Proteomic study of vitreous in proliferative diabetic retinopathy patients after treatment with aflibercept:a quantitative analysis based on 4D label-free technique

AIM:To identify different metabolites,proteins and related pathways to elucidate the causes of proliferative diabetic retinopathy(PDR)and resistance to anti-vascular endothelial growth factor(VEGF)drugs,and to provide biomarkers for the diagnosis and treatment of PDR.METHODS:Vitreous specimens from patients with diabetic retinopathy were collected and analyzed by Liquid Chromatography-Mass Spectrometry(LC-MS/MS)analyses based on 4D label-free technology.Statistically differentially expressed proteins(DEPs),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway representation and protein interactions were analyzed.RESULTS:A total of 12 samples were analyzed.The proteomics results showed that a total of 58 proteins were identified as DEPs,of which 47 proteins were up-regulated and 11 proteins were down-regulated.We found that C1q and tumor necrosis factor related protein 5(C1QTNF5),Clusterin(CLU),tissue inhibitor of metal protease 1(TIMP1)and signal regulatory protein alpha(SIRPα)can all be specifically regulated after aflibercept treatment.GO functional analysis showed that some DEPs are related to changes in inflammatory regulatory pathways caused by PDR.In addition,protein-protein interaction(PPI)network evaluation revealed that TIMP1 plays a central role in neural regulation.In addition,CD47/SIRPαmay become a key target to resolve anti-VEGF drug resistance in PDR.CONCLUSION:Proteomic analysis is an approach of choice to explore the molecular mechanisms of PDR.Our data show that multiple proteins are differentially changed in PDR patients after intravitreal injection of aflibercept,among which C1QTNF5,CLU,TIMP1 and SIRPαmay become targets for future treatment of PDR and resolution of anti-VEGF resistance.

4D label-free proteomic analysis of vitreous from patients with rhegmatogenous retinal detachment

AIM:To identify metabolites,proteins,and related pathways involved in the etiology of rhegmatogenous retinal detachment(RRD)for use as biomarkers in diagnosing and treating RRD.METHODS:Vitreous specimens were collected and liquid chromatography-tandem mass spectrometry analysis was per formed using the four-dimensional label-free technique.Statistically significant differentially expressed proteins,gene ontology(GO)terms,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway representations,and protein interactions were analyzed.RESULTS:Nine specimens were subjected to proteomic analysis.In total,161 proteins were identified as differentially expressed proteins(DEPs),including 53 upregulated proteins and 108 downregulated proteins.GO functional analysis revealed that some DEPs were enriched in neuron-related terms and membrane protein terms.Moreover,KEGG analysis indicated that the cell adhesion molecule metabolic pathway was associated with the greatest number of DEPs.Finally,the evaluation of protein-protein interaction network revealed that DEPs were clustered in neuronal adhesion,apoptosis,inflammation and immune responses,correct protein folding,and glycolysis.CONCLUSION:Proteomic profiling is useful for the exploration of molecular mechanisms that underlie RRD.This study reveals increased expression levels of proteins related to heat shock protein content,glycolysis,and inflammatory responses in RRD.Knowledge regarding biomarkers of RRD pathogenesis may help to prevent the occurrence of RRD in the future.

Stigma-Specific Comparative Proteomic Analysis Reveals the Distyly Response to Self-Incompatibility in Plumbago auriculata Lam

