Thioredoxin-dependent Peroxide Reductase Research Articles

Identification of Candidate Biomarkers of Alzheimer's Disease via Multiplex Cerebrospinal Fluid and Serum Proteomics.

Alzheimer's disease (AD) is the most prevalent form of dementia among elderly people worldwide. Cerebrospinal fluid (CSF) is the optimal fluid source for AD biomarkers, while serum biomarkers are much more achievable. To search for novel diagnostic AD biomarkers, we performed a quantitative proteomic analysis of CSF and serum samples from AD and normal cognitive controls (NC). CSF and serum proteomes were analyzed via data-independent acquisition quantitative mass spectrometry. Our bioinformatic analysis was based on Gene Ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. In comparison to the controls, 8 proteins were more abundant in AD CSF, and 60 were less abundant in AD CSF, whereas 55 proteins were more and 10 were less abundant in the serum samples. ATPase-associated activity for CSF and mitochondrial functions for CSF and serum were the most enriched GO terms of the DEPs. KEGG enrichment analysis showed that the most significant pathways for the differentially expressed proteins were the N-glycan biosynthesis pathways. The area under the curve (AUC) values for CSF sodium-/potassium-transporting ATPase subunit beta-1 (AT1B1), serglycin (SRGN), and thioredoxin-dependent peroxide reductase, mitochondrial (PRDX3) were 0.867 (p = 0.004), 0.833 (p = 0.008), and 0.783 (p = 0.025), respectively. A panel of the above three CSF proteins accurately differentiated AD (AUC = 0.933, p = 0.001) from NC. The AUC values for serum probable phospholipid-transporting ATPase IM (AT8B4) and SRGN were moderate. The AUC of the CSF SRGN + serum SRGN was 0.842 (p = 0.007). These novel AD biomarker candidates are mainly associated with inflammation, ATPase activity, oxidative stress, and mitochondrial dysfunction. Further studies are needed to investigate the molecular mechanisms by which these potential biomarkers are involved in AD.

Space Omics and Tissue Response in Astronaut Skeletal Muscle after Short and Long Duration Missions.

The molecular mechanisms of skeletal muscle adaptation to spaceflight are as yet not fully investigated and well understood. The MUSCLE BIOPSY study analyzed pre and postflight deep calf muscle biopsies (m. soleus) obtained from five male International Space Station (ISS) astronauts. Moderate rates of myofiber atrophy were found in long-duration mission (LDM) astronauts (~180 days in space) performing routine inflight exercise as countermeasure (CM) compared to a short-duration mission (SDM) astronaut (11 days in space, little or no inflight CM) for reference control. Conventional H&E scout histology showed enlarged intramuscular connective tissue gaps between myofiber groups in LDM post vs. preflight. Immunoexpression signals of extracellular matrix (ECM) molecules, collagen 4 and 6, COL4 and 6, and perlecan were reduced while matrix-metalloproteinase, MMP2, biomarker remained unchanged in LDM post vs. preflight suggesting connective tissue remodeling. Large scale proteomics (space omics) identified two canonical protein pathways associated to muscle weakness (necroptosis, GP6 signaling/COL6) in SDM and four key pathways (Fatty acid β-oxidation, integrin-linked kinase ILK, Rho A GTPase RHO, dilated cardiomyopathy signaling) explicitly in LDM. The levels of structural ECM organization proteins COL6A1/A3, fibrillin 1, FBN1, and lumican, LUM, increased in postflight SDM vs. LDM. Proteins from tricarboxylic acid, TCA cycle, mitochondrial respiratory chain, and lipid metabolism mostly recovered in LDM vs. SDM. High levels of calcium signaling proteins, ryanodine receptor 1, RyR1, calsequestrin 1/2, CASQ1/2, annexin A2, ANXA2, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) pump, ATP2A, were signatures of SDM, and decreased levels of oxidative stress peroxiredoxin 1, PRDX1, thioredoxin-dependent peroxide reductase, PRDX3, or superoxide dismutase [Mn] 2, SOD2, signatures of LDM postflight. Results help to better understand the spatiotemporal molecular adaptation of skeletal muscle and provide a large scale database of skeletal muscle from human spaceflight for the better design of effective CM protocols in future human deep space exploration.

