High expression of APC is an unfavorable prognostic biomarker in T4 gastric cancer patients
医学英语写作与翻译
第三部分医学英语的写作任务一标题的写作（Title）标题的结构1. 名词+介词Blindness（视觉缺失）after Treatment for Malignant Hypertension 2. 名词+分词Unilateral Neurogenic Pruritus Following Stroke中风后单侧神经性瘙痒3. 名词+不定式Suggestion to Abolish Icterus Index Determination（黄疸指数测定）where Quantitative Bilirubin Assay（胆红素定量）is Available建议能做胆红素定量的化验室不再做黄疸指数测定4. 名词+同位语Gentamicine, a Selelctive Agent for the isolation of Betahemolytic Streptocc ociβ-溶血性链球菌庆大霉素是分离β-溶血性链球菌的选择性药物5. 名词+从句Evidence that the V-sis Gene Product Transforms by Interaction with the Receptor for Platelet-derived Growth Factor血小板源性生长因子.V-sis 基因产物由血小板生成因子受体相互作用而转化的依据6. 动名词短语Preventing Stroke in patients with Atrial Fibrillation心房纤维性颤动心旁纤颤患者中风预防Detecting Acute Myocardial Infarction（急性心肌梗死）byRadio-immunoassay for Creative Kinase（酐激酶）用放射免疫法测定酐激酶诊断急性心肌梗死7. 介词短语On Controlling Rectal Cancer8. 陈述句Dietary Cholesterol is Co-carcinogenic协同致癌因素for Human Colon Cancer9. 疑问句Home or Hospital BirthsIs Treatment of Borderline Hypertension Good or Bad?注意副标题的作用1．数目：Endoluminal Stent-graft 带支架腔内搭桥for Aortic Aneurysms动脉瘤: A report of 6 cases带支架腔内搭桥治疗动脉瘤的六例报告2．重点：Aorto-arteritis 大动脉炎Chest X-ray Appearance and Its Clinical Significance大动脉炎胸部X线表现及临床意义3．方法：Gallstone Ileus（胆结石梗阻）: A Retrospective Study 4．作用：Carcinoembryonic Antigen in Breast-cancer Tissue: A useful prognostic indictor乳腺癌组织中癌胚抗原——一种有用的预后指示5．疑问：Unresolved—Do drinkers have less coronary heart disease? 6．连载顺序：Physical and Chemical Studies of Human Blood Serum: II. A study of miscellaneous Disease conditions人类血清的理论研究：II. 多种病例的研究7．时间：A Collaborative 综合Study of Burn Nursing in China: 1995-1999常见标题句式举例1. 讨论型：Discussion of/ on; An approach to; A probe into; Investigation of; Evaluation of / on汉语中的“初步体会”、“试论”、“浅析”之类的谦辞可以不译。
High-Level Expression of
High-Level Expression of a Soluble Functional Antimicrobial Peptide,Human -Defensin2,in Escherichia coliZhinan Xu,*,†Li Peng,†,§Zhixia Zhong,†Xiangming Fang,‡and Peilin Cen†Institute of Bioengineering,Department of Chemical and Biochemical Engineering,Zhejiang University,Hangzhou310027, China,and The Central Laboratory of Sir Run Run Shaw Hospital,School of Medical Science,Zhejiang University, Hangzhou310016,ChinaIn this work,taking human -defensin-2(HBD2)as a demonstrative molecule,the strategiesfor high efficient production of functional human -defensins in E.coli were studied.Fusionmature HBD2(TrxA-mHBD2)showed high solubility and productivity without the need forlowering the cultivation temperature.The solubility of target fusion protein could attain81.3%even at37°C with a volumetric productivity as high as235mg/L in a rich medium MBL at thesame temperature and reached346mg/L at28°C.The His-Tag in the fusion protein enabledthe application of affinity chromatography separation to obtain high purity of the overexpressedrecombinant fusion protein.After digestion by enterokinase,purification via cationic exchangechromatography,and desalting by ultrafiltration,mature HBD2product was obtained with apurity of95%in an overall recovery of29.2%.The antimicrobial activity of the recombinantmature HBD2and the influence factors were tested using E.coli K12D31as a sensitive strain.IntroductionHuman -defensins are cationic antimicrobial peptides with three conserved intramolecular cysteine disulfide bonds and exhibit a broad microorganism-killing spectrum.They play important roles along with human R-defensins in human primary defense system against infection and are also reported to link with the innate and adaptive immunity by attracting immature dendritic cells and memory T cells(1,2).So far,various members of this family were found with different function and distribution in human body(3-5).Due to their unique mech-anism of action,human -defensins are expected to be ideal therapeutic agents as peptide antibiotics mitigating the problem of acquired resistance(3,4,6).Producing these cationic antimicrobial peptides with recombinant techniques will ac-celerate the research on their pharmaceutical potential and clinical application.During the past decade,many small cationic antimicrobial peptides have been synthesized successfully by recombinant DNA methods.The most commonly used host cell is Escheri-chia coli because of its fast growth rate and well-established expression systems.However,there are two major barriers in using E.coli as the host cell for cationic antibiotic peptides expression,including the host-killing activity and the suscep-tibility to degradation of the product(7).The fusion expression of the target peptide with a partner might alleviate the above obstacles.But there is another problem that most fusion products were inactive or in insoluble form,a low-recovery renaturation process is usually indispensable(7,8).LaVallie et al.reported a fusion expression system of thioredoxin(TrxA)(9),and an increased yield of soluble products was observed for several target proteins.This strategy opened a way to high-level production of soluble functional heterologous protein in E.coli. Human -defensin-2(HBD2)is a cysteine-rich cationic antimicrobial peptide with41amino acids discovered in1997 in human skin(3).It was selected as a model peptide in our laboratory to study the recombinant production of human -defensins in E.coli.In our previous work,two precursor HBD2genes were obtained:one was obtained by RT-PCR from human skin,and another was synthesized with preferential codon of E.coli(10,11).About9-fold improvement of expression level was achieved by the codon optimization(11).Then further efforts were made to improve the productivity,including the tests of different expression plasmids,different fusion partners, and the multiple joined gene expression(12-14).The highest yield was achieved in soluble fusion expression of plasmid pET32-sHBD2with a volumetric productivity of1.3g/L TrxA-HBD2,i.e.,208mg/L mature HBD2,and a soluble fusion protein percentage of41.6%in total soluble proteins(12).One cell-free protein biosynthesis system was also developed to produce soluble HBD2fusion protein,but the high cost of this process restricted its practical use for large-scale production(15). In this work,the prepro-peptide sequence in the sHBD2gene was deleted and a new plasmid pET32-smHBD2(synthetic mature HBD2gene)was constructed and overexpressed in E. coli BL21(DE3).Then the product,mature HBD2,was purified and its antimicrobial activity was analyzed.Methods and MaterialsStrains and Media.E.coli TG1(supE,hsd∆5,thi,∆(lac-proAB)/F′(traD36,proAB+,lacI q,lacZ∆M15))was used as the host for gene manipulation.E.coli BL21(DE3)(F-ompT hsdS B (r B-m B-)gal dcm(DE3))was used as the host for expression of heterologous protein.E.coli K12D31was used in antimi-crobial assay of recombinant HBD2.Luria-Bertani(LB)medium(w/v),containing0.5%yeast extract,1%tryptone,and1%NaCl,was used for manipulation*To whom correspondence should be addressed.Tel:+86-571-87951220.Fax:+86-571-87951358.E-mail:znxu@.†Department of Chemical and Biochemical Engineering.‡School of Medical Science.§Current Address:Department of Molecular and Medical Pharmacology,University of California at Los Angeles,Los Angeles,CA90095.382Biotechnol.Prog.2006,22,382−38610.1021/bp0502680CCC:$33.50©2006American Chemical Society and American Institute of Chemical EngineersPublished on Web01/17/2006of molecular clone,simple recombinant expression,and seed culture.MBL medium (w/v),containing 0.5%glucose,3%yeast extract,2%tryptone,0.35%(NH4)2HPO 4,0.35%KH 2PO 4,0.5%K 2HPO 4,0.7%MgSO 4‚7H 2O,and 2.1%NaCl,was used for fermentation (16).Mueller-Hutton (M-H)medium (w/v),con-taining 5g/L beef extract,17.5g/L casamino acid and 1.5g/L starch,was used for antimicrobial assay.Genes and Plasmids.A coding sequence of HBD2precursor,sHBD2(GenBank accession no.AY155577),was designed using the preferential codon of E.coli according to the Codon Usage Table and synthesized by Sangon (Shanghai,China).It had shown 9-fold improvement of recombinant protein expres-sion compared to human source HBD2gene (11).The synthetic mature HBD2gene,smHBD2(prepro sequence removed sHBD2gene),was obtained by a PCR reaction with pET32-sHBD2plasmid as a template.Plasmid pGEM-T (Promega,Madison,WI)was used to construct the cloning vector.Plasmid pET32-sHBD2was constructed in our previous work (11).Plasmid pET-32a(+)(Novagen,Madison,WI)was used to construct the expression vector.Primers for the PCR reaction were synthesized by Sangon (Shanghai,China).All restriction enzymes and T 4DNA ligase used in gene manipulation were purchased from Takara Biotech Co.Ltd.(Dalian,China).Expression Vector Construction and Protein Expression.Standard molecular biology techniques were employed in vector construction.The cloning vector containing smHBD2gene,pGEM-smHBD2,was cleaved by Nco I and Bam HI,and the smHBD2fragment was inserted into similarly digested pET-32a(+)vector to construct the expression plasmid pET32-smHBD2(Figure 1).A fresh clone of E.coli BL21(DE3),harboring the pET32-smHBD2vector,was grown in LB medium containing 50µg/mL ampicillin.When the cells had been cultured (37°C,250rpm)to an optical density (OD 600)of 0.4,they were inoculated,with a ratio of 5%(v/v),into 30mL of MBL medium (with 50µg/mL ampicillin)in 250mL flasks and cultured following the optimized condition of pET-sHBD2(12):250rpm,induce at OD 6007.5with 0.8mM IPTG and harvest at 8h postinduction.Simple comparison of heterologous expression between pET32-sHBD2and pET32-smHBD2was carried out in LB medium.The heterologous expression was induced by 0.8mM IPTG at OD 6001.0for 4h in LB medium.Product Analysis and Recombinant HBD2Purification.After harvest,cells were centrifuged at 5,000g for 20min and then resuspended in 20mM Tris-HCl (pH 7.5)for sonication.Following the centrifugation (14,000g ,30min),the supernatant (soluble protein fraction)was isolated.The cell debris was washed twice and dissolved in 8M urea to yield the fraction of insoluble protein.The soluble fraction of cellular protein was subjected to a four-step process of purification.After nickel-affinity chroma-tography,the 6His-tagged fusion product,TrxA-mHBD2,was obtained and then digested with recombinant enterokinase (Huadong Gene Technology Institute,Hangzhou,China)at 25°C for 12h.The released mature HBD2was purified to homogeneity by cationic exchange chromatography and desalted by ultrafiltration.All chromatographic operations were per-formed on the A ¨KTA purifier system (Amersham Pharmacia Biotech,UmeåPlant,Sweden),with Ni-NTA Agarose (QIAGEN GmbH,Hilden,Germany)for