In plants,heteromorphic self-incompatibility(HetSI)is a strategy for avoiding self-pollination and promoting outcrossing,and during this process,numerous protein-protein interaction events occur between the pistil and pollen.Previous studies in Primula and Fagopyrum that focused on HetSI systems have provided interesting insights;however,the molecular mechanism underlying HetSI remains largely unknown.In this study,we profiled the proteome of Plumbago auriculata stigmas before and after self-incompatible(SI)and self-compatible(SC)pollination.Comparative analyses were conducted by 4D-DIA(Four-dimensional data independent acquisition),a promising technology that increases the sensitivity and reduces the spectral complexity of proteomic analysis by adding a fourth dimension,ion mobility.The results revealed 33387 peptides and 5311 proteins in all samples.The pathways in which the differentially expressed proteins(DEPs)identified in the P×P(Pin style self-pollinated with pin pollen)vs.PS(Pin style)and T×T(Thrum style self-pollinated with thrum pollen)vs.TS(Thrum style)comparisons were significantly enriched were biosynthesis of secondary metabolites and pentose and glucuronate interconversions.In the P×T(Pin style cross-pollinated with thrum pollen)vs.PS and T×P(Thrum style cross-pollinated with pin pollen)vs.TS comparison,the top three pathways were biosynthesis of secondary metabolites,pentose and glucuronate interconversions,and phenylpropanoid biosynthesis.The phenylpropanoid biosynthesis,cutin,suberine and wax biosynthesis,and flavonoid biosynthesis pathways were enriched in the P×T vs.P×P comparison,and starch and sucrose metabolism,glycerophospholipid metabolism,and alpha-linolenic acid metabolism were abundant in the T×T vs.T×P comparison.The enriched pathways between PS and TS were the biosynthesis of secondary metabolites,phenylpropanoid biosynthesis,and pentose and glucuronate interconversion.Self-incompatibility protein S1(SI S1),Mitogen-activated protein kinase 3/4(MPK3/4),Mitogen-activated protein kinase kinase 2/3(M2K2/3),Exocyst complex component EXO70A1(E70A1)and Thioredoxin H1/2(TRXH1/2)were found to be HetSI-related candidates,and O-fucosyltransferase 23(OFT23),3-ketoacyl-CoA synthase 6(KCS6),Receptor-like protein kinase FERONIA(FERON),Fimbrin-5(FIMB5),Pollen-specific leucine-rich repeat extensin-like protein 4(PLRX4),Transcription initiation factor IIB-2(TF2B2)and Pectinesterase 1(AL11A),etc.,were identified as other regulatory transducers.These findings combined with our morphological and reactive oxygen species(ROS)intensity analyses indicate that P.auriculata has typical dry-stigmas and that the HetSI mechanism might differ between the pin and thrum.SI S1 might be the key factor in HetSI,and ROS are overexpressed during SC pollination to rapidly activate the mitogen-activated protein kinase(MAPK)-mediated phosphorylation of E70A1 to maintain stigma receptivity in plants with HetSI.

Neuroprotective effects of acteoside in a glaucoma mouse model by targeting Serta domain-containing protein 4

AIM:To explore the therapeutic effect and main molecular mechanisms of acteoside in a glaucoma model in DBA/2J mice.METHODS:Proteomics was used to compare the differentially expressed proteins of C57 and DBA/2J mice.After acteoside administration in DBA/2J mice,anterior segment observation,intraocular pressure(IOP)monitoring,electrophysiology examination,and hematoxylin and eosin staining were used to analyze any potential effects.Immunohistochemistry(IHC)assays were used to verify the proteomics results.Furthermore,retinal ganglion cell 5(RGC5)cell proliferation was assessed with cell counting kit-8(CCK-8)assays.Serta domain-containing protein 4(Sertad4)mRNA and protein expression levels were measured by qRT-PCR and Western blot analysis,respectively.RESULTS:Proteomics analysis suggested that Sertad4 was the most significantly differentially expressed protein.Compared with the saline group,the acteoside treatment group showed decreased IOP,improved N1-P1 wave amplitudes,thicker retina,and larger numbers of cells in the ganglion cell layer(GCL).The IHC results showed that Sertad4 expression levels in DBA/2J mice treated with acteoside were significantly lower than in the saline group.Acteoside treatment could improve RGC5 cell survival and reduce the Sertad4 mRNA and protein expression levels after glutamate injury.CONCLUSION:Sertad4 is differentially expressed in DBA/2J mice.Acteoside can protect RGCs from damage,possibly through the downregulation of Sertad4,and has a potential use in glaucoma treatment.

