iTRAQ-based Quantitative Proteomics Research Articles

SOX4 maintains the stemness of cancer cells via transcriptionally enhancing HDAC1 revealed by comparative proteomics study

BackgroundCancer stem cells (CSCs) are the root of human cancer development and the major cause of treatment failure. Aberrant elevation of SOX4, a member of SOX (SRY-related HMG-box) family transcription factors, has been identified in many types of human cancer and promotes cancer development. However, the role of SOX4 in CSCs, especially at a proteome-wide level, has remained elusive. The aim of this study is to investigate the effect of SOX4 on the stemness of CSCs and reveal the underlying mechanisms by identification of SOX4-induced proteome changes through proteomics study.ResultsOverexpression of SOX4 promotes sphere formation and self-renewal of colorectal cancer cells in vitro and in vivo and elevates the expression levels of CSCs markers. Through iTRAQ-based quantitative proteomics analysis, 215 differentially expressed proteins (128 upregulated, 87 downregulated) in SOX4-overexpressing HCT-116 spheres were identified. The bioinformatic analysis highlighted the importance of HDAC1 as the fundamental roles of its impacted pathways in stem cell maintenance, including Wnt, Notch, cell cycle, and transcriptional misregulation in cancer. The mechanistic study showed that SOX4 directly binds to the promoter of HDAC1, promotes HDAC1 transcription, thereby supporting the stemness of colorectal cancer cells. HDAC1 hallmarks colorectal cancer stem cells and depletion of HDAC1 abolished the stimulatory effect of SOX4. Furthermore, SOX4-HDAC1 axis is conserved in multiple types of cancer.ConclusionsThe results of this study reveal SOX4-induced proteome changes in HCT-116 spheres and demonstrates that transcriptional activation of HDAC1 is the primary mechanism underlying SOX4 maintaining CSCs. This finding suggests that HDAC1 is a potential drug target for eradicating SOX4-driven human CSCs.

Proteomic Analysis of Perihematoma Tissue from Patients with Intracerebral Hemorrhage Using iTRAQ-Based Quantitative Proteomics.

Intracerebral hemorrhage is a complicated disorder with limited proven prognostic and therapeutic targets and elusive mechanisms. With proteomic methods, we aimed to explore the global protein expression profile of perihematomal tissue from ICH patients and identify potential pathophysiological pathways and protein markers. Using iTRAQ-labeling quantitative proteomics technology, four ICH brain sample and four non-ICH brain samples were analyzed. Among the 3740 quantifiable proteins, 884 were dysregulated in the patients compared to those in the controls (p < 0.05). After bioinformatics analysis, the differentially expressed proteins were found to be mostly involved in hemostatic processes, nutrient metabolism signaling pathways, and antioxidation pathways. Moreover, fibronectin 1 was revealed to be at the center of the protein-protein interaction networks. In summary, the potential pathways and brain protein markers that could potentially be used to predict the prognosis of ICH were obtained from the altered proteomic profile of perihematomal tissue. Thus, these data may yield novel insights into the mechanisms of ICH-induced secondary brain injury.

Quantitative analysis of proteins secreted by Leishmania (Viannia) braziliensis strains associated to distinct clinical manifestations of American Tegumentary Leishmaniasis