affinity chromatography and CM Sepharose Fast Flow (Pharmacia Biotech)for the cationic exchange chromatography.A standard SDS -PAGE method (15%)was applied for fusion protein assay and the Tricine-SDS -PAGE (18%gel)was used for mature HBD2product assay (17).The images of gels were scanned by GEL-DOC 2000gel documentation system (Bio-Rad,USA)and analyzed using Quantity One software,Version 4.4.0(Bio-Rad,USA).The Bradford protein assay (18)was used for quantitative analysis of protein.Antimicrobial Activity Assay of Recombinant HBD2.The concentration killing curve of recombinant HBD2was used to determine its antimicrobial activity.About 106cfu (colony formation units)E.coli K12D31growing in log phase was inoculated into 500µL of half-strength M-H medium.Purified recombinant HBD2was added at various concentrations,and 20mM Tris-HCl buffer (pH8.0)was added in negative controls.After shaking for 12h (37°C,250rpm),bacterial growth was determined by measuring OD 600.Because HBD2was reported to be a salt-sensitive antimicrobial peptide (19),various concentration of NaCl (from 0to 150mM)was used in the culture with 1µg/mL recombinant HBD2to analyze the effect of NaCl concentration on the antimicrobial activity.Results and DiscussionsSolubility Comparison of Fusion Proteins.Two recombi-nant strains,BL21(DE3)/pET32-sHBD2and BL21(DE3)/pET32-smHBD2(without prepro-peptide sequence),were cultivated in LB medium at both 30and 37°C.After IPTG-induction,the products,TrxA-HBD2and TrxA-mHBD2(Figure 2),were analyzed by SDS -PAGE as illustrated in Figure 3.It is obvious that the solubility of BL21(DE3)/pET32-smHBD2product was greatly improved by the deletion of prepro-peptide.The fusion mature peptide still retained high solubility (81.3%)at 37°C,which is a less than 8%decrease compared with that at 30°C.However,the fusion precursor exhibited a much lower solubil-ity:less than 50%at 30°C and almost insoluble at 37°C (13.2%).(Triple experiments were carried out,and average values are presented here with variations within 10%.)As for human defensins,the anionic prepro-sequence at the N-terminal of their precursors (composed of prepro-sequence and mature peptide)is generally considered to be the natural ideal fusion partner,which can stabilize the peptide and neutralize their cationic charges (7).Thus the prepro-peptide of human -defensin-2was retained in HBD2expression in our previous work.However,in this work,after removing the prepro-peptide,higher solubility of fusion proteinTrxA-mHBD2Figure 1.Schematic representation of the expression vector,pET32-smHBD2.was achieved in E.coli .To find possible reasons,we first tried the Wilkinson-Harrison statistical solubility model (20,21),but our experiment result was just opposite to the calculation result of that model,which showed that the TrxA-mHBD2was less likely to be soluble than TrxA-HBD2.Unlike the Wilkinson-Harrison model that considered only eight amino acids,we then analyzed all of the amino acids of the prepro-peptide,the major difference between the two fusion proteins.The prepro-peptide was specifically rich in hydrophobic amino acid (60.87%)and low in charged amino acid (8.7%).So the high hydrophobicity of prepro-peptide resulted in the lower solubility of sHBD2gene expression as the exposure of the hydrophobic part might induce the aggregation of heterologous protein.Another possible explanation is that the prepro-peptide between TrxA (thio-redoxin)and mature HBD2reduced the folding rate or accuracy of disulfide bonds in mature HBD2.As TrxA protein was reported to assist the formation of accurate disulfide bonds in the foreign protein fused with it,the distance between the TrxA protein and according disulfide bonds might also be an influence factor.Productivity of Fusion ing a more nutrient-rich medium,MBL instead of LB medium,the recombinant strain BL21(DE3)/pET32-smHBD2and BL21(DE3)/pET32-sHBD2were cultured and induced at both 37and 28°C (12).The volumetric productivities of soluble mature HBD2at different conditions are compared in Table 1.Purification of Recombinant HBD2.Recombinant HBD2was purified according to the following five steps:(1)cell disruption to release soluble fusion protein of TrxA-mHBD2;(2)nickel-affinity chromatography to separate the fusion protein from other soluble proteins;(3)enterokinase treatment to release the mature HBD-2from the fusion protein;(4)cationic exchangeto purify the mature HBD2,due to its high isoelectric point (pI )9.3);and (5)ultrafiltration and freeze-dry to refine the mature HBD2peptide.After affinity chromatography,the recombinant fusion protein was purified to a purity of 86.2%with a recovery of 81.7%(Figure 4).The obtained fusion protein was subjected to enterokinase (EK)digestion at 25°C for 12h,and the released mature HBD2was examined by Tricine-SDS -PAGE (Figure 5).Because of a high pI of 9.30(pI TrxA )5.49,pI EK )5.20),the mature HBD2peptide can be purified from the digestion mixture at pH 8.0by cationic exchange chromatography to a purity of 95.0%with a recovery of 64.0%(Figure 6).In the final step,the product peptide was processed by ultrafiltration and freeze-drying.The total recovery of recombinant mature HBD2is 29.2%.Antimicrobial Activity Assay of Recombinant HBD2.To examine the antimicrobial