Silencing ribosomal protein L4 enhances the inhibitory effects of triptolide on non-small cell lung cancer cells by disrupting the mouse double minute 2 protein–P53 tumor suppressor pathway

Non-small cell lung cancer(NSCLC)is a malignant tumor with high incidence worldwide.Triptolide(TP),extracted from Tripterygium wilfordii Hook F,exhibits potent broad-spectrum antitumor activity.Although some mechanisms through which TP inhibits NSCLC are well understood,those that involve ribosomal proteins remain yet to be understood.In this study,the transcriptome and proteome were integrated and analyzed.Our data indicated ribosomal protein L4(RPL4)to be a core hub protein in the protein-protein interaction network.RPL4 is overexpressed in NSCLC tissues and cells.Transfection with siRPL4 or TP treatment alone arrested the cell cycle in the G1 phase,induced cell apoptosis,and repressed cell invasion.Compared to treating cells with TP alone or siRPL4,treating them with siRPL4–TP enhanced the inhibition of NSCLC cells.Reduced RPL4 expression reinforced the inhibitory effects of TP on NSCLC cells by disrupting the MDM2-P53 pathway and by altering the expression of PARP1/Snail/cyclin D1.In vivo assays verified that TP induced cell apoptosis and reduced RPL4 expression in xenografts.These findings provide clues to facilitate the development of effective TP-based therapeutic strategies to kill NSCLC cells.

基于4D-DIA蛋白质组学探讨散偏汤对慢性偏头痛模型大鼠的作用机制

为探讨经典名方散偏汤对慢性偏头痛的作用机制,该研究采用4D-DIA蛋白质组学技术分析其对慢性偏头痛模型大鼠的干预影响,并对关键差异蛋白进行实验验证。首先将SD雄性大鼠随机分组,反复注射硝酸甘油制备慢性偏头痛模型。连续灌胃7 d后,采用大鼠痛苦面容量表(RGS)评估药效。取三叉神经节进行4D-DIA相对定量蛋白质组学检测,筛选各组差异蛋白,结合多个数据库进行基因本体论(Gene Ontology,GO)功能和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析;使用STRING数据库及Cytoscape软件进行蛋白-蛋白互作(PPI)网络分析,并对关键差异表达蛋白TRPV1进行蛋白免疫印迹法(Western blot)检测。结果显示,空白组与模型组间有517个差异表达蛋白,模型组与散偏汤中剂量组间有221个差异表达蛋白。GO功能和KEGG通路富集分析显示,差异表达蛋白主要与炎症反应、伤害性感受刺激、甘油三酯代谢、免疫调节等相关,主要集中在炎症相关TRP通路、AMPK信号通路、PI3K-Akt信号通路、TGF-β信号通路等。PPI网络显示,IGF、TOP2A、APOA1、CDK1、TTN、RYR1、CSRP3等靶蛋白具有较高的度值(degree)。Western blot结果显示,与模型组比较,散偏汤中、高剂量组TRPV1表达水平差异有统计学意义(P<0.05)。综上,散偏汤可能通过调节TRP、AMPK、PI3K-Akt等炎症相关通路治疗慢性偏头痛,其对TRPV1蛋白的调节发挥了重要作用,并对伤害性刺激感受、脂代谢和免疫反应等具有潜在的调节作用。

高氧暴露早产大鼠肺组织线粒体蛋白质组学初步研究以及ND4动态表达

目的初步分析高氧暴露下早产大鼠肺组织线粒体蛋白质表达谱的改变,并对还原型尼克酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位4(ND4)的动态表达进行研究。方法建立高氧暴露早产大鼠动物模型,采用双向电泳检测线粒体蛋白质差异性表达,运用RT-PCR和Western blot方法分别检测ND4mRNA及其蛋白表达。结果与空气组比较,高氧暴露4d导致46种肺组织线粒体蛋白质出现差异性表达;高氧暴露1、4和7d,早产大鼠肺组织ND4mRNA和蛋白表达均较空气对照组显著降低(均P<0.01),10d时,差异则无显著性意义(P>0.05)。结论高氧暴露导致肺细胞线粒体(包括呼吸链)功能状态改变可能是促使肺损伤发生发展的重要因素。