The role of Leishmania braziliensis in the development of different clinical forms of American Tegumentary Leishmaniasis (ATL) is unclear, but it has been suggested that molecules secreted/released by parasites could modulate the clinical outcome. Here, we analyzed the infection rate and cytokine profile of macrophages pretreated with the secretome of two L. braziliensis strains associated with polar clinical forms of ATL: one associated with localized self-healing cutaneous leishmaniasis (LCL) and other associated with the disseminated form (DL). Besides, we use an iTRAQ-based quantitative proteomics approach to compare the abundance of proteins secreted by those strains. In vitro infection demonstrated that pretreatment with secretome resulted in higher number of infected macrophages, as well as higher number of amastigotes per cell. Additionally, macrophages pretreated with LCL secretome exhibited a proinflammatory profile, whereas those pretreated with the DL one did not. These findings suggest that secretomes made macrophages more susceptible to infection and that molecules secreted by each strain modulate, differentially, the macrophages' cytokine profile. Indeed, proteomics analysis showed that the DL secretome is rich in molecules involved in macrophage deactivation, while is poor in proteins that activate proinflammatory pathways. Together, our results reveal new molecules that may contribute to the infection, persistence and dissemination of the parasite. SignificanceLeishmania braziliensis is associated to localized self-healing cutaneous lesions (LCL), disseminated leishmaniasis (DL), and mucocutaneous lesions (MCL). To understand the role of the parasite in those distinct clinical manifestations we evaluated infection rates and cytokine profiles of macrophages pre-treated with secretomes of two L. braziliensis strains associated with DL and LCL, and quantitatively compared these secretomes. The infection index of macrophages pretreated with the DL secretome was significantly higher than that exhibited by non-treated cells. Interestingly, whereas the LCL secretome stimulated a proinflammatory setting, favoring an effector cell response that would explain the proper resolution of the disease caused by this strain, the DL strain was not able to elicit such response or has mechanisms to prevent this activation. Indeed, DL secretome is rich in peptidases that may deactivate cell pathways crucial for parasite elimination, while is poor in proteins that could activate proinflammatory pathways, favoring parasite infection and persistence.

Comparative proteomics of three Chinese potato cultivars to improve understanding of potato molecular response to late blight disease

BackgroundLate blight disease (LBD) caused by the pathogen Phytophthora infestans (PI), is the most devastating disease limiting potato (Solanum tuberosum) production globally. Currently, this disease pathogen is re-emerging and appearing in new areas at a very high intensity. A better understanding of the natural defense mechanisms against PI in different potato cultivars especially at the protein level is still lacking. Therefore, to elucidate potato proteome response to PI, we investigated changes in the proteome and leaf morphology of three potato cultivars, namely; Favorita (FA), Mira (MA), and E-malingshu N0.14 (E14) infected with PI by using the iTRAQ-based quantitative proteomics analysis.ResultsA total of 3306 proteins were found in the three potato genotypes, and 2044 proteins were quantified. Cluster analysis revealed MA and E14 clustered together separately from FA. The protein profile and related functions revealed that the cultivars shared a typical hypersensitive response to PI, including induction of elicitors, oxidative burst, and suppression of photosynthesis in the potato leaves. Meanwhile, MA and E14 deployed additional specific response mechanism different from FA, involving high induction of protease inhibitors, serine/threonine kinases, terpenoid, hormone signaling, and transport, which contributed to MA tolerance of LBD. Furthermore, inductions of pathogenesis-related proteins, LRR receptor-like kinases, mitogen-activated protein kinase, WRKY transcription factors, jasmonic acid, and phenolic compounds mediate E14 resistance against LBD. These proteins were confirmed at the transcription level by a quantitative polymerase chain reaction and at the translation level by western-blot.ConclusionsWe found several proteins that were differentially abundant among the cultivars, that includes common and cultivar specific proteins which highlighted similarities and significant differences between FA, MA, and E14 in terms of their defense response to PI. Here the specific accumulation of mitogen-activated protein kinase, Serine/threonine kinases, WRKY transcription played a positive role in E14 immunity against PI. The candidate proteins identified reported in this study will form the basis of future studies and may improve our understanding of the molecular mechanisms of late blight disease resistance in potato.