Dichloroacetate as a metabolic modulator of heart mitochondrial proteome under conditions of reduced oxygen utilization

Myocardial compensatory mechanisms stimulated by reduced oxygen utilization caused by streptozotocin-induced diabetes mellitus (DM) and treated with dichloroacetate (DCA) are presumably associated with the regulation of mitochondria. We aimed to promote the understanding of key signaling pathways and identify effectors involved in signal transduction. Proteomic analysis and fluorescence spectroscopy measurements revealed significantly decreased membrane potential and upregulated protein amine oxidase [flavin-containing] A (AOFA) in DM mitochondria, indicative of oxidative damage. DCA in diabetic animals (DM + DCA) downregulated AOFA, increased membrane potential, and stimulated thioredoxin-dependent peroxide reductase, a protein with antioxidant function. Furthermore, the DM condition was associated with mitochondrial resistance to calcium overload through mitochondrial permeability transition pores (mPTPs) regulation, despite an increased protein level of voltage-dependent anion-selective protein (VDAC1). In contrast, DM + DCA influenced ROS levels and downregulated VDAC1 and VDAC3 when compared to DM alone. The diabetic myocardium showed an identical pattern of mPTP protein interactions as in the control group, but the interactions were attenuated. Characterization of the combined effect of DM + DCA is a novel finding showing that DCA acted as an effector of VDAC protein interactions, calcium uptake regulation, and ROS production. Overall, DM and DCA did not exhibit an additive effect, but an individual cardioprotective pathway.

Extracellular H2O2, peroxiredoxin, and glutathione reductase alter Neospora caninum invasion and proliferation in Vero cells

Neospora caninum is a protozoan member of the Apicomplexa phylum and is closely connected with abortion in cattle. The development of the parasite in host cells is characterized by the active secretion of proteins, allied to the tight control of the redox status. In this sense, elucidating the mechanisms related to the role of the redox agents and enzymes during the invasion and proliferation of N. caninum may contribute to developing novel forms of neosporosis control. In this study we verified the effects of the recombinant forms of N. caninum glutathione reductase (rNcGR) and thioredoxin-dependent peroxide reductase (rNcPrx), as well as H2O2 in the tachyzoite invasion and proliferation. rNcPrx interfered in the N. caninum invasion in a redox state manner. Oxidized rNcPrx inhibited the N. caninum invasion and proliferation with no toxic effects observed in Vero cells. In contrast, lower concentrations of H2O2 (10 μM) stimulated the N. caninum invasion, which was reverted in higher doses (>100 μM). H2O2 inhibited the parasite proliferation in lower concentrations than cytotoxicity in host cells, resulting in a positive selectivity index (1.8). Besides, rNcPrx (reduced and non-reduced) and rNcGR inhibited the parasite proliferation without affecting the host cell. Our results indicate the connection between the N. caninum development and the redox state, contributing to the elucidation of parasite propagation and control mechanisms.

Ractopamine-induced remodeling in the mitochondrial proteome of postmortem longissimus lumborum muscle from feedlot steers

Dietary ractopamine causes a muscle fiber shift in cattle, and the biochemistry of mitochondria in postmortem muscles is influenced by fiber type. Nonetheless, the influence of ractopamine on beef skeletal muscle mitochondrial proteome has not been evaluated. Therefore, the objective of this study was to examine the effects of dietary ractopamine on mitochondrial proteome of postmortem longissimus lumborum (LL) from feedlot crossbred steers. Pen-housed crossbred steers were fed either a corn-based basal diet (CON) or a diet top-dressed with Optaflexx 45 (Elanco Animal Health) to provide 400 mg of ractopamine hydrochloride/steer per day (RAC). Ractopamine was fed the last 28 days prior to the harvest. The LL muscle samples were obtained from nine (n = 9) RAC and nine (n = 9) CON carcasses. The mitochondrial proteome was analyzed using two-dimensional gel electrophoresis and mass spectrometry. Seven differentially abundant proteins (P < 0.05) were identified. Three proteins over-abundant in RAC were complement component 1 Q subcomponent-binding protein (C1QBP), very long-chain specific acyl-CoA dehydrogenase (ACADVL), and aconitate hydratase (ACO2). On the other hand, four proteins, ATP synthase subunit beta (ATP5B), prohibitin (PHB), cytochrome b-c1 complex subunit (UQCRC1), and thioredoxin-dependent peroxide reductase (PRDX3), were over-abundant in CON. The differentially abundant proteins belong to four functional groups – energy metabolism (ATP5B, UQCRC1, and ACO2); chaperone activity (C1QBP and PHB); redox metabolism (PRDX3); and fatty acid degradation (ACADVL). The increased protein synthesis and leanness reported in ractopamine-fed cattle may be attributed to the increased expression of enzyme involved in fatty acid degradation and the decreased expression of enzymes involved in oxidative phosphorylation. Additionally, the decreased tenderness previously reported in beef from ractopamine-fed cattle may be attributed to the increased expression of antiapoptotic protein (C1QBP) and decreased expression of proapoptotic protein (PHB) resulted from ractopamine supplement.