activity of recombinant HBD2,it was added into the culture of the sensitive E.coli strain in the half-strength M-H medium .The concentration killing-curve is shown in Figure 7A.The growth of E.coli K12D31was dramatically suppressed with the increasing concentration of recombinant HBD2.It was obvious that the recombinant HBD2was bioactive and very effective in killing the sensitive strain.The effect of NaCl concentration on its bactericidal activity is shown in Figure 7B.The result showed that the antimicrobial effectwasFigure parison of HBD2-expressing cassettes between pET32-sHBD2and pET32-smHBD2.The bold letters are sequence of prepro-peptide,the shaded letters are sequences of natural matureHBD2.Figure 3.SDS -PAGE analysis of fusion mature HBD2and fusion HBD2precursor at different temperatures.The solubility of TrxA-mhBD2is 89.1%at 37°C and 81.3%at 30°C.The solubility of TrxA-shBD2is 47.2%at 30°C and 13.2%at 37°nes 1and 2:insoluble and soluble fractions of BL21(DE3)/pET32-smHBD2culture at 30°nes 3and 4:insoluble and soluble fractions of BL21(DE3)/pET32-sHBD2culture at 30°nes 5and 6:insoluble and soluble fractions of BL21(DE3)/pET32-smHBD2culture at 37°nes 7and 8:insoluble and soluble fractions of BL21(DE3)/pET32-sHBD2culture at 37°C.Table parison of Volumetric Productivities (mg/L)of Soluble Mature HBD2Produced by BL21(DE3)/pET32-smHBD2and BL21(DE3)/pET32-sHBD2temp BL21(DE3)/pET32-smHBD2BL21(DE3)/pET32-sHBD228°C 34620837°C235103suppressed by increasing NaCl concentration,and the peptide was almost totally inactive when the concentration is higher than 150mM.This salt-sensitive property was in accordance with the reported behavior of human source HBD2(19).Furtherinvestigations of the purified recombinant HBD2against some more pathogenic bacteria are needed before its practical clinic application.ConclusionsIn this work,by removing the prepro-peptide,high-level expression of soluble mature HBD2fusion was achieved in E.coli with a volumetric productivity of 346mg/L.Both the solubility and volumetric productivity of this fusion protein were better than those of the former product,with prepro-peptide,from the pET32-shBD2plasmid.In the downstream purification,after nickel-affinity chromatography,enterokinase cleavage,cationic ion exchange chromatography,ultrafiltration,and freeze-drying,the final product containing 95%mature HBD2was obtained with the whole recovery of 29.2%.The recom-binant HBD2showed antimicrobial activity against sensitive strain E.coli K12D31and the salt-sensitive property was in accordance with the isolated peptide from human source.As most reported production of cationic antimicrobial peptides was inactive,our strategies of functional HBD2expression including the soluble expression and the fusion expression with TrxA protein was promising and valuable.The strategiesofFigure 4.Separation of fusion protein TrxA-mHBD2from other soluble proteins by affinity chromatography.(A)Affinity chromatog-raphy curves of UV 280nm and concentration of imidazol.(B)SDS -PAGE analysis of collected samples of affinity ne 1:the loading sample of soluble cellular ne 2:the ne 3:the eluate.The purity of the purified fusion protein in Lane 3is 86.2%and the recovery is81.7%.Figure 5.SDS -PAGE analysis of enterokinase digested ne 1:the start point of digestion (0h).Lane 2:the end point of digestion (12h).Figure 6.Separation of released mature HBD2from the mixture of enterokinase digested samples by cationic exchange chromatography.(A)Cationic exchange chromatography curves of UV 280nm and concentration of elution buffer.(B)SDS -PAGE analysis of collected samples of cationic exchange ne 1:the loading sample of digestion ne 2:the ne 3:the eluate.The purity of the purified mature HBD2in Lane 3is 95.0%and the recovery is 64.0%.producing recombinant HBD2can also be used in other defensins and have been applied in HBD3and HBD4expression successfully in our laboratory (21).This work opened a way to the production of functional defensins at a large scale to hopefully meet the need for a large amount of human defensins in medical and pharmaceutical research.AcknowledgmentThis work was financially supported by National Natural Science Foundation of China (no.20276066)and the Department of Science and Technology (no.413491030J-30007001103241),Zhejiang Provincial People’s Government,People’s Republic of China.References and Notes(1)Ganz,T.Defensin and host defense.Science 1999,286,420.(2)Yang,D.;Chertov,O.;Oppenheim,J.J.Participation of mammalian defensins and cathelicidins in anti-microbial immunity:receptorsand activities of human defensins and aathelicidin (LL-37).J.Leukocyte Biol.2001,69,691-697.(3)Harder,J.;Bartels,J.;Christophers,E.;Schro ¨der,J.M.A peptide antibiotic from human skin.Nature 1997,387,861.(4)Harder,J.;Bartels,J.;Christophers,E.;Schro ¨der,J.M.Isolation and characterization of human beta-defensin-3,a novel human inducible peptide antibiotic.J.Biol.Chem.2001,276(8),5707-5713.(5)Garcia,J.R.;Krause,A.;Schulz,S.;Rodriguez-Jimenez,F.J.;Kluver,E.;Adermann,K.;Forssmann,U.;Frimpong-Boateng,A.;Bals,R.;Forssmann W.G.Human beta-defensin 4:a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity.FASEB J .2001,15,1819-1821.(6)Epand,R.M.;Vogel,H.J.Diversity of antimicrobial peptides and their mechanisms of action.Biochim.Biophys.Acta 1999,1462,11-28.