Serum proteins differentially expressed in gestational diabetes mellitus assessed using isobaric tag for relative and absolute quantitation proteomics

BACKGROUND As a well-known fact to the public,gestational diabetes mellitus(GDM)could bring serious risks for both pregnant women and infants.During this important investigation into the linkage between GDM patients and their altered expression in the serum,proteomics techniques were deployed to detect the differentially expressed proteins(DEPs)of in the serum of GDM patients to further explore its pathogenesis,and find out possible biomarkers to forecast GDM occurrence.METHODS Subjects were divided into GDM and normal control groups according to the IADPSG diagnostic criteria.Serum samples were randomly selected from four cases in each group at 24-28 wk of gestation,and the blood samples were identified by applying iTRAQ technology combined with liquid chromatography-tandem mass spectrometry.Key proteins and signaling pathways associated with GDM were identified by bioinformatics analysis,and the expression of key proteins in serum from 12 wk to 16 wk of gestation was further verified using enzyme-linked immunosorbent assay (ELISA).RESULTS Forty-seven proteins were significantly differentially expressed by analyzing the serum samples between the GDMgravidas as well as the healthy ones. Among them, 31 proteins were found to be upregulated notably and the rest16 proteins were downregulated remarkably. Bioinformatic data report revealed abnormal expression of proteinsassociated with lipid metabolism, coagulation cascade activation, complement system and inflammatory responsein the GDM group. ELISA results showed that the contents of RBP4, as well as ANGPTL8, increased in the serumof GDM gravidas compared with the healthy ones, and this change was found to initiate from 12 wk to 16 wk ofgestation.CONCLUSION GDM symptoms may involve abnormalities in lipid metabolism, coagulation cascade activation, complementsystem and inflammatory response. RBP4 and ANGPTL8 are expected to be early predictors of GDM.

Isobaric Tag for Relative and Absolute Quantitation-Based Proteomics for Investigating the Effect of Guasha on Lumbar Disc Herniation in Rats

Objective:The objective of the study is to examine the possible mechanism by which Guasha(scraping therapy)affects the expression profiles of proteins in a lumbar disc herniation(LDH)rat model using isobaric tags for relative and absolute quantitation(iTRAQ)-based proteomics.Methods:Thirty-six rats were used in this study.LDH rats were subjected to noncompressive LDH surgeries.Rats were randomly divided into the model and Guasha groups.Guasha was applied on alternate days for a total of nine times(three courses).At the end of each course,six rats were randomly selected from each group and their blood samples were collected.iTRAQ labeling was used to examine the mechanism of action of Guasha against LDH.The molecular functions,cellular components,and biological processes were analyzed using gene ontology analysis.The Ingenuity Pathway Analysis database was used to identify canonical pathways involving these proteins.Results:Compared to the model group,198,182,and 170 proteins were identified as differentially expressed at the three respective Guasha treatment courses.Pathways,including focal adhesion kinase signaling,acute-phase response signaling,and the LXR/RXR activation pathway,were closely related to the effects of Guasha in LDH rats.Furthermore,Rac1,Orm1,and Hpx were validated by western blotting,and the results were consistent with the protein expression levels observed using the iTRAQ method.Conclusion:Guasha could not only regulate the pathological changes related to LDH,but also achieve therapeutic effects by stimulating physiological changes.Our results offer a better understanding of the effects of Guasha on LDH.

Proteomic analysis of trans-hemispheric motor cortex reorganization following contralateral C7 nerve transfer

Nerve transfer is the most common treatment for total brachial plexus avulsion injury. After nerve transfer, the movement of the injured limb may be activated by certain movements of the healthy limb at the early stage of recovery, i.e., trans-hemispheric reorganization. Pre- vious studies have focused on functional magnetic resonance imaging and changes in brain-derived neurotrophic factor and growth asso- ciated protein 43, but there have been no proteomics studies. In this study, we designed a rat model of total brachial plexus avulsion injury involving contralateral C7 nerve transfer. Isobaric tags for relative and absolute quantitation and western blot assay were then used to screen differentially expressed proteins in bilateral motor cortices. We found that most differentially expressed proteins in both cortices of upper limb were associated with nervous system development and function （including neuron differentiation and development, axonogenesis, and guidance）, microtubule and cytoskeleton organization, synapse plasticity, and transmission of nerve impulses. Two key differentially expressed proteins, neurofilament light （NFL） and Thy-1, were identified. In contralateral cortex, the NFL level was upregulated 2 weeks after transfer and downregulated at 1 and 5 months. The Thy-1 level was upregulated from 1 to 5 months. In the affected cortex, the NFL level increased gradually from 1 to 5 months. Western blot results of key differentially expressed proteins were consistent with the proteom- ic findings. These results indicate that NFL and Thy-1 play an important role in trans-hemispheric organization following total brachial plexus root avulsion and contralateral C7 nerve transfer.