Ractopamine-Induced Changes in the Mitochondrial Proteome of Postmortem Beef Longissimus Lumborum

ObjectivesRactopamine is a β-adrenergic agonist approved as growth promotant in beef cattle, and it increases muscle deposition while limiting fat deposition. Dietary ractopamine causes a muscle fiber shift in cattle, and the biochemistry of mitochondria in postmortem beef skeletal muscles is influenced by fiber type. Therefore, dietary ractopamine may potentially affect mitochondrial functionality. Nonetheless, the influence of ractopamine on beef skeletal muscle mitochondrial proteome has not been evaluated. Therefore, the objective of this study was to examine the effects of ractopamine on mitochondrial proteome of postmortem longissimus lumborum (LL) from beef cattle.Materials and MethodsPen-housed crossbred steers were fed either a corn-based basal diet (CON) or a diet top-dressed with Optaflexx 45 (Elanco Animal Health) to provide 400 mg of ractopamine hydrochloride/steer per day (RAC). Ractopamine was fed the last 28 d prior to the harvest. The animals were harvested, and carcasses were chilled for 24 h. The LL muscle samples were obtained from nine (n = 9) RAC and nine (n = 9) CON carcasses. Mitochondrial proteome was analyzed using two-dimensional electrophoresis, and the digital gel images were analyzed. The protein spots exhibiting more than 1.5-fold intensity differences (P &lt; 0.10) between RAC and CON were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsSeven differentially abundant proteins were identified in the mitochondrial proteome. Three proteins were over-abundant (P &lt; 0.10) in RAC, whereas four spots were over-abundant in CON. The proteins over-abundant in RAC mitochondrial proteome was complement component 1 Q subcomponent-binding protein, very long-chain specific acyl-CoA dehydrogenase, and aconitate hydratase. On the other hand, ATP synthase subunit β, prohibitin, Cytochrome b-c1 complex subunit, and thioredoxin-dependent peroxide reductase were over-abundant in CON samples. The differentially abundant proteins belong to four functional groups; i.e., energy metabolism (ATP synthase subunit β, Cytochrome b-c1 complex subunit 1, and aconitate hydratase), chaperone activity (complement component 1 Q subcomponent-binding protein and prohibitin), antioxidant activity (thioredoxin-dependent peroxide reductase), and lipid metabolism (very long-chain specific acyl-CoA dehydrogenase).ConclusionDietary ractopamine impacts mitochondrial proteome in postmortem beef LL muscle and influences the abundance of proteins involved in cellular metabolism and protective mechanisms. The increased protein synthesis and leanness previously reported in ractopamine-fed cattle may be attributed to the decreased expression of enzymes involved in respiratory electron transport pathways and the increased expression of enzymes involved in lipolysis.

Supranutritional Supplementation of Vitamin E Influences the Abundance of Antioxidant Proteins in Postmortem Longissimus Lumborum from Heifers