ITRAQ-based quantitative proteomics analysis reveals the molecular mechanism of controlled atmosphere storage for Tibetan hull-less barley (Hordeum vulgare L) preservation

Controlled atmosphere storage (CAS) is an effective method for cereal grain preservation, but its mechanism is yet to be completely elucidated. In this study, the molecular mechanism of CAS was evaluated for Tibetan hull-less barley preservation using iTRAQ quantitative proteomics, Western blot, and physicochemical and individual volatiles analyses. In barley stored at 25 °C, compared with non-controlled atmosphere storage (NCAS), CAS altered the expression of 116 proteins by > 2.0 fold. Among these proteins, 82 downregulated proteins were mainly involved in carbon metabolism, protein and amino acid metabolism, and lipid and fatty acid degradation. Physicochemical analyses suggest that CAS could reduce the severity of abiotic stress. Bioinformatic and molecular biology analyses reveal that CAS could decrease the rates of carbon metabolism, protein and amino acid metabolism, and lipid and fatty acid degradation by downregulating some key proteins (Beta-amylase, 6-phosphofructokinase 1, isocitrate dehydrogenase, glutamate decarboxylase, 1-pyrroline-5-carboxylate dehydrogenase, seryl-tRNA synthetase, leucyl-tRNA synthetase, aspartyl-tRNA synthetase, aldehyde dehydrogenase (NAD+), and alcohol dehydrogenase), accompanied by significant reductions in oxidative dehydrogenation reactions (p < 0.05). Together, a favorable environment provided by CAS ultimately mitigated physiological abnormalities, nutrient and energy consumption, and flavor deterioration in the barley. The proteomics analysis results were confirmed by Western blot, and physicochemical and individual volatiles analyses.

Tumor suppressor ATP4B serve as a promising biomarker for worsening of gastric atrophy and poor differentiation.

Hydrogen/potassium ATPase β (ATP4B) is a proton pump acting an essential role in gastric acid secretion. This study aimed to investigate the diagnostic performance of ATP4B and its biological role in tumor progression in gastric cancer. The correlations between ATP4B expression level and clinicopathologic parameters, as well as the relevance of ATP4B expression with overall survival were assessed. The functional roles of ATP4B in gastric cancer were verified by gain- and loss-of-function cell models and tumor xenograft models. The possible downstream effects of ATP4B were analyzed by iTRAQ-based quantitative proteomics analysis. A dramatic decrease in ATP4B was associated with malignant transformation in gastric mucosa lesions and correlated with poor differentiation. Restoration of ATP4B expression in gastric cancer cells significantly suppressed cell proliferation, cell viability, migration, invasion, tumorigenicity and induced apoptosis, whereas ATP4B silencing exerted the opposite effects. Mechanistically, we found a quality control on mitochondrial metabolism and functions in ATP4B-overexpression GC cells. Our data suggest that decreasing ATP4B is an indicator for gastric mucosa malignant transformation and GC aggressive phenotype and it plays an inhibitory role in gastric cancer as a tumor suppressor via regulating mitochondrial metabolism and apoptosis pathway.

Quantitative proteomics revealed energy metabolism pathway alterations in human epithelial ovarian carcinoma and their regulation by the antiparasite drug ivermectin: data interpretation in the context of 3P medicine.