病毒性心肌炎差异表达蛋白MYL4的筛选鉴定及表达研究

目的:调查心肌肌球蛋白轻链4(myosin light polypeptide 4,MYL4)在病毒性心肌炎损伤中的作用。方法 :随机将Balb/c小鼠分为实验组(n=30)和对照组(n=10),实验组用于建立柯萨奇病毒B3病毒性心肌炎小鼠模型,分别在病毒感染后第3、7、14天留取心脏及血清标本。利用差异蛋白质组学的方法鉴定了部分的病毒性心肌炎差异表达蛋白,并对其中一个分子MYL4在病毒性心肌炎发病中的作用进行研究。结果:质谱鉴定6个差异表达分子分别为:MYL4、热休克蛋白B1、异柠檬酸脱氢酶a亚单位、电压依赖的阴离子通道蛋白、蛋白酶体a亚单位1型和巨噬细胞封端蛋白。Western blot及ELISA验证发现MYL4在病毒性心肌炎小鼠心脏组织及血清中表达明显升高(P<0.01),且ELISA结果显示MYL4的表达与疾病严重程度呈正相关(r=0.97,P<0.00)。结论:MYL4在病毒性心肌炎组织及血清中表达升高,参与了病毒性心肌炎的发生发展。

Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤)ameliorates alcoholic fatty liver in mice by regulating lipid and bile acid metabolism and with exertion of antioxidant stress based on 4DLabel-free quantitative proteomic study

OBJECTIVE:To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤,GJDD)on alcoholic fatty live disease(AFLD)by using proteomic methods.METHODS:The male C57BL/6J mouse were randomly divided into four groups:control group,model group,GJDD group and resveratrol group.After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method,the GJDD group and resveratrol group were intragastrically administered with GJDD(4900 mg/kg)and resveratrol(400 mg/kg)respectively,once a day for 9 d.The fat deposition of liver tissue was observed and evaluated by oil red O(ORO)staining.4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group.The differentially expressed proteins were screened according to protein expression differential multiples,and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment.Finally,expression validation of the differentially co-expressed proteins from control group,model group and GJDD group were verified by targeted proteomics quantification techniques.RESULTS:In semiquantitative analyses of ORO,all kinds of steatosis(ToS,MaS,and MiS)were evaluated higher in AFLD mice compared to those in GJDD or resveratroltreated mice.4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified,of which 3763 proteins were quantified and 946 differentially expressed proteins were screened.Compared with the control group,145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group.In addition,compared with the model group,92 proteins were up-regulated and 135 proteins were downregulated in the liver tissue of the GJDD group.15 differentially co-expressed proteins were found between every two groups(model group vs control group,GJDD group vs model group and GJDD group vs control group),which were involved in many biological processes.Among them,11 differentially co-expressed key proteins(Aox3,H1-5,Fabp5,Ces3a,Nudt7,Serpinb1a,Fkbp11,Rpl22l1,Keg1,Acss2 and Slco1a1)were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis.CONCLUSIONS:Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression,likely through the modulation of lipid metabolism,bile acid metabolism and with exertion of antioxidant stress.

Differential protein expression in substantia nigra induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine in a mouse model of chronic Parkinson’s disease

BACKGROUND： To date, a complete protein expression profile of the midbrain substantia nigra in a mouse model of chronic Parkinson＇s disease, induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine （MPTP）, does not exist. In addition, there are no reports of analysis of differential protein expression. OBJECTIVE： To separate and evaluate MPTP-induced differential protein expression through the use of proteomics in the substantia nigra of a mouse model of chronic Parkinson＇s disease. DESIGN： Randomized controlled animal study. SETTING： Department of Neurology, the First Affiliated Hospital, Chongqing Medical University. MATERIALS： Sixteen 8-10-week old, healthy, male, C57BL mice, weighing 20-25 g, and of clean grade, were provided by the Experimental Animal Center of Chongqing Medical University. The experimental animals were disposed according to ethical criteria. MPTP was provided by Sigma Company, USA; Pdquest 2D image analysis software and gelatum/irradiance image analysis system （ChemiDoc XRS） by Bio-Rad, USA; and Voyager DE-PROMALD1-TOF-MS mass spectroscopy analyzer by AB1 Company, USA. METHODS： This study was performed in Chongqing Neurological Laboratory between November 2006 and July 2007. Mice were randomly divided into model and control groups, with 8 mice in each group. Mice in the model group were received a subcutaneous injection of MPTP （25 mg＆g）, twice a week, for five successive weeks, to establish a chronic Parkinson＇s disease model. Mice in the control group received the same volume of a subcutaneous saline injection at the same time points. Mice were sacrificed by anesthesia to rapidly obtain the midbrain for protein separation of the substantia nigra. MAIN OUTCOME MEASURES： （1） 2-ED handbook （Bio-Rad Company） was referenced for two-dimensional electrophoresis, （2） PDQUEST8,0 analytical electrophoresis pattern was adopted to evaluate differential protein expression. （3） Peptide mass finger print map and data were retrieved on http：//www.prospector.ucsf.edu to compare differential substantia nigral protein expression in the two groups. RESULTS： Two-dimensional gel electrophoresis of substantia nigra tissue indicated that there were 33 differential protein expressions between the two groups. Three new proteins were evaluated, including α -enolase, which exhibited regulated expression, tumor necrosis factor ligand superfamily member 4, and cyclin-dependent kinase inhibitor 1B. CONCLUSION： There are three proteins that exhibit differential expression in the substantia nigra- α -enolase, tumor necrosis factor ligand superfamily member 4, and cyclin-dependent kinase inhibitor 1B.

S100A4 over-expression underlies lymph node metastasis and poor prognosis in colorectal cancer

AIM:To develop lymph node metastasis(LNM)-associated biomarkers for colorectal cancer(CRC) using quantitative proteome analysis.METHODS:Differences in protein expression between primary CRC with LNM(LNM CRC) and without LNM(non-LNM CRC) were assessed using methyl esterification stable isotope labeling coupled with 2D liquid chromatography followed by tandem mass spectrometry(2D-LC-MS/MS).The relationship to clinicopathological parameters and prognosis of candidate biomarkers was examined using an independent sample set.RESULTS:Forty-three proteins were found to be differentially expressed by at least 2.5-fold in two types of CRC.S100A4 was significantly upregulated in LNM CRC compared with non-LNM CRC,which was confirmed by Western blotting,immunohistochemistry and real-time quantitative polymerase chain reaction.Further immunohistochemistry on another 112 CRC cases showed that overexpression of S100A4 frequently existed in LNM CRC compared with non-LNM CRC(P < 0.001).Overexpression of S100A4 was significantly associated with LNM(P < 0.001),advanced TNM stage(P < 0.001),increased 5-year recurrence rate(P < 0.001) and decreased 5-year overall survival rate(P < 0.001).Univariate and multivariate analyses indicated that S100A4 expression was an independent prognostic factor for recurrence and survival of CRC patients(P < 0.05).CONCLUSION:S100A4 might serve as a powerful biomarker for LNM and a prognostic factor in CRC.

Proteomic identification of tumor biomarkers associated with primary gallbladder cancer

AIM: To identify potential biomarkers of primary gallbladder cancer (PGC).

Proteomic Analysis of Chrysanthemum Lateral Buds after Removing Apical Dominance Based on Label-Free Technology

Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.Quantitative real-time PCR(qPCR)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qPCR indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.

Study on Relationship Between Differential Proteins of Bacillus cereus LBR-4 and Its Salt Tolerance Mechanism