ObjectivesVitamin E is a lipid-soluble antioxidant that can inhibit lipid oxidation and improve beef color stability. The effect of vitamin E on fresh beef color, from the standpoint of lipid oxidation-induced myoglobin oxidation, have been extensively studied. However, the influence of vitamin E on sarcoplasmic proteome profile of beef skeletal muscles is yet to be investigated. Therefore, the objective of this study was to examine the effect of dietary vitamin E on sarcoplasmic proteome of postmortem beef longissimus lumborum (LL) muscle.Materials and MethodsCrossbred heifers, managed with a GrowSafe feeding system, were fed ad libitum corn-based diet containing either no supplemental (CONT) or 1000 IU vitamin E/heifer per day (VITE) for 89 d. The animals were harvested, and carcasses were chilled. The LL muscle samples were obtained from the carcasses of nine (n = 9) VITE and nine (n = 9) CONT heifers 24 h postmortem. The muscle samples were individually vacuum-packaged and frozen at –80°C for proteome analysis. Sarcoplasmic proteome was analyzed using two-dimensional electrophoresis, employing immobilized pH gradient strips (pH 3–10; 17 cm) in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The gels were scanned, and the digital gel images were analyzed. The protein spots exhibiting more than 1.5-fold intensity differences (P &lt; 0.10) between VITE and CONT were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsFive differentially abundant spots were identified using mass spectrometry, and all the spots were over-abundant in CONT. The proteins in the differentially abundant spots were antioxidant proteins (thioredoxin-dependent peroxide reductase, peroxiredoxin-6, and serum albumin) and glycolytic enzymes (β-enolase and triosephosphate isomerase). The antioxidant proteins minimize oxidation of lipids and proteins in muscle matrix, whereas the glycolytic enzymes generate NADPH, which helps maintain the antioxidant proteins in their reduced forms.ConclusionThe strong antioxidant protection offered by vitamin E could have possibly led to less expression of antioxidant proteins as well as glycolytic enzymes that generate antioxidant metabolites in the VITE group, whereas the lack of such protection in CONT group may have led to increased expression of these proteins in the skeletal muscles.

Endpoint Temperature Influences Sarcoplasmic Proteome Profile of Cooked Beef Longissimus Lumborum

ObjectivesCooking ensures safety and enhances the palatability attributes of meat. Denaturation of myoglobin results in the dull-brown color of cooked meats. The denaturation of sarcoplasmic proteins is influenced by the degree of heat treatment, and their solubility is decreased with an increase in the endpoint cooking temperature. While previous studies examined the relationship between myoglobin denaturation, cooked color, and internal temperature in beef, investigations are yet to be undertaken to characterize the association between endpoint temperature, sarcoplasmic proteome, and color attributes in cooked steaks. Therefore, the objective of the present study was to examine the influence of endpoint cooking temperature (60 and 71°C) on sarcoplasmic proteome and internal color of beef longissimus lumborum (LL) steaks.Materials and MethodsEight (n = 8) beef LL muscles (14 d postmortem; USDA Choice) were obtained from a commercial packing plant. Two 2.5-cm thick steaks were fabricated from the center of the muscles and were cooked to internal endpoint temperature of 60°C (C-60) or 71°C (C-71) in a clam-shell grill. Cooked steaks were immediately cooled in slushed ice, sliced parallel to the grilled surface, and internal redness (a* value) and color stability (R630/580) were evaluated instrumentally. Sarcoplasmic proteome from the interiors of the cooked steaks was analyzed using 2-dimensional electrophoresis, and the gel images were digitally analyzed. The protein spots exhibiting more than 2.5-fold intensity differences (P &lt; 0.05) between C-60 and C-71 were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsThe C-60 steaks demonstrated greater (P &lt; 0.05) a* and R630/580 than their C-71 counterparts. Seven differentially abundant proteins were identified and were over-abundant (P &lt; 0.05) in C-60 compared to C-71. The differentially abundant proteins belong to 6 functional groups, i.e., transport proteins (serum albumin and hemoglobin), energy metabolism (adenylate kinase isoenzyme 1), chaperones (heat shock protein β-1), antioxidant (thioredoxin-dependent peroxide reductase), glycolytic enzymes (fructose-bisphosphate aldolase B), and protease (cytosol aminopeptidase).ConclusionThe findings indicated that the endpoint cooking temperature influences the internal cooked color and the sarcoplasmic proteome profile of beef LL steaks. The overabundant proteins in steaks cooked to 60°C may be utilized as potential biomarkers for undercooked beef, which is a source for foodborne infections.