Inflammation and apoptosis accelerate progression to irreversible atrophy in denervated intrinsic muscles of the hand compared with biceps: proteomic analysis of a rat model of obstetric brachial plexus palsy

In treating patients with obstetric brachial plexus palsy,we noticed that denervated intrinsic muscles of the hand become irreversibly atrophic at a faster than denervated biceps.In a rat model of obstetric brachial plexus palsy,denervated intrinsic musculature of the forepaw entered the irreversible atrophy far earlier than denervated biceps.In this study,isobaric tags for relative and absolute quantitation were examined in the intrinsic musculature of forepaw and biceps on denervated and normal sides at 3 and 5 weeks to identify dysregulated proteins.Enrichment of pathways mapped by those proteins was analyzed by Kyoto Encyclopedia of Genes and Genomes analysis.At 3 weeks,119 dysregulated proteins in denervated intrinsic musculature of the forepaw were mapped to nine pathways for muscle regulation,while 67 dysregulated proteins were mapped to three such pathways at 5 weeks.At 3 weeks,27 upregulated proteins were mapped to five pathways involving inflammation and apoptosis,while two upregulated proteins were mapped to one such pathway at 5 weeks.At 3 and 5 weeks,53 proteins from pathways involving regrowth and differentiation were downregulated.At 3 weeks,64 dysregulated proteins in denervated biceps were mapped to five pathways involving muscle regulation,while,five dysregulated proteins were mapped to three such pathways at 5 weeks.One protein mapped to inflammation and apoptotic pathways was upregulated from one pathway at 3 weeks,while three proteins were downregulated from two other pathways at 5 weeks.Four proteins mapped to regrowth and differentiation pathways were upregulated from three pathways at 3 weeks,while two proteins were downregulated in another pathway at 5 weeks.These results implicated inflammation and apoptosis as critical factors aggravating atrophy of denervated intrinsic muscles of the hand during obstetric brachial plexus palsy.All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Fudan University,China(approval No.DF-325)in January 2015.

Effects of Space Flight on Expression of Key Proteins in Rice Leaves

As a unique form of abiotic stress, the environmental conditions of outer space are expected to induce changes in plant genomes, proteomes and metabolic pathways. However, the effect of outer space conditions on the overall physiology of plants at the protein level has yet to be reported. To investigate the effects of outer space conditions on the growth-and development-related physiological processes and metabolic pathways of rice different stages, the seeds of rice variety DN423 were sent into orbit for 12.5 d aboard the SJ-10 Returning Satellite, and then the seedlings of both treated and control rice were compared at the three-leaf stage(TLS) and tillering stage(TS). In addition to comparing plant growth and reactive oxygen species(ROS) levels, seedling proteomes were also compared using isobaric tags for relative and absolute quantitation(i TRAQ). Space flight increased TLS plant height by 20%, reduced and increased ROS levels of the TLS and TS seedlings, respectively, and affected the expression of 36 and 323 proteins in TLS and TS leaves, respectively. Furthermore, the functions of the differentially abundant proteins were mainly associated with metabolism, energy, and protein synthesis and degradation. These results suggested that the exposure of seeds to outer space conditions affects the subsequent abundance of key signaling proteins, gene expression, and the processes of protein synthesis and degradation, thereby affecting metabolic processes and promoting adaptation to the abiotic stress of outer space. As such, the present study sheds light on the effects of space flight on plants and contributes to a more comprehensive understanding of extraterrestrial biology.

Immune Priming Induced by Heat-inactivated White Spot Syndrome Virus (WSSV) in Fenneropenaeus chinensis Cultured at Different Temperature

[ Objective] This study was designed to explore whether heat-inactivated white spot syndrome virus （ WSSV ） could induce immune priming in Fenneropenaeus chinensis. [Method] Shrimps cultured at 15 （group E15℃ ）, 23 （group E23℃ ）, 28 （group E28℃ ） and 32 （group E32℃ ）℃ were fed for six successive days with the meat of WSSV-infected shrimps, which had been treated at 60℃ for 1 h. Meanwhile, the shrimps of positive control were fed with a diet containing active WSSV at 23℃ （ group C23℃ ）, and the shrimps of negative control were fed with commercial compound feed only at 23℃ （ group CF23℃ ）. On Day 13 of the experiment, the survivors were re-challenged with active WSSV, and then the survival time, survival rate, mortality rate and viral load in each group were measured. The results showed that no death occurred among the shrimps fed with a diet containing heat-inactivated WSSV before the secondary challenge, while all the shrimps fed with a diet containing active WSSV （ group CF23℃ ） died, suggesting that WSSV was completely inactivated by treatment at 60℃ for 1 h. On day 19 of the experiment, the survival rate in groups E15℃ , E23℃, E28℃, E32℃ and CF23℃ was 80.41% , 33.29%, 8.47% , 16.43% and 8.89%, respectively. There was an extremely significant difference in survival rate between group E15℃ and other groups （ P 〈 0.01 ）, a significant difference in survival rate between group E23℃ and CF23℃ , E28℃, E32℃ （P 〈 0. 05）, and no significant difference in survival rate between groups E28℃ and E32℃ （ P 〉 0. 05 ）. RT-qPCR revealed WSSV replicated most rapidly at 28℃ after the secondary challenge, while high （32℃） and low （ 15℃ ） temperature inhibited the multiplication of WSSV. In summary, heat-inactivated WSSV can induce immune priming response and provide some protection to shrimps against WSSV infection. The multiplication rate of WSSV is closely related to water temperature.