In order to explore the salt tolerance mechanism of Bacillus cereus LBR-4 with salinity of 14%NaCl,differential proteomic analysis of the whole protein of LBR-4 strain expressed under 14%NaCl high salinity condition and normalculture condition(1%NaCl)was studied by two-dimensional electrophoresis and mass spectrometry.The isoelectric point of most detected proteins was between pH 4-7 and the molecular weight distribution was 10-70 ku.Compared with the normal culture condition,the expression level of 118 protein spots in the whole protein expression map changed significantly(accounting for 25.2%of the total protein spots).The expression level of 78 protein spots increased significantly,including 22 new protein spots that appeared under high salt stress.The expression levels of 40 protein spots decreased significantly,including 18 protein spots that disappeared under high salt stress.By mass spectrometry,six distinct differentially expressed protein spotswere dihydroxy acid dehydratase,cell division protein FtsZ,iron sulfur cluster synthesis protein SufD,unknown carboxylase YngE,hypothetical acetaldehyde dehydrogenase DhaS and phenylalanine acid tRNA ligase alpha subunit.It was speculated that under high salt stress,the cells had protective measures and the secretion of intracellular compatible solutes increased.The iron and sulfur clusters involved in various physiological reactions also activated the stressful suf synthesis pathway,and therate of cell division and reproduction was also slowed down and ensured the normal progress of physiological reactions inthe cells.

A proteomic analysis of the effect of radiation therapy on wound healing in women reconstructed with the TRAM flap

The incidence of breast cancer is still increasing, and with improved cancer treatment, more women live longer with the side effects of their treatment. The response of normal tissue to radiation continues for years after the treatment is completed. The influence of radiotherapy on the outcome of breast reconstructtive surgery remains unpredictable. The combination of two surgical sites of which one is previously irradiated, is rarely encountered in humans and thus compiles a unique opportunity to study the implications of irradiation followed by surgery. The aim of this study was to examine the long-term effect of radiation therapy on the proteins expressed in the wound tissue after a breast reconstruction. Ten patients were included in the study, all treated with radiotherapy after a mastectomy and breast reconstruction with a contralateral pedicled TRAM flap. Expanded poly-tetrafluoretylene polymer tubes were implanted for 10 days, subcutaneously, below the inframammary fold and below the donor site. The protein from the newly synthesized granulation tissue in the tubes was extracted and analyzed for differences in protein expression with 2D gel electrophoresis and mass spectrometry. A total of 676 proteins were detected;of these, 4 proteins changed significantly and were successfully identified. TPM4 and APOA4 from the radiation treated tissue were shown to be significantly decreased, whereas IGKC and VDAC1 were found to be significantly increased. The proteomic technique combined with the ePTFE tube wound model can elucidate some of the molecular alterations in the wound healing induced by radiation therapy. The protein modifications of TPM4, APOA4, IGKC and VDAC1 may influence the cell proliferation, apoptosis and the inflammation of the tissue repair process.

Transcriptomic,proteomic,and phosphoproteomic analyses reveal dynamic signaling networks influencing long-grain rice development

The LGS1(Large grain size 1)gene,also known as GS2/GL2/Os GRF4,is involved in regulating grain size and quality in rice,but the mechanism governing grain size has not been elucidated.We performed transcriptomic,proteomic,and phosphoproteomic analyses of young rice panicles in Samba(a wild-type cultivar with extra-small grain)and NIL-LGS1(a nearly isogenic line of LGS1 with large grain in the Samba genetic background)at three developmental stages(4–6)to identify internal dynamic functional networks determining grain size that are mediated by LGS1.Differentially expressed proteins formed seven highly functionally correlated clusters.The concordant regulation of multiple functional clusters may be key features of the development of grain length in rice.In stage 5,16 and 24 phosphorylated proteins were significantly up-regulated and down-regulated,and dynamic phosphorylation events may play accessory roles in determining rice grain size by participating in protein–protein interaction networks.Transcriptomic analysis in stage 5 showed that differentially expressed alternative splicing events and dynamic gene regulatory networks based on 39 transcription factors and their highly correlated target genes might contribute to rice grain development.Integrative multilevel omics analysis suggested that the regulatory network at the transcriptional and posttranscriptional levels could be directly manifested at the translational level,and this analysis also suggested a regulatory mechanism,regulation of protein translation levels,in the biological process that extends from transcript to protein to the development of grain.Functional analysis suggested that biological processes including MAPK signaling,calcium signaling,cell proliferation,cell wall,energy metabolism,hormone pathway,and ubiquitin-proteasome pathway might be involved in LGS1-mediated regulation of grain length.Thus,LGS1-mediated regulation of grain size is affected by dynamic transcriptional,posttranscriptional,translational and posttranslational changes.