(7)Piers,K.L.;Brown,M.H.;Hancock,R.E.W.Recombinant DNA procedures for producing small antimicrobial cationic peptides in bacteria.Gene 1993,134,7-13.(8)Lee,J.H.;Kim,J.H.;Hwang,S.W.;Lee,W.J.;Yoon,H.K.;Lee,H.S.;Hong,S.S.High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion mun.2000,277,575-580.(9)LaVaillie,E.R.;Lu,Z.J.;Diblasio-Smith,E.A.;Collins-Racie,L.A.;McCoy,J.M.Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli .Methods Enzymol.2000,326,322-340.(10)Fang,X.;Peng,L.;Xu,Z.;Wu,J.M.;Cen,P.Cloning and expression of human beta-defensin-2gene in Escherichia coli .Protein Pept.Lett.2002,9,31-37.(11)Peng,L.;Xu,Z.;Fang,X.;Wang,F.;Yang,S.;Cen,P.Preferential codons enhancing the expression level of human beta-defensin-2.Protein Pept.Lett .2004,11,339-344.(12)Peng,L.;Xu,Z.;Fang,X.;Wang, F.;Cen,P.High-level expression of soluble human beta-defensin-2in E.coli.Process Biochem .2004,39:2199-2205.(13)Wang,F.;Fang,X.;Xu,Z.;Peng,L.;Cen P.Fusion expression of human beta-defensin-2from multiple jointed genes in E.coli .Prep .Biochem.Biotechnol .2004,34,215-225.(14)Xu,Z.;Wang,F.;Peng,L.;Fang,X.;Cen,P.Expression of human -defensin-2with multiple joined genes in Escherichia coli .Appl.Biochem.Biotechnol.2005,120,1-14.(15)Chen,H.;Xu,Z.;Xu,N.;Cen P.;Peng,L.Efficient expression of soluble fusion protein containing human beta-defensin-2in E.coli cell-free system.J.Biotechnol.2005,115(3),307-315.(16)SivaKesava,S.;Xu,Z.N.;Chen,Y.H.;Hackett,J.;Huang,R.C.;Lam,E.;Lam,T.L.;Siu,K.L.;Wong,R.S.C.;Wong,W.K.R.;Production of excreted human epidermal growth factor (hEGF)by an efficient recombinant Escherichia coli system.Process Biochem.1999,34(9),893-900.(17)Schagger,H.;von Jagow,G.Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1to 100kDa.Anal.Biochem .1987,166,368-379.(18)Bradford,M.M.A rapid and sensitive method for the qualititation of microgram quantities of protein utilizing the principle of protein-dying binding.Anal.Biochem .1976,72,248-m254.(19)Bals,R.;Wang,X.;Wu,Z.;Freeman,T.;Bafna,V.;Zasloff,M.;Wilson,J.M.Human -defensin 2is a salt-sensitive peptide antibiotic expressed in human lung.J.Clin.In V est.1998,102,874-880.(20)Wilkinson,D.L.;Harrison,R.G.Predicting the solubility of recombinant proteins in Escherichia coli.Biotechnology 1991,9,443-448.(21)Xu,Z.;Zhong,Z.;Huang,L.;Peng,L.;Wang,F.High-level production of bioactive human beta-defensin-4in Escherichia coli by soluble fusion expression.Appl.Microbiol.Biot.In press.Accepted for publication December 21,2005.BP0502680Figure 7.Antimicrobial activity assay of the recombinant mature HBD2in liquid culture (half-strength M-H medium).Triple experiments were carried out,and the average values and variations (error bars)are presented.(A)Microbial-killing curve.(B)Effect of NaCl concentration on the antimicrobial activity of HBD2.。
课件：U4 Useful Expressions
2) small and with only small spaces between parts 紧密的，
密集的
e.g. He received a compact package.
他收到一个扎得很紧的包裹。
to support sales. 企图 4. The bids for this contract were all disappointingly high.
投标
integrity
• Identify the meanings of the following phrases.
1. a man of integrity
• 全部木材砍伐量 • all harvested trees • 定期种植 • plant on a regular basis • 造纸厂 • paper mills • 对空气污染最严重的污染源/者 • the worst polluter to air
• 报纸比笔记本电脑更方便之处 • the convenience of the paper over the laptop • 外卖食品 • takeaway food • 打苍蝇 • swat flies • 不会轻易消亡 • won’t die without a struggle • 对生活方式新闻的持续开发利用 • the continued exploitation of lifestyle journalism
• 自称每天看报
• claim to read a daily newspaper • 逐分逐秒地监视
• monitor minute by minute • 发行量
• circulation figure • 互联网可提供的更多的窗口
畜牧兽医专业英语
Embryo Transfer
The process of transferring embryos from one animal to another
Vocabulary related to veterinary medicine and vaccinVeetserinary Diagnostics
02
Professional English vocabulary for animal husbandry and veterinary medicine
Vocabulary related to animal diseases
Infectious Diseases
Tuberculosis, Pneumonia, Salmonellosis, Ringworm, Distemper
Learning Methods for Animal Husbandry and Veterinary Professional English
• Specialized Textbooks: It is recommended to use English textbooks or translated materials that are specifically designed for animal husbandry and veterinary medicine to ensure accuracy and authentication of language usage
Professional English for Animal Husbandry and Veterinary Medicine
目录
• Overview of English for Animal Husbandry and Veterinary Medicine
DRUGSUSEDFORANESTHESIA,MUSCLERELAXATION用于麻醉药物,肌肉松弛
• When a nerve loses depolarization and capacity to propagate an impulse, the individual loses sensation in the area supplied by the nerve.
• Example: An injectable local anesthetic which combines capsaicin with QX-314, a variation of lidocaine
c. cocaine
• Cocaine is 2 times as potent as procaine. It is used for ear, nose and throat procedures.
d. tetracaine(Pontocaine)
• Used for:
• spinal anesthesia requiring 2 to 3 hours of anesthesia
• It is 4 times more potent than procaine and has replaced procaine in the area of dental anesthesia.
c. mepivacaine (Carbocaine)
• This local anesthetic is used for dental procedures, surgical procedures and during labor and delivery.
• Procaine is the prototype drug of the local anesthetics. It has the lowest potency (except for Benzocaine).