Effects of electroacupuncture on rats with cognitive impairment:An iTRAQ-based proteomics analysis

Objective The study explores the effects of electroacupuncture(EA)at the governing vessel(GV)on proteomic changes in the hippocampus of rats with cognitive impairment.Methods Healthy male rats were randomly divided into 3 groups:sham,model and EA.Cognitive impairment was induced by left middle cerebral artery occlusion in the model and EA groups.Rats in the EA group were treated with EA at Shenting(GV24)and Baihui(GV20)for 7 d.Neurological deficit was scored using the Longa scale,the learning and memory ability was detected using the Morris water maze(MWM)test,and the proteomic profiling in the hippocampus was analyzed using protein-labeling technology based on the isobaric tag for relative and absolute quantitation(iTRAQ).The Western blot(WB)analysis was used to detect the proteins and validate the results of iTRAQ.Results Compared with the model group,the neurological deficit score was significantly reduced,and the escape latency in the MWM test was significantly shortened,while the number of platform crossings increased in the EA group.A total of 2872 proteins were identified by iTRAQ.Differentially expressed proteins(DEPs)were identified between different groups:92 proteins were upregulated and 103 were downregulated in the model group compared with the sham group,while 142 proteins were upregulated and 126 were downregulated in the EA group compared with the model group.Most of the DEPs were involved in oxidative phosphorylation,glycolipid metabolism and synaptic transmission.Furthermore,we also verified 4 DEPs using WB technology.Although the WB results were not exactly the same as the iTRAQ results,the expression trends of the DEPs were consistent.The upregulation of heat-shock proteinβ1(Hspb1)was the highest in the EA group compared to the model group.Conclusion EA can effect proteomic changes in the hippocampus of rats with cognitive impairment.Hspb1 may be involved in the molecular mechanism by which acupuncture improves cognitive impairment.

Lactobacillus gasseri LA39 promotes hepatic primary bile acid biosynthesis and intestinal secondary bile acid biotransformation

A growing body of evidence has linked the gut microbiota to liver metabolism.The manipulation of intestinal microflora has been considered as a promising avenue to promote liver health.However,the effects of Lactobacillus gasseri LA39,a potential probiotic,on liver metabolism remain unclear.Accumulating studies have investigated the proteomic profile for mining the host biological events affected by microbes,and used the germ-free(GF)mouse model to evaluate host-microbe interaction.Here,we explored the effects of L.gasseri LA39 gavage on the protein expression profiles of the liver of GF mice.Our results showed that a total of 128 proteins were upregulated,whereas a total of 123 proteins were downregulated by treatment with L.gasseri LA39.Further bioinformatics analyses suggested that the primary bile acid(BA)biosynthesis pathway in the liver was activated by L.gasseri LA39.Three differentially expressed proteins(cytochrome P450 family 27 subfamily A member 1(CYP27A1),cytochrome P450 family 7 subfamily B member 1(CYP7B1),and cytochrome P450 family 8 subfamily B member 1(CYP8B1))involved in the primary BA biosynthesis pathway were further validated by western blot assay.In addition,targeted metabolomic analyses demonstrated that serum and fecalβ-muricholic acid(a primary BA),dehydrolithocholic acid(a secondary BA),and glycolithocholic acid-3-sulfate(a secondary BA)were significantly increased by L.gasseri LA39.Thus,our data revealed that L.gasseri LA39 activates the hepatic primary BA biosynthesis and promotes the intestinal secondary BA biotransformation.Based on these findings,we suggest that L.gasseri LA39 confers an important function in the gut‒liver axis through regulating BA metabolism.