Ractopamine-Induced Changes in the Mitochondrial Proteome of Postmortem Beef Longissimus Lumborum

ObjectivesRactopamine is a β-adrenergic agonist approved as growth promotant in beef cattle, and it increases muscle deposition while limiting fat deposition. Dietary ractopamine causes a muscle fiber shift in cattle, and the biochemistry of mitochondria in postmortem beef skeletal muscles is influenced by fiber type. Therefore, dietary ractopamine may potentially affect mitochondrial functionality. Nonetheless, the influence of ractopamine on beef skeletal muscle mitochondrial proteome has not been evaluated. Therefore, the objective of this study was to examine the effects of ractopamine on mitochondrial proteome of postmortem longissimus lumborum (LL) from beef cattle.Materials and MethodsPen-housed crossbred steers were fed either a corn-based basal diet (CON) or a diet top-dressed with Optaflexx 45 (Elanco Animal Health) to provide 400 mg of ractopamine hydrochloride/steer per day (RAC). Ractopamine was fed the last 28 d prior to the harvest. The animals were harvested, and carcasses were chilled for 24 h. The LL muscle samples were obtained from nine (n = 9) RAC and nine (n = 9) CON carcasses. Mitochondrial proteome was analyzed using two-dimensional electrophoresis, and the digital gel images were analyzed. The protein spots exhibiting more than 1.5-fold intensity differences (P < 0.10) between RAC and CON were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsSeven differentially abundant proteins were identified in the mitochondrial proteome. Three proteins were over-abundant (P < 0.10) in RAC, whereas four spots were over-abundant in CON. The proteins over-abundant in RAC mitochondrial proteome was complement component 1 Q subcomponent-binding protein, very long-chain specific acyl-CoA dehydrogenase, and aconitate hydratase. On the other hand, ATP synthase subunit β, prohibitin, Cytochrome b-c1 complex subunit, and thioredoxin-dependent peroxide reductase were over-abundant in CON samples. The differentially abundant proteins belong to four functional groups; i.e., energy metabolism (ATP synthase subunit β, Cytochrome b-c1 complex subunit 1, and aconitate hydratase), chaperone activity (complement component 1 Q subcomponent-binding protein and prohibitin), antioxidant activity (thioredoxin-dependent peroxide reductase), and lipid metabolism (very long-chain specific acyl-CoA dehydrogenase).ConclusionDietary ractopamine impacts mitochondrial proteome in postmortem beef LL muscle and influences the abundance of proteins involved in cellular metabolism and protective mechanisms. The increased protein synthesis and leanness previously reported in ractopamine-fed cattle may be attributed to the decreased expression of enzymes involved in respiratory electron transport pathways and the increased expression of enzymes involved in lipolysis.

Endpoint Temperature Influences Sarcoplasmic Proteome Profile of Cooked Beef Longissimus Lumborum

ObjectivesCooking ensures safety and enhances the palatability attributes of meat. Denaturation of myoglobin results in the dull-brown color of cooked meats. The denaturation of sarcoplasmic proteins is influenced by the degree of heat treatment, and their solubility is decreased with an increase in the endpoint cooking temperature. While previous studies examined the relationship between myoglobin denaturation, cooked color, and internal temperature in beef, investigations are yet to be undertaken to characterize the association between endpoint temperature, sarcoplasmic proteome, and color attributes in cooked steaks. Therefore, the objective of the present study was to examine the influence of endpoint cooking temperature (60 and 71°C) on sarcoplasmic proteome and internal color of beef longissimus lumborum (LL) steaks.Materials and MethodsEight (n = 8) beef LL muscles (14 d postmortem; USDA Choice) were obtained from a commercial packing plant. Two 2.5-cm thick steaks were fabricated from the center of the muscles and were cooked to internal endpoint temperature of 60°C (C-60) or 71°C (C-71) in a clam-shell grill. Cooked steaks were immediately cooled in slushed ice, sliced parallel to the grilled surface, and internal redness (a* value) and color stability (R630/580) were evaluated instrumentally. Sarcoplasmic proteome from the interiors of the cooked steaks was analyzed using 2-dimensional electrophoresis, and the gel images were digitally analyzed. The protein spots exhibiting more than 2.5-fold intensity differences (P < 0.05) between C-60 and C-71 were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsThe C-60 steaks demonstrated greater (P < 0.05) a* and R630/580 than their C-71 counterparts. Seven differentially abundant proteins were identified and were over-abundant (P < 0.05) in C-60 compared to C-71. The differentially abundant proteins belong to 6 functional groups, i.e., transport proteins (serum albumin and hemoglobin), energy metabolism (adenylate kinase isoenzyme 1), chaperones (heat shock protein β-1), antioxidant (thioredoxin-dependent peroxide reductase), glycolytic enzymes (fructose-bisphosphate aldolase B), and protease (cytosol aminopeptidase).ConclusionThe findings indicated that the endpoint cooking temperature influences the internal cooked color and the sarcoplasmic proteome profile of beef LL steaks. The overabundant proteins in steaks cooked to 60°C may be utilized as potential biomarkers for undercooked beef, which is a source for foodborne infections.