#x #
ppic9k_man
pPIC9KVersion E03040225-0106pPIC9KA Pichia Vector for Multicopy Integration and Secreted Expression Catalog no. V175-20tech_service@iiINDIVIDUAL PICHIA EXPRESSION KIT LICENSE AGREEMENTThe Pichia Expression Kit is based on the yeast Pichia pastoris. Pichia pastoris was developed into an expression system by scientists at Salk Institute Biotechnology/Industry Associates (SIBIA) for high-level expression of recombinant proteins. All patents for Pichia pastoris and licenses for its use as an expression system are owned by Research Corporation Technologies, Inc. Tucson, Arizona. Invitrogen has an exclusive license to sell the Pichia Expression Kit to scientists for research purposes only, under the terms described below. Use of Pichia pastoris by commercial corporations requires the user to obtain a commercial license as detailed below. Before using the Pichia Expression Kit, please read the following license a greement. If you do not agree to be bound by its terms, contact Invitrogen within 10 days for authorization to return the unused Pichia Expression Kit and to receive a full credit. If you do agree to the terms of this Agreement, please complete the User Registration Card and return it to Invitrogen before using the kit.INDIVIDUAL PICHIA EXPRESSION KIT LICENSE AGREEMENTInvitrogen Corporation (INVITROGEN) grants you a non-exclusive license to use the enclosed Pichia Expression Kit (EXPRESSION KIT) for academic research or for evaluation purposes only. The EXPRESSION KIT is being transferred to you in furtherance of, and reliance on, such license. You may not use the EXPRESSION KIT, or the materials contained therein, for any commercial purpose without a license for such purpose from RESEARCH CORPORATION TECHNOLOGIES, INC., Tucson, Arizona. Commercial purposes include the use in or sale of expressed proteins as a commercial product, or use to facilitate or advance research or development of a commercial product. Commercial entities may conduct their evaluation for one year at which time this license automatically terminates. Commercial entities will be contacted by Research Corporation Technologies during the evaluation period regarding the purchase of a commercial license.Access to the EXPRESSION KIT must be limited solely to those officers, employees and students of your institution who need access thereto in order to perform the above-described research or evaluation. You must inform each of such officer, employee and student of the provisions of this Agreement and require them to agree, in writing, to be bound by the provisions of this Agreement. You may not distribute the EXPRESSION KIT to others, even those within your own institution. You may transfer modified, altered or original material from the EXPRESSION KIT to a third party following notification of INVITROGEN such that the recipient can be licensed. You may not assign, sub-license, rent lease or otherwise transfer this License or any of the rights or obligation hereunder, except as expressly permitted.This License is effective until terminated. You may terminate it at any time by destroying all Pichia expression products in your control. It will also terminate automatically if you fail to comply with the terms and conditions of the Agreement. You shall, upon termination of the License, destroy all Pichia Expression Kits in your control, and so notify INVITROGEN in writing.This License Shall be governed in its interpretation and enforcement by the laws of the State of California.Product User Registration CardPlease complete and return the enclosed Product User Registration Card for each Pichia Expression Kit that you purchase. This will serve as a record of your purchase and registration and will allow Invitrogen to provide you with technical support and manual updates. It will also allow Invitrogen to update you on future developments of and improvements to the Pichia Expression Kit. The agreement outlined above becomes effective upon our receipt of your User Registration Card or 10 days following the sale of the Pichia Expression Kit to you. Use of the kit at any time results in immediate obligation to the terms and conditions stated in this Agreement.Technical ServicesInvitrogen provides Technical Services to all of our registered Pichia Expression Kit users. Please contact us if you need assistance with the Pichia Expression Kit.United States Headquarters:Japanese HeadquartersEuropean Headquarters:Invitrogen Corporation1600 Faraday AvenueCarlsbad, CA 92008 USATel: 1 760 603 7200Tel (Toll Free): 1 800 955 6288 Fax: 1 760 602 6500E-mail:tech_service@ Invitrogen Japan K.K.Nihonbashi Hama-Cho Park Bldg. 4F2-35-4, Hama-Cho, NihonbashiTel: 81 3 3663 7972Fax: 81 3 3663 8242E-mail: jpinfo@Invitrogen Ltd3 Fountain DriveInchinnan Business ParkPaisley PA4 9RF, UKTel (Free Phone Orders): 0800 269 210Tel (General Enquiries): 0800 5345 5345Fax: +44 (0) 141 814 6287E-mail: eurotech@iiiivTable of ContentsTable of Contents (v)Important Information (vi)Introduction (1)Overview (1)Materials (4)pPIC9K (6)Methods (8)Cloning into pPIC9K (8)Analysis of E. coli Transformants (11)Transformation into Pichia (12)pPIC9K: In Vivo Screening of Multiple Inserts (14)Appendix (18)Recipes (18)Pichia Genomic DNA Isolation (19)Easy-DNA™ Protocol for Isolation of DNA from Pichia (21)Determination of Copy Number of Multiple Integrants (22)Technical Service (24)Purchaser Notification (26)References (28)vImportant InformationContents 20 µg of lyophilized pPIC9K is supplied.Add 20 µl of sterile, deionized water to resuspend to a final concentration of 1 µg/µl.Store at -20°C.Storage Conditions Lyophilized vectors are stored at -20°C.Resuspend in sterile, deionized water and store at -20°C.Product Qualification pPIC9K is qualified by restriction digest. Restriction digests must demonstrate the correct banding pattern when electrophoresed on an agarose gel. The table below lists the restriction enzymes used to digest the vector and the expected fragments.Vector Restriction Enzyme Expected Fragments (bp) pPIC9K Eco R IXho IBgl IIHin d III92764759, 45176874, 24024959, 3596, 370, 339, 12vi1IntroductionOverviewIntroduction Multiple copy integration of recombinant genes in Pichia has been demonstrated to increase expression of the desired gene in some cases (Brierley, et al., 1994; Clare, et al.,1991a; Cregg, et al., 1993; Romanos, et al., 1991; Scorer, et al., 1993; Scorer, et al., 1994; Thill, et al., 1990; Vedvick, et al., 1991.). The vector included in this kit allows isolation of multicopy inserts by an in vivo method, in order to test whether increasing the copy number of your recombinant gene will lead to a subsequent increase in secreted protein expression. This in vivo method utilizes resistance to Geneticin ® (G418 sulfate) to screen for possible multicopy inserts. Frequency of Multicopy Inserts Multiple plasmid integration