Study on the Application of iTRAQ Technology in Retinal Apoptosis Protein in Diabetic Retinopathy Mice

Objective:Using isobaric tags for relative and absolute quantitation(i TRAQ)technology to study differential protein expression in the retinal tissue of DR mouse models,providing proteomic evidence at the protein level for the pathogenesis of DR.Methods:Firstly,establish diabetic mice and DR mice models,use i TRAQ technology to detect the retinal tissue samples of normal control group and DR model group mice,label the total retinal proteins of mice with i TRAQ reagent,and analyze them using mass spectrometry technology.Evaluate the differential proteins of the two groups using BioWorks TM 3.0 software,and conduct interaction feature analysis on the different proteins using the STRING website.Results:The i TRAQ technology detected a total of 406 different proteins between the diabetes and normal control groups,with 19 of them closely related to retinal cell apoptosis.Among them,significantly different proteins include acyl-CoA dehydrogenase short chain specific(ACADS),ataxin-10(ATXN10),BCL-2-associated X protein(BAX),caspase-3(CASP3),collagen type IIα1 chain(COL4A2),glycyl-tRNA synthetase(GARS),glial fibrillary acidic protein(GFAP),legumain(LGMN),mucin-4(MUC4),N-myc downstream-regulated gene 1 protein(NDRG1),with ratios to internal controls in the normal group of 1.67,2.06,1.76,2.16,1.53,1.87,1.24,1.61,0.42,0.56,respectively;and there is a potential functional association between GFAP,CASP3,and BAX proteins.Conclusion:In DR mice retinas,there are abnormal changes in the expression of a large number of apoptosis-related proteins.i TRAQ technology can effectively screen out key apoptosis proteins,among which GFAP,CASP3,and BAX may have adverse effects on the progression of DR by participating in the apoptosis process.The application of i TRAQ technology can provide new technical support for proteomic research on apoptosis proteins.

Cardiac proteomics reveals the potential mechanism of microtubule associated protein 4 phosphorylation-induced mitochondrial dysfunction

Background:Our previous work suggested that microtubule associated protein 4(MAP4)phosphorylation led to mitochondrial dysfunction in MAP4 phosphorylation mutant mice with cardiomyopathy,but the detailed mechanism was still unknown.Thus,the aim of this study was to investigate the potential mechanism involved in mitochondrial dysfunction responsible for cardiomyopathy.Methods:The present study was conducted to explore the potential mechanism underlying the mitochondrial dysfunction driven by MAP4 phosphorylation.Strain of mouse that mimicked constant MAP4 phosphorylation(S737 and S760)was generated.The isobaric tag for relative and absolute quantitation(iTRAQ)analysis was applied to the heart tissue.Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),and protein-protein interaction(PPI)were all analyzed on the basis of differential expressed proteins(DEPs).Results:Among the 72 cardiac DEPs detected between the two genotypes of mice,12 were upregulated and 60 were downregulated.GO analysis showed the biological process,molecular function,and cellular component of DEPs,and KEGG enrichment analysis linked DEPs to 96 different biochemical pathways.In addition,the PPI network was also extended on the basis of DEPs as the seed proteins.Three proteins,including mitochondrial ubiquitin ligase activator of NF-κB 1,reduced form of nicotinamide adenine dinucleotide(NADH)-ubiquinone oxidoreductase 75 kDa subunit,mitochondrial and growth arrest,and DNA-damage-inducible proteins-interacting protein 1,which play an important role in the regulation of mitochondrial function,may correlate with MAP4 phosphorylationinduced mitochondrial dysfunction.Western blot was used to validate the expression of the three proteins,which was consistent with iTRAQ experiments.Conclusions:These findings revealed that the DEPs caused by MAP4 phosphorylation in heart tissue using iTRAQ technique and may provide clues to uncover the potential mechanism of MAP4 phosphorylation-induced mitochondrial dysfunction.

Quantitative Proteomic Studies on TNBC in Premenopausal Patients

The molecular mechanism of triple-negative breast cancer（TNBC） remains unclear, and there has been no effective targeted therapy for it. A better understanding of the mechanisms of TNBC is urgently needed to identify new therapeutic targets. In this study, eight cases of premenopausal TNBC patients were collected, and a comparative proteomic analysis of their breast cancer tissues and matched paraneoplastic ones was performed via isobaric tags for relative and absolute quantitation（iTRAQ） technology coupled with two-dimensional liquid chromatography-tandem mass spectrumetry（2D LC-MS/MS）. The researches result in the identification of 1254 nonredundant proteins, of which 1243 proteins reached the strict quantitative standard. The quantitative comparison reveal that among the 214 proteins, 81 proteins significantly increased and 133 proteins decreased in TNBC tissues compared to corresponding ones in control. The Gene Ontology（GO） annotations and pathway analysis show their distributions in GO and the marked functions, as well as the closely related signal transduction pathways involved in extra cellular matrix （ECM）-receptor interaction, protein digestion and absorption, renin-angiotensin system, complement and coagulation cascades and focal adhesion. This pilot study will lay a foundation for further searching for therapeutic targets of TNBC and exploring the molecular mechanism, which can also be extended as a part of a large scale biomarker discovery plan.