Anemoside B4 inhibits SARS-CoV-2 replication in vitro and in vivo

Objective: Anemoside B4(AB4), the most abundant triterpenoidal saponin isolated from Pulsatilla chinensis, inhibited influenza virus FM1 or Klebsiella pneumoniae-induced pneumonia. However, the anti-SARS-CoV-2 effect of AB4 has not been unraveled. Therefore, this study aimed to determine the antiviral activity and potential mechanism of AB4 in inhibiting human coronavirus SARS-CoV-2 in vivo and in vitro.Methods: The cytotoxicity of AB4 was evaluated using the Cell Counting Kit-8(CCK8) assay. SARS-CoV-2 infected HEK293T, HPAEpiC, and Vero E6 cells were used for in vitro assays. The antiviral effect of AB4 in vivo was evaluated by SARS-CoV-2-infected hACE2-IRES-luc transgenic mouse model. Furthermore,label-free quantitative proteomics and bioinformatic analysis were performed to explore the potential antiviral mechanism of action of AB4. Type Ⅰ IFN signaling-associated proteins were assessed using Western blotting or immumohistochemical staining.Results: The data showed that AB4 reduced the propagation of SARS-CoV-2 along with the decreased Nucleocapsid protein(N), Spike protein(S), and 3C-like protease(3CLpro) in HEK293T cells. In vivo antiviral activity data revealed that AB4 inhibited viral replication and relieved pneumonia in a SARS-CoV-2 infected mouse model. We further disclosed that the antiviral activity of AB4 was associated with the enhanced interferon(IFN)-β response via the activation of retinoic acid-inducible gene Ⅰ(RIG-1) like receptor(RLP) pathways. Additionally, label-free quantitative proteomic analyses discovered that 17 proteins were significantly altered by AB4 in the SARS-CoV-2 coronavirus infections cells. These proteins mainly clustered in RNA metabolism.Conclusion: Our results indicated that AB4 inhibited SARS-CoV-2 replication through the RLR pathways and moderated the RNA metabolism, suggesting that it would be a potential lead compound for the development of anti-SARS-CoV-2 drugs.

Large-Scale Proteomics Data Reveal Integrated Prognosis-Related Protein Signatures and Role of SMAD4 and RAD50 in Prognosis and Immune Infiltrations of Prostate Cancer Microenvironment

As prostate cancer(PCa)is one of the most commonly diagnosed cancer worldwide,identifying potential prognostic bio-markers is crucial.In this study,the survival information,gene expression,and protein expression data of 344 PCa cases were collected from the Cancer Proteome Atlas(TCPA)and the Cancer Genome Atlas(TCGA)to investigate the potential prognostic biomarkers.The integrated prognosis-related proteins(IPRPs)model was constructed based on the risk score of each patients using machine-learning algorithm.IPRPs model suggested that Elevated RAD50 expression(p=0.016)and down-regulated SMAD4 expression(p=0.017)were significantly correlated with unfavorable outcomes for PCa patients.Immunohistochemical(IHC)staining and western blot(WB)analysis revealed significant differential expression of SMAD4 and RAD50 protein between tumor and normal tissues in validation cohort.According to the overall IHC score,patients with low SMAD4(p<0.0001)expression and high RAD50 expression(p=0.0001)were significantly correlated with poor outcomes.Besides,expression of SMAD4 showed significantly negative correlation with most immune checkpoint molecules,and the low SMAD4 expression group exhibited significantly high levels of LAG3(p<0.05),TGFβ(p<0.001),and PD-L1(p<0.05)compared with the high SMAD4 expression group in the validation cohort.Patients with low SMAD4 expression had significantly higher infiltration of memory B cells(p=0.002),CD8+T cells(p<0.001),regulatory T cells(p=0.006),M2-type macrophages(p<0.001),and significantly lower infiltration of naïve B cells(p=0.002),plasma cells(p<0.001),resting memory CD4+T cells(p<0.001)and eosinophils(p=0.045).Candidate proteins were mainly involved in antigen processing and presentation,stem cell differentiation,and type I interferon pathways.