events occur spontaneously in Pichia at a frequency between 1 and 10% of all His + transformants. The in vivo method allows you to screenfor the His + transformants that may have multiple inserts of your gene.Generation of Multicopy Inserts in vivo pPIC9K contains the bacterial kanamycin gene (kan from Tn 903) that confers resistance to Geneticin ® in Pichia . Note that kan does not confer resistance to kanamycin in Pichia . The level of Geneticin ® resistance roughly depends on the number of kanamycin genesintegrated. A single copy of pPIC9K integrated into the Pichia genome confers resistance to Geneticin ® to a level of ~0.25 mg/ml. Multiple integrated copies of pPIC9K canincrease the Geneticin ® resistance level from 0.5 mg/ml (1-2 copies) up to 4 mg/ml (7-12 copies). Because of the genetic linkage between the kanamycin gene and the "expression cassette" (P AOX1 and your gene of interest), one can infer that Geneticin ® resistant clones contain multiple copies of your gene. Secreted protein expression may increase because of a gene dosage effect. Thus, the presence of the kan gene on pPIC9K can be used as a tool to detect pPIC9K transformants that harbor multiple copies of the your gene. The graphic below shows multiple insertion of your expression cassette linked to the kan gene.(2nd Insertion Event3rd Insertion Event, etc.((continued on next pageScreening on Geneticin®Direct selection of Geneticin® resistance in yeast does not work well because newly transformed cells need time to express sufficient amounts of the resistance factor. Since yeast grow much more slowly than bacteria, significant numbers of recombinant yeast are killed before they accumulate enough of the resistance factor to survive direct plating on antibiotic. Do not use Geneticin® resistance as a selectable marker. The procedure to generate Geneticin® resistant clones requires an initial selection of His+ transformants followed by a screen for varying levels of Geneticin® resistance. Resistance to Geneticin® conferred by the kanamycin gene present on pPIC9K is used as a SCREEN, not as a SELECTION for multicopy integrants.Alternatives for Generating Multicopy Inserts In addition to this vector for secreted expression, Invitrogen has available two othervectors, pPIC3.5K and pAO815. These vectors are designed for intracellular expressionof recombinant proteins. pPIC3.5K also uses Geneticin® resistance to screen formulticopy inserts. pAO815 is used to construct multiple copies of your gene in vitro priorto transformation into Pichia. Multiple copies are cloned in tandem into pAO815, thentransformed into Pichia. When His+ transformants are selected, they will contain multiplecopies of your gene. A summary of the advantages and disadvantages of each method ispresented in the table below. The "best" method is the one that works for your protein; unfortunately, there is no way to predict beforehand which method will work for you.In vitro Method (pAO815)Advantages Disadvantages • Quantitative--construction of adefined number of multimers• More work up front to clone definednumber of multimers• Most of the His+ transformants willcontain the proper, defined number ofinserts• Size of the vector may become quitelarge depending on the size of yourgene and the number of copies youcreate• Isolation of recombinants withmultiple inserts is easier because mostof the His+ transformants will containmultiple copies of your gene• RearrangementsinE. coli may occur• In vitro construction allows step-wiseanalysis of copy number effects onprotein expression• Multiple inserts are located at a singlelocus• No need for a second drug resistancemarker in the vectorcontinued on next page2Alternatives for Generating Multicopy Inserts, continuedIn vivo Method (pPIC3.5K and pPIC9K)Advantages Disadvantages• Easier to initiate experiment because only one copy of your gene is clonedinto pPIC3.5K or pPIC9K beforetransforming into Pichia • Qualitative screen-- Geneticin®resistance roughly correlates with thenumber of copies of your gene.• Identifies the 1-10% of spontaneous His+ transformants that have multipleinserts • ScreeningHis+ transformants may involve more work because you willneed thousands of His+ transformantsto generate enough Geneticin®resistant colonies to test• Average size of vector is similar to other Pichia expression vectors • The number of multiple inserts is unknown (although this can bedetermined through Southern or dotblot analysis)• Multiple inserts are located at a single locus • Screening on Geneticin® is sensitiveto the density of the cells and mayresult in the isolation of false positives3MaterialsContents 20 µg of lyophilized pPIC9K is supplied.Add 20 µl of sterile, deionized water to resuspend to a final concentration of 1 µg/µl.Store at -20°C.Materials Supplied by the User For the procedures described in this manual, you will need:• Manual from the Pichia Expression System• Microbiological equipment• Electrocompetent or chemically competent E. coli (must be rec A, end A) for transformation. You will need 3-4 tubes of competent cells per experiment. For protocols to prepare competent E. coli and transformation protocols, see Current Protocols (Ausubel, et al., 1990) or Molecular Biology: A Laboratory Manual (Sambrook, et al., 1989)• Sterile water• Phenol/chloroform• 3 M sodium acetate• 100% ethanol• 80% ethanol• T4 Ligase (2.5 units/µl)• 10X Ligation Buffer (with ATP)• LB medium• LB-ampicillin plates (50-100 µg/ml ampicillin)• 16°C, 37°C, and 65°C water baths or temperature blocks• Geneticin®• YPD- Geneticin® plates (see Recipes, page 18)• 50 ml conical centrifuge tubes• Hemacytometer• 30°C and 37°C incubator• Microtiter plates (optional)Important Registered Pichia users should already have the Pichia Expression System and the current manual. Procedures for transformation into E. coli and Pichia, analysis of recombinants, and expression are described in the Pichia manual. This manual is available for downloading from our Web site () or by contacting Technical Service (page 24).continued on next page4Materials, continuedOther PichiaProductsOther Pichia products available from Invitrogen are described below:Item PurposeReactionsorAmountCatalog no.Pichia Expression Kit Complete Kit for GeneExpression in Pichia pastoris10-50 K1701-01Pichia Spheroplast Module Preparation of Pichiaspheroplasts10-50 K1720-015´ and 3´ AOX1 Primers PCR to confirm Pichiarecombinants2 µg each N740-02pPIC3.5K Forin vivo isolation of multiplecopy inserts for intracellularexpression20 µg V173-20 pAO815 Forin vitro construct ofmultiple copy inserts forintracellular expression20 µg V180-20pPIC9KFeatures of pPIC9K The table below describes the features of the pPIC9K expression vector.Feature Description Benefit5´ AOX1An ~1000 bp fragment containingthe AOX1 promoterAllows methanol-inducible high levelexpression in PichiaTargets plasmid integration to theAOX1 locus.α-FactorSignalSequence269 bp fragment encoding theα-factor signal sequence forsecretion in PichiaAllows secretion of desired proteininto the mediumMCS Multiple Cloning Site Allows insertion of your gene into theexpression vectorTT Native transcription terminationand polyadenylation signal fromAOX1 gene (~260 bp)Permits efficient transcriptiontermination and polyadenylation ofthe mRNAHIS4Pichia wild-type gene coding forhistidinol dehydrogenase (~2.4 kb)and used to complement Pichiahis4 strainsProvides a selectable marker to isolatePichia recombinant strains3´ AOX1Sequences from the AOX1 genethat are further 3´ to the TTsequences (~650 bp)Targets plasmid integration at theAOX1 geneAmppBR322originAmpicillin resistance geneE. coli origin of replicationAllows selection, replication, andmaintenance in E. coliNot IBgl IISac ISal IUnique restriction sites Permits linearization of vector forefficient integration into the Pichiagenome and generation of either Mut+or Mut S recombinantskan Kanamycin resistance gene fromTn903 which confers resistance toGeneticin® in Pichia andkanamycin resistance in E. coliAllows in vivo screening formulticopy inserts by increasedresistance to Geneticin®Also allows selection for kanamycinresistance in E. coliThere is no yeast origin of replication in any of the Pichia expression vectors included in this kit. His+ transformants can only be isolated if recombination occurs between the plasmid and the Pichia genome.continued on next pagepPIC9K, continuedDescription The vector pPIC9K is identical to pPIC9 except for the presence of the kanamycinresistance gene for in vivo screening of multiple copy inserts. pPIC9K is functional inPichia strains GS115 and KM71. Other details are:• 9276 bp fusion vector• Four unique restriction sites for cloning in frame with the α-factor secretion signal:Sna B I, Eco R I, Avr II, Not I• Secreted expression of your gene using the α-factor secretion signal• For expression, your gene must be cloned in frame with the initiation codon of thesignal sequence.• HIS4 selection in Pichia• For gene replacement at AOX1 in GS115, linearize with Bgl II (generates His+ Mut S)• For insertion at AOX1 in GS115 or KM71, linearize with Sac I (generates His+ Mut+in GS115 and His+ Mut S in KM71)• For insertion at HIS4, linearize with Sal I (generates His+ Mut+ in GS115 and His+Mut S in KM71)See page 12 for alternate restriction sites if your insert DNA has a Bgl II, Sac I, or Sal Isite.Map of pPIC9K The figure below shows the map of pPIC9K. Details of the multiple cloning site and the α-factor secretion signal are shown on page 10. The complete sequence of pPIC9K isavailable for downloading from our Web site () or bycontacting Technical Service (page 24).Comments for pPIC9K:9276 nucleotides5´ AOX1 promoter fragment: bases 1-9485´ AOX1 primer site: bases 855-875a-Factor secretion signal(s): bases 949-1218 a-Factor primer site: bases 1152-1172 Multiple Cloning Site: bases 1192-12413´ AOX1 primer site: bases 1327-13473´ AOX1 transcriptiontermination (TT): bases 1253-1586 HIS4 ORF: bases 4514-1980Kanamycin resistance gene: bases 5743-4928 3´ AOX1 fragment: bases 6122-6879pBR322 origin: bases 7961-7288 Ampicillin resistance gene: bases 8966-8106aBIoRIrIItISal IMethodsCloning into pPIC9KIntroduction It is important to clone your gene in frame with the α-factor signal sequence. Below are some guidelines to consider when developing a cloning strategy for this vector. Refer topage 10 for the multiple cloning site of pPIC9K.We recommend that you transform pPIC9K into E. coli, so that you have a permanentstock and a way to make more plasmid.• Dilute 1 µl of the plasmid (1 µg/µl) to 10-100 pg/µl using sterile water or TE buffer.• Transform competent E. coli with 1-2 µl of the diluted plasmid and select on LB with50-100 µg/ ml ampicillin (LB-Amp).GeneralConsiderationsThe following are some general considerations applicable to pPIC9K.• The codon usage in Pichia is believed to be the same as Saccharomyces cerevisiae.• Many Saccharomyces genes have proven to be cross-functional in Pichia.• Plasmid constructions should be maintained in a rec A, end A mutant E. coli strain suchas TOP10. Electrocompetent TOP10 cells are available from Invitrogen.Item AmountCatalogno.10 x 50 µl (500 µl total) C4040-50One Shot® Top 10 Electrocomp™20 x 50 µl (1 ml total) C4040-525 x 80 µl (400 µl total) C664-55TOP10 Electrocomp™10 x 80 µl (800 µl total) C664-11• The native 5´ end of the AOX1 mRNA is noted in the multiple cloning site (see page10). This is needed to calculate the size of the expressed mRNA of the gene of interestif you need to analyze mRNA for any reason.• Translation termination is determined by either stop codons in the gene of interest or inthe 3´ AOX1 sequence. The stop codons in the 3´ AOX1 sequence are noted in themultiple cloning site (see page 10).• The premature termination of transcripts because of "AT rich regions" has beenobserved in Pichia and other eukaryotic systems (Henikoff and Cohen, 1984; Irniger,et al., 1991; Scorer, et al., 1993; Zaret and Sherman, 1984). If you have problemsexpressing your gene, check for premature termination and AT rich regions. It may benecessary to change the sequence in order to expressed your gene (Scorer, et al.,1993).• The predicted protease cleavage sites for the α-factor signal sequences are indicated inin the multiple cloning site (see page 10).• The open reading frame (ORF) of the mature gene of interest should be cloned inframe and downstream of the α-factor signal sequence.continued on next pageGeneral Cloning Strategies Strategies generally fall into three different categories:1. Ligation of a compatible restriction fragment:a) Forced (directional) insertion involving the use of two different sites in themultiple cloning site.b) Ligation of the fragment with the same restriction end on both ends into asingle, compatible site.2. PCR amplification of the fragment containing the gene of interest in such a way thatcompatible restriction ends are generated for ligation into the appropriate vector. 3. Direct cloning of an amplified fragment containing the gene of interest via the TACloning® Kit (Catalog no. K2000-01), followed by subcloning of a compatiblefragment into pPIC9K.Cloning Procedures Refer to (Ausubel, et al., 1990), pages 3.16.1 to 3.17.3. or (Sambrook, et al., 1989), pages 5.10 to 5.13. for help with cloning.Signal Sequence Processing The processing of the α-factor mating signal sequence in pPIC9K occurs in two steps: 1. The preliminary cleavage of the signal sequence by the KEX2 gene product, withthe final Kex2 cleavage occurring between arginine and glutamine in the sequence Glu-Lys-Arg * Glu-Ala-Glu-Ala, where * is the site of cleavage.2. The Glu-Ala repeats are further cleaved by the STE13 gene product.Optimization of Signal Cleavage In Saccharomyces cerevisiae, