus radiata Somatic Embryos Reveals a Combined Response of Sugar Metabolism and Translational Regulation Mechanisms.

Somatic embryogenesis is the process by which bipolar structures with no vascular connection with the surrounding tissue are formed from a single or a group of vegetative cells, and in conifers it can be divided into five different steps: initiation, proliferation, maturation, germination and acclimatization. Somatic embryogenesis has long been used as a model to study the mechanisms regulating stress response in plants, and recent research carried out in our laboratory has demonstrated that high temperatures during initial stages of conifer somatic embryogenesis modify subsequent phases of the process, as well as the behavior of the resulting plants ex vitro. The development of high-throughput techniques has facilitated the study of the molecular response of plants to numerous stress factors. Proteomics offers a reliable image of the cell status and is known to be extremely susceptible to environmental changes. In this study, the proteome of radiata pine somatic embryos was analyzed by LC-MS after the application of high temperatures during initiation of embryonal masses [(23°C, control; 40°C (4 h); 60°C (5 min)]. At the same time, the content of specific soluble sugars and sugar alcohols was analyzed by HPLC. Results confirmed a significant decrease in the initiation rate of embryonal masses under 40°C treatments (from 44 to 30.5%) and an increasing tendency in the production of somatic embryos (from 121.87 to 170.83 somatic embryos per gram of embryogenic tissue). Besides, heat provoked a long-term readjustment of the protein synthesis machinery: a great number of structural constituents of ribosomes were increased under high temperatures, together with the down-regulation of the enzyme methionine-tRNA ligase. Heat led to higher contents of heat shock proteins and chaperones, transmembrane transport proteins, proteins related with post-transcriptional regulation (ARGONAUTE 1D) and enzymes involved in the synthesis of fatty acids, specific compatible sugars (myo-inositol) and cell-wall carbohydrates. On the other hand, the protein adenosylhomocysteinase and enzymes linked with the glycolytic pathway, nitrogen assimilation and oxidative stress response were found at lower levels.