Supranutritional Supplementation of Vitamin E Influences the Abundance of Antioxidant Proteins in Postmortem Longissimus Lumborum from Heifers

ObjectivesVitamin E is a lipid-soluble antioxidant that can inhibit lipid oxidation and improve beef color stability. The effect of vitamin E on fresh beef color, from the standpoint of lipid oxidation-induced myoglobin oxidation, have been extensively studied. However, the influence of vitamin E on sarcoplasmic proteome profile of beef skeletal muscles is yet to be investigated. Therefore, the objective of this study was to examine the effect of dietary vitamin E on sarcoplasmic proteome of postmortem beef longissimus lumborum (LL) muscle.Materials and MethodsCrossbred heifers, managed with a GrowSafe feeding system, were fed ad libitum corn-based diet containing either no supplemental (CONT) or 1000 IU vitamin E/heifer per day (VITE) for 89 d. The animals were harvested, and carcasses were chilled. The LL muscle samples were obtained from the carcasses of nine (n = 9) VITE and nine (n = 9) CONT heifers 24 h postmortem. The muscle samples were individually vacuum-packaged and frozen at –80°C for proteome analysis. Sarcoplasmic proteome was analyzed using two-dimensional electrophoresis, employing immobilized pH gradient strips (pH 3–10; 17 cm) in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The gels were scanned, and the digital gel images were analyzed. The protein spots exhibiting more than 1.5-fold intensity differences (P < 0.10) between VITE and CONT were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsFive differentially abundant spots were identified using mass spectrometry, and all the spots were over-abundant in CONT. The proteins in the differentially abundant spots were antioxidant proteins (thioredoxin-dependent peroxide reductase, peroxiredoxin-6, and serum albumin) and glycolytic enzymes (β-enolase and triosephosphate isomerase). The antioxidant proteins minimize oxidation of lipids and proteins in muscle matrix, whereas the glycolytic enzymes generate NADPH, which helps maintain the antioxidant proteins in their reduced forms.ConclusionThe strong antioxidant protection offered by vitamin E could have possibly led to less expression of antioxidant proteins as well as glycolytic enzymes that generate antioxidant metabolites in the VITE group, whereas the lack of such protection in CONT group may have led to increased expression of these proteins in the skeletal muscles.

Proteomic differences between focal and diffuse traumatic brain injury in human brain tissue

The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.

Proteomic analysis to investigate color changes of chilled beef longissimus steaks held under carbon monoxide and high oxygen packaging

This study investigated the proteome basis for color stability variations in beef steaks packaged under two modified atmosphere packaging (MAP) methods: HiOx-MAP (80% O2/20% CO2) and CO-MAP (0.4% CO/30% CO2/69.6% N2) during 15 days of storage. The color stability, pH, and sarcoplasmic proteome analysis of steaks were evaluated on days 0, 5, 10 and 15 of storage. Proteomic results revealed that the differential expression of the sarcoplasmic proteome during storage contributed to the variations in meat color stability between the two MAP methods. Compared with HiOx-MAP steaks, some glycolytic and energy metabolic enzymes important in NADH regeneration and antioxidant processes, antioxidant peroxiredoxins (thioredoxin-dependent peroxide reductase, peroxiredoxin-2, peroxiredoxin-6) and protein DJ-1 were more abundant in CO-MAP steaks. The over-expression of these proteins could induce CO-MAP steaks to maintain high levels of metmyoglobin reducing activity and oxygen consumption rate, resulting in CO-MAP steaks exhibiting better color stability than HiOx-MAP steaks during storage.

Deletion of protein kinase C-ε attenuates mitochondrial dysfunction and ameliorates ischemic renal injury.

Previously, we documented that activation of protein kinase C-ε (PKC-ε) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of PKC-ε decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC-ε levels in mitochondria of ischemic kidneys increased 24 h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44 and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC-ε-deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO but not in WT animals. PKC-ε deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC-ε deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC-ε activation in hypoxic RPTC in primary culture exacerbated, whereas PKC-ε inhibition reduced, decreases in: 1) complex I- and complex II-coupled state 3 respirations and 2) activities of complexes I, III, and IV. We conclude that PKC-ε activation mediates 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia.

Postmortem changes in sarcoplasmic proteins associated with color stability in lamb muscle analyzed by proteomics

The aim of this research was to correlate the postmortem changes of sarcoplasmic proteins with instrumental color and various biochemical parameters relating to color attributes. Two-dimensional electrophoresis was used to investigate sarcoplasmic protein changes in cardiac muscle during refrigerated storage. Eleven differentially abundant protein spots were identified between storage time at day 1 and day 5 (P < 0.05). The functional category of proteins consisted of metabolic enzymes, binding proteins, chaperone proteins, and antioxidant proteins. These proteins demonstrated positive correlation with a*-value (creatine kinase M-type and malate dehydrogenase), metmyoglobin-reducing activity (aconitate hydratase, creatine kinase, and thioredoxin-dependent peroxide reductase), tissue oxygen consumption rate (creatine kinase and malate dehydrogenase), and mitochondrial respiration control ratio (thioredoxin-dependent peroxide reductase and mitochondrial superoxide dismutase). The protein patterns were likely to have impact on postmortem metabolism, thereby protecting muscle against discoloration.

The influence of chronic ibuprofen treatment on proteins expressed in the mouse hippocampus

Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID), treatment with which has been shown to delay the onset, slows the cognitive decline, and decreases the incidence of Alzheimer׳s disease (AD) in epidemiological and clinical studies. However, a comprehensive understanding of its mechanism of action remains unclear. To elucidate the prophylactic effect of ibuprofen on the onset of the learning and memory disturbances of AD, we performed proteomic analysis of the hippocampus of chronic ibuprofen-treated mice using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. Twenty-eight proteins and seven phosphoproteins were identified to be significantly changed in the hippocampus of chronic ibuprofen-treated mice: translationally controlled tumor protein, thioredoxin-dependent peroxide reductase, and peroxiredoxin 6 were increased, and glial fibrillary acidic protein, dihydropyrimidinase-related protein 2, EF-hand domain-containing protein D2, and 14-3-3ζ were decreased. These identified proteins and phosphoproteins could be classified as cytoskeletal, neuronal development, chaperone, metabolic, apoptosis, neurotransmitter release, ATP synthase, deubiquitination, proteasome, NOS inhibitor, adapter, vesicle transport, signal transduction, antioxidant enzyme, proton transport, synaptogenesis, and serine/threonine phosphatase types. Western blot analysis showed the changes in dihydropyrimidinase-related protein 2, heat shock protein 8, ubiquitin carboxyl-terminal hydrolase PGP9.5, and γ-enolase levels in the hippocampus of chronic ibuprofen-treated mice. These findings showed that the chronic treatment with ibuprofen changed the levels of some proteins and phosphoproteins in the hippocampus. We propose that these identified proteins and phosphoproteins play an important role in decreasing the incidence of AD, especially impaired learning and memory functions.

MTor Is a Signaling Hub in Cell Survival: A Mass-Spectrometry-Based Proteomics Investigation

mTor plays a central role in controlling protein homeostasis and cell survival. Recently, we have demonstrated that perturbations of mTor signaling are implicated in Alzheimer's disease (AD) and that mTor complex 1 (mTorC1) is involved in the formation of toxic phospho-tau. Therefore, we employed mass-spectrometry-based proteomics to identify specific protein expression changes in relation with cell survival in human neuroblastoma SH-SY5Y cells expressing genetically modified mTor. Cell death in SH-SY5Y cells was induced by moderate serum deprivation. Using flow cytometry we observed that up-regulated mTor complex 2 (mTorC2) increases the number of viable cells. By using a combination approach of proteomic and enrichment analysis we have identified several proteins (Thioredoxin-dependent peroxide reductase, Peroxiredoxin-5, Cofilin 1 (non-muscle), Annexin A5, Mortalin, and 14-3-3 protein zeta/delta) involved in mitochondrial integrity, apoptotosis, and pro-survival functions (caspase inhibitor activity and anti-apoptosis) that were significantly altered by mTor activity modulation. The major findings of this study are the implication of mTorC2 but not mTorC1 in cell viability modulation by activating the pro-survival machinery. Taken together, these results suggest that up-regulated mTorC2 might be playing an important role in promoting cell survival by suppressing the mitochondria-caspase-apoptotic pathway in vitro.