Energy metabolism abnormality is the hallmark in epithelial ovarian carcinoma (EOC). This study aimed to investigate energy metabolism pathway alterations and their regulation by the antiparasite drug ivermectin in EOC for the discovery of energy metabolism pathway-based molecular biomarker pattern and therapeutic targets in the context of predictive, preventive, and personalized medicine (PPPM) in EOC. iTRAQ-based quantitative proteomics was used to identify mitochondrial differentially expressed proteins (mtDEPs) between human EOC and control mitochondrial samples isolated from 8 EOC and 11 control ovary tissues from gynecologic surgery of Chinese patients, respectively. Stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics was used to analyze the protein expressions of energy metabolic pathways in EOC cells treated with and without ivermectin. Cell proliferation, cell cycle, apoptosis, and important molecules in energy metabolism pathway were examined before and after ivermectin treatment of different EOC cells. In total, 1198 mtDEPs were identified, and various mtDEPs were related to energy metabolism changes in EOC, with an interesting result that EOC tissues had enhanced abilities in oxidative phosphorylation (OXPHOS), Kreb's cycle, and aerobic glycolysis, for ATP generation, with experiment-confirmed upregulations of UQCRH in OXPHOS; IDH2, CS, and OGDHL in Kreb's cycle; and PKM2 in glycolysis pathways. Importantly, PDHB that links glycolysis with Kreb's cycle was upregulated in EOC. SILAC-based quantitative proteomics found that the protein expression levels of energy metabolic pathways were regulated by ivermectin in EOC cells. Furthermore, ivermectin demonstrated its strong abilities to inhibit proliferation and cell cycle and promote apoptosis in EOC cells, through molecular networks to target PFKP in glycolysis; IDH2 and IDH3B in Kreb's cycle; ND2, ND5, CYTB, and UQCRH in OXPHOS; and MCT1 and MCT4 in lactate shuttle to inhibit EOC growth. Our findings revealed that the Warburg and reverse Warburg effects coexisted in human ovarian cancer tissues, provided the first multiomics-based molecular alteration spectrum of ovarian cancer energy metabolism pathways (aerobic glycolysis, Kreb's cycle, oxidative phosphorylation, and lactate shuttle), and demonstrated that the antiparasite drug ivermectin effectively regulated these changed molecules in energy metabolism pathways and had strong capability to inhibit cell proliferation and cell cycle progression and promote cell apoptosis in ovarian cancer cells. The observed molecular changes in energy metabolism pathways bring benefits for an in-depth understanding of the molecular mechanisms of energy metabolism heterogeneity and the discovery of effective biomarkers for individualized patient stratification and predictive/prognostic assessment and therapeutic targets/drugs for personalized therapy of ovarian cancer patients.

ITRAQ-Based Quantitative Proteomics Identifies AQP8, KLK11, and BCLAF1 as a Predictive Panel for Pancreatic Adenocarcinoma

iTRAQ-Based Quantitative Proteomics Identifies AQP8, KLK11, and BCLAF1 as a Predictive Panel for Pancreatic Adenocarcinoma

The iTRAQ-based quantitative proteomics reveals metabolic changes in Scylla paramamosain under different light intensities during indoor overwintering

Light intensity is one of the ecological factors that appreciably affects the metabolism of Scylla paramamosain during overwintering. This study adopted the isobaric tag for relative and absolute quantitation (iTRAQ) method to investigate metabolic changes of S. paramamosain under three illumination levels (0, 1.43 and 40.31 μmol m−2·s−1) for four months during indoor overwintering. The iTRAQ identified 3282 proteins, among which 267 exhibited significant differential expression (122 upregulated and 145 downregulated) in the low light group, and 299 with significant differential expression (252 upregulated and 47 downregulated) in the high light group. Analysis of these results showed that there were different metabolic regulatory patterns under different light intensities. Low light is more conducive to the survival of S. paramamosain, which needs to produce and consume relatively less energy to sustain physiological activities. Thus, the essential proteins associated with physiological activities were significantly upregulated, while those related to energy production were significantly downregulated. In contrast, high light exerts a certain stress on the survival of S. paramamosain and required more energy to cope with this stress, which forced a significant upregulation of proteins related to stress response and energy production. The findings of this study highlighted the metabolic regulatory mechanisms of S. paramamosain under different light intensities.

TonB-Dependent Receptors Affect the Spontaneous Oxytetracycline Resistance Evolution in Aeromonas hydrophila.

It is well known that most microbial populations develop their intrinsic antibiotics resistance at low concentrations in antibiotics environments, but the factors influencing spontaneous resistance are still largely unknown. In this study, Aeromonas hydrophila strains with different resistance levels to oxytetracycline (OXY) were induced by sublethal antibiotic selection pressure, and differential expression of proteins was compared among them using iTRAQ-based quantitative proteomics. Our following bioinformatic analysis showed that energy metabolism-related proteins were downregulated, while several iron-related proteins were upregulated in high OXY-resistant strains. To further investigate the role of spontaneous OXY resistance evolution, four TonB-dependent receptor-coded genes were deleted, and their OXY susceptibility capabilities and antibiotic evolutionary assays were performed, respectively. Our results showed that the deletion of these genes did not affect the susceptibility to OXY, but showed different evolution rates in the spontaneous OXY evolution compared with wild-type strain, especially for AHA_0971 and AHA_4251. Therefore, this study indicates the important role of TonB-dependent receptor proteins during the bacterial antibiotics resistance evolution and may provide a new prophylactic strategy against the development of antibiotic resistance.

ITRAQ-based quantitative proteomics analysis reveals inhibitory mechanismsof the antimicrobial peptide MDAP-2 against Salmonella gallinarum

MDAP-2 is a new AMP with high inhibitory activity on Salmonella gallinarum, which may be developed as an antimicrobial agent in the agricultural industry and food preservation. To investigate the underlying the action mechanism of MDAP-2 on Salmonella gallinarum, impacts of MDAP-2 on the growth curve and bacterial morphology of Salmonella gallinarum were studied. iTRAQ-based proteomics analysis was also performed on proteins extracted from treated and untreated Salmonella gallinarum cells. The differentially expressed proteins were then analyzed using the KEGG and GO databases. Finally, the function of some differentially expressed proteins was verified. The results showed that 150 proteins (41 up-regulated and 109 down-regulated) were found differentially expressed (fold > 1.8, p⟨0.05). The results indi- cate that MDAP-2 kills Salmonella gallinarum mainly through two mechanisms: (i) direct inhibi- tion of cell wall/ membrane/ envelope biogenesis, energy production/ conversion, carbohydrate transport/ metabolism, and DNA transcription/ translation through regulation of special protein levels; (ii) indirect effects on the same pathway through the accumulation of Reactive oxygen species (O2 ▪-, H2O2 and OH▪-).

ITRAQ and PRM-based comparative proteomic profiling in gills of white shrimp Litopenaeus vannamei under copper stress

Crustaceans are particularly sensitive to heavy metal pollution. Copper (Cu) is one of typical heavy metal pollutants in aquatic ecosystems. However, limited attention has been paid on the proteomic responses of shrimp under Cu stress. White shrimp Litopenaeus vannamei held in 5‰ seawater were exposed to 5 mg L−1 Cu for 3 h, and the regulatory mechanism in the gills was elucidated using iTRAQ-based quantitative proteomics. The results showed that a total of 5034 proteins were identified, 385 differentially expressed proteins (DEPs), including 147 differentially up-regulated proteins (DUPs) and 238 differentially down-regulated proteins (DDPs) were found. Bioinformatics analysis indicated the DEPs responding to Cu stress mainly involved in cytoskeleton, immune response, stress response, protein synthesis, detoxification, ion homeostasis and apoptosis. Furthermore, we still performed PRM analysis on sarcoplasmic calcium binding protein (SCP), serine proteinase inhibitor B3 (SPIB3), C-type lectin 4 (CTL4), cathepsin L (CATHL), JHE-like carboxylesterase 1 (CXE1) and paramyosin (PMY), and biochemical analysis on Cu/Zn-superoxide dismutase (Cu/Zn-SOD) to validate the iTRAQ results, respectively. The present proteome analysis revealed that Cu stress disrupted the ion homeostasis and protein synthesis, and L.vannamei mainly regulates a series of molecular pathways which contained many key proteins involved in the immune process to protect the organism from Cu stress. Our data provides more insight about the underlying mechanisms that related to the stress response of Cu exposure in crustacean.

Inhibition of orf virus replication in goat skin fibroblast cells by the HSPA1B protein, as demonstrated by iTRAQ-based quantitative proteome analysis.

Orf virus (ORFV) infects sheep and goat tissues, resulting in severe proliferative lesions. To analyze cellular protein expression in ORFV-infected goat skin fibroblast (GSF) cells, we used two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ). The proteomics approach was used along with quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect differentially expressed proteins in ORFV-infected GSF cells and mock-infected GSF cells. A total of 282 differentially expressed proteins were identified. It was found that 222 host proteins were upregulated and 60 were downregulated following viral infection. We confirmed that these proteins were differentially expressed and found that heat shock 70-kDa protein 1B (HSPA1B) was differentially expressed and localized in the cytoplasm. It was also noted that HSPA1B caused inhibition of viral proliferation, in the middle and late stages of viral infection. The differentially expressed proteins were associated with the biological processes of viral binding, cell structure, signal transduction, cell adhesion, and cell proliferation.