Lymphocyte mitochondria: toward identification of peripheral biomarkers in the progression of Alzheimer disease

Alzheimer disease (AD) is an age-related neurodegenerative condition. AD is histopathologically characterized by the presence of three main hallmarks: senile plaques (rich in amyloid-β peptide), neuronal fibrillary tangles (rich in phosphorylated tau protein), and synapse loss. However, definitive biomarkers for this devastating disease in living people are still lacking. In this study, we show that levels of oxidative stress markers are significantly increased in the mitochondria isolated from lymphocytes of subjects with mild cognitive impairment (MCI) compared to cognitively normal individuals. Further, an increase in mitochondrial oxidative stress in MCI is associated with MMSE score, vitamin E components, and β-carotene. Further, a proteomics approach showed that alterations in the levels of thioredoxin-dependent peroxide reductase, myosin light polypeptide 6, and ATP synthase subunit β might be important in the progression and pathogenesis of AD. Increased understanding of oxidative stress and protein alterations in easily obtainable peripheral tissues will be helpful in developing biomarkers to combat this devastating disorder.

Extremely low-frequency electromagnetic fields induce neural differentiation in bone marrow derived mesenchymal stem cells

Extremely low-frequency electromagnetic fields (ELF-EMF) affect numerous biological functions such as gene expression, cell fate determination and even cell differentiation. To investigate the correlation between ELF-EMF exposure and differentiation, bone marrow derived mesenchymal stem cells (BM-MSCs) were subjected to a 50-Hz electromagnetic field during invitro expansion. The influence of ELF-EMF on BM-MSCs was analysed by a range of different analytical methods to understand its role in the enhancement of neural differentiation. ELF-EMF exposure significantly decreased the rate of proliferation, which in turn caused an increase in neuronal differentiation. The ELF-EMF-treated cells showed increased levels of neuronal differentiation marker (MAP2), while early neuronal marker (Nestin) was down-regulated. In addition, eight differentially expressed proteins were detected in two-dimensional electrophoresis maps, and were identified using ESI-Q-TOF LC/MS/MS. Among them, ferritin light chain, thioredoxin-dependent peroxide reductase, and tubulin β-6 chain were up-regulated in the ELF-EMF-stimulated group. Ferritin and thioredoxin-dependent peroxide reductase are involved in a wide variety of functions, including Ca(2+) regulation, which is a critical component of neurodegeneration. We also observed that the intracellular Ca(2+) content was significantly elevated after ELF-EMF exposure, which strengthens the modulatory role of ferritin and thioredoxin-dependent peroxide reductase, during differentiation. Notably, western blot analysis indicated significantly increased expression of the ferritin light chain in the ELF-EMF-stimulated group (0.60 vs. 1.08; P < 0.01). These proteins may help understand the effect of ELF-EMF stimulation on BM-MSCs during neural differentiation and its potential use as a clinically therapeutic option for treating neurodegenerative diseases.

Proteomic analysis of endothelial progenitor cells exposed to oxidative stress

Endothelial progenitor cells (EPCs) repair vascular damage and participate in neovascularization. Accumulating evidence has demonstrated that EPCs have therapeutic potential in reactive oxygen species (ROS)-mediated vascular diseases. In this study, to investigate the effects of oxidative stress on EPCs, EPCs were treated with H2O2 at different final concentrations for 3 h. MTT assay, scratch-wound assay and Matrigel invasion assay revealed that cell proliferation, migration and tubule formation and function, respectively, were impaired under H2O2 stress in a concentration-dependent manner. To determine protein response to H2O2 stress, two-dimensional differential in-gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry were performed. The results revealed that triosephosphate isomerase and ADP-sugar pyrophosphatase were downregulated, while peroxiredoxin-2, thioredoxin-dependent peroxide reductase, mitochondrial (Prx‑3), peroxiredoxin-6, EGF-containing fibulin-like extracellular matrix protein 1, vimentin and Rab GDP dissociation inhibitor α were upregulated in the H2O2-treated EPCs. To further confirm the results from mass spectrometry, the expression pattern of Prx-3 in response to H2O2 stress was examined by western blot analysis. The data presented in this study provide novel insight into the defensive mechanisms of EPCs and the pathways of oxidative damage in an oxidative environment.