Functional characterization and regulatory mechanism of wheat CPK34 kinase in response to drought stress

BackgroundDrought is one of the most adverse environmental factors limiting crop productions and it is important to identify key genetic determinants for food safety. Calcium-dependent protein kinases (CPKs) are known to be involved in plant growth, development, and environmental stresses. However, biological functions and regulatory mechanisms of many plant CPKs have not been explored. In our previous study, abundance of the wheat CPK34 (TaCPK34) protein was remarkably upregulated in wheat plants suffering from drought stress, inferring that it could be involved in this stress. Therefore, here we further detected its function and mechanism in response to drought stress.ResultsTranscripts of the TaCPK34 gene were significantly induced after PEG-stimulated water deficiency (20% PEG6000) or 100 μM abscisic acid (ABA) treatments. The TaCPK34 gene was transiently silenced in wheat genome by using barley stripe mosaic virus-induced silencing (BSMV-VIGS) method. After 14 days of drought stress, the transiently TaCPK34-silenced wheat seedlings showed more sensitivity compared with control, and the plant biomasses and relative water contents significantly decreased, whereas soluble sugar and MDA contents increased. The iTRAQ-based quantitative proteomics was employed to measure the protein expression profiles in leaves of the transiently TaCPK34-silenced wheat plants after drought stress. There were 6103 proteins identified, of these, 51 proteins exhibited significantly altered abundance, they were involved in diverse function. And sequence analysis on the promoters of genes, which encoded the above identified proteins, indicated that some promoters harbored some ABA-responsive elements. We determined the interactions between TaCPK34 and three identified proteins by using bimolecular fluorescent complementation (BiFC) method and our data indicated that TaCPK34directly interacted with the glutathione S-transferase 1 and prx113, respectively.ConclusionsOur study suggested that the TaCPK34 gene played positive roles in wheat response to drought stress through directly or indirectly regulating the expression of ABA-dependent manner genes, which were encoding identified proteins from iTRAQ-based quantitative proteomics. And it could be used as one potential gene to develop crop cultivars with improved drought tolerance.

Construction of a Conditionally Asporogenous Bacillus thuringiensis Recombinant Strain Overproducing Cry Protein by Deletion of the leuB Gene.

One of the common shortcomings with Bacillus thuringiensis (Bt) biopesticides in field application is their instability under UV irradiation. In Bt, the leuB gene encodes the 3-isopropylmalate dehydrogenase. In addition to its role in leucine biosynthesis, LeuB would be likely recruited to catalyze the dehydrogenation of malate in the final step of tricarboxylic acid cycle during sporulation. In this study, we constructed a Bt recombinant strain in which the gene leuB was deleted by using the markerless gene deletion system. The ΔleuB mutant strain showed a conditionally asporogenous phenotype while overproducing insecticidal crystal proteins and retaining its insecticidal activity well in both fermentation and LB media. Furthermore, the metabolic regulation mechanisms of LeuB was elucidated by iTRAQ-based quantitative proteomics approach. Evidences from proteomics data suggested that the inhibited supply of pyruvate (carbon source) was an important factor related to the conditionally asporogenous feature of the mutant. Consistently, the mutant regained its ability to sporulate in LB medium by adding 1% glucose or 1% sodium pyruvate. Taken together, our study demonstrated that deletion of the leuB gene resulted in delayed or completely blocked mother cell lysis, allowing the crystals encapsulated within cells, which makes this recombinant strain a good candidate for developing Bt preparations with better UV-stability.

Integration of metabolomics and proteomics to highlight altered neural development related pathways in the adult offspring after maternal folic acid supplement.

Maternal folic acid (FA) supplement (FolS) programs the early development of an offspring. The onset of complex diseases at a later stage of life has been evidently linked with maternal FA ingestion. However, little is known regarding the underlying molecule fingerprints of the offspring. Here, we analyze the influence of maternal FolS on the metabolism of the adult offspring rats using the integrated metabolomics-proteomics. Twenty pregnant female rats were randomly assigned to a FA supplement (FolS group) or control group which were fed AIN93G diet with 2 or 5mg/kg FA, respectively. The blood samples from the offspring at 0, 3 and 7 weeks after birth were collected. The brain samples were obtained from the offspring at 7 weeks after birth. Serum and brain metabolite profiles were performed by UPLC-MS/MS and the brain proteomics analysis was obtained using iTRAQ-based quantitative proteomics. The metabolic change of the offspring for the maternal FA supplement is characterized by the phospholipids, fatty acid and amino acids, which are involved in linoleic acid, docosahexaenoic acid, glycerophosphocholine, lysophosphatidylcholine, tryptophan, glycine, arachidonic acid, γ-aminobutyric acid, and so on. Using iTRAQ-based quantitative proteomics analysis, 51 differential proteins in the brain are identified, which provides valuable insight into the underlying mechanisms of the offspring after the maternal FolS. These results demonstrate neural development related metabolites and proteins, such as docosahexaenoic acid, glycine, tryptophan, γ-aminobutyric acid, dopaminergic synapse related proteins including G protein, PPP1R1B and CAMK2G, are significantly altered, which suggests that the active neural conduction occurs in the offspring after maternal FA supplement. The behavioral testing demonstrates that the high level of memory is observed in rats with FA supplement. We conceive that the alterations of metabolites and protein in the offspring are associated with the maternal FA supplement and these alterations are involved in the neural development, although such animal data are limited in their ability to mimic metabolic outcomes in humans.

ITRAQ-based Quantitative Proteomics Analysis Identifies Host Pathways Modulated during Toxoplasma gondii Infection in Swine.

Toxoplasma gondii is a leading cause of foodborne illness and consumption of undercooked pig meat is a major risk factor for acquiring toxoplasmosis, which causes a substantial burden on society. Here, we used isobaric tags for relative and absolute quantification (iTRAQ) labelling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify cellular proteins and pathways altered during T. gondii infection in pigs. We also used parallel reaction monitoring-based LC-MS/MS to verify the levels of protein expression of infected spleens and mesenteric lymph nodes (MLNs). At 6 days post-infection (dpi), 156, 391, 170, 292, and 200 differentially expressed proteins (DEPs) were detected in the brain, liver, lung, MLNs and spleen, respectively. At 18 dpi, 339, 351, 483, 388, and 303 DEPs were detected in the brain, liver, lung, MLNs and spleen, respectively. Although proteins involved in immune responses were upregulated in all infected tissues, protein expression signature in infected livers was dominated by downregulation of the metabolic processes. By weighted gene co-expression network analysis, we could further show that all proteins were clustered into 25 co-expression modules and that the pink module significantly correlated with the infection status. We also identified 163 potential anti-T. gondii proteins (PATPs) and provided evidence that two PATPs (HSP70.2 and PDIA3) can reduce T. gondii burden in porcine macrophages in vitro. This comprehensive proteomics analysis reveals new facets in the pathogenesis of T. gondii infection and identifies key proteins that may contribute to the pig’s defense against this infection.

Proteomics Analysis Reveals that Warburg Effect along with Modification in Lipid Metabolism Improves In Vitro Embryo Development under Low Oxygen.

The molecular mechanism regulating embryo development under reduced oxygen tension remains elusive. This study aimed to identify the molecular mechanism impacting embryo development under low oxygen conditions. Buffalo embryos were cultured under 5% or 20% oxygen and were evaluated according to their morphological parameters related to embryo development. The protein profiles of these embryos were compared using iTRAQ-based quantitative proteomics. Physiological O2 (5%) significantly promoted blastocyst yield, hatching rate, embryo quality and cell count as compared to atmospheric O2 (20%). The embryos in the 5% O2 group had an improved hatching rate of cryopreserved blastocysts post-warming (p < 0.05). Comparative proteome profiles of hatched blastocysts cultured under 5% vs. 20% O2 levels identified 43 differentially expressed proteins (DEPs). Functional analysis indicated that DEPs were mainly associated with glycolysis, fatty acid degradation, inositol phosphate metabolism and terpenoid backbone synthesis. Our results suggest that embryos under physiological oxygen had greater developmental potential due to the pronounced Warburg Effect (aerobic glycolysis). Moreover, our proteomic data suggested that higher lipid degradation, an elevated cholesterol level and a higher unsaturated to saturated fatty acid ratio might be involved in the better cryo-survival ability reported in embryos cultured under low oxygen. These data provide new information on the early embryo protein repertoire and general molecular mechanisms of embryo development under varying oxygen levels.

ITRAQ-based quantitative proteomics reveals a ferroptosis-like programmed cell death in plants infected by a highly virulent tobacco mosaic virus mutant 24A+UPD

Plants trigger a highly orchestrated defence mechanism in response to viral infection. In this study, we aimed at understanding the molecular events that lead to more accelerated cell death in Nicotiana benthamiana plants infected with the fast-replicating TMV 24A + UPD in comparison to TMV. TMV 24A + UPD is an artificial mutant that induces more severe symptoms leading to precocious death in plants. We employed the iTRAQ-based quantitative proteomics approach to identify and map the proteomes of TMV and TMV 24A + UPD infected plants at time points that correlate with initiation of early cell death symptoms. TMV 24A + UPD proteome profile revealed 183 highly abundant proteins versus 71 for TMV infected plants. KEGG analysis revealed differentially abundant proteins in the two proteome profiles under cell death, stress signalling, protein folding, sorting, degradation, transport and catabolism. We identified unique differentially abundant proteins in the TMV 24A + UPD profile, in particular under the ferroptosis and glutathione metabolism pathways. For validation, we varied the amount of intracellular iron by supplementing plants with Fe3+, employing iron chelators and by virus induced gene silencing of iron storage protein ferritin gene. We also employed potent ferroptosis inhibitors ferostatin-1, liprostatin-1, and transiently silenced glutathione peroxidase 4 gene. TMV 24A + UPD infected plants showed accelerated cell death symptoms when intracellular iron was increased. Decreasing intracellular iron protected the plants from accelerated cell death. We also observed a decrease in TMV 24A + UPD induced cell death when we applied ferroptosis inhibitors. Glutathione peroxidase 4 gene-silenced plants showed enhanced cell death compared to non-silenced control plants. Our study uncovered a link between intracellular iron and accelerated lipid ROS-induced cell death in TMV 24A + UPD infected plants. We propose that the fast-replicating mutant of TMV induces a distinct and potent form of cell death akin to ferroptosis.

ITRAQ-based quantitative proteomics and target-fishing strategies reveal molecular signatures on vasodilation of Compound Danshen Dripping Pills

Angina pectoris can be used as an early warning for coronary artery disease. Vasodilation is an important mechanism of angina pectoris. Traditional Chinese medicine - Compound Danshen Dripping Pill (CDDP) is widely used to improve the symptoms of cardiovascular diseases (CVDs). To investigate the influence of vasodilation effect and underlying mechanisms of CDDP, we determined the vasodilation effect of thoracic aorta ring on rat induced by norepinephrine (NE). Then targets-fishing method was used to predict the potential mechanism of CDDP on vasodilation, based on the structures of the main components. Then, iTRAQ-based quantitative proteomics analysis was used for verification of the candidate target proteins and pathways to illustrate the underlying mechanisms. Furthermore, the differentially expressed proteins in the enriched pathways were validated by western blotting. In this study, we found that CDDP could significantly inhibit NE induced aortic contraction tension, and the mechanism may be related to platelet activation, cGMP – PKG signaling pathway and vascular smooth muscle contraction. The method provides a new way to uncover the vasodilation mechanism of CDDP, as well as other multi-component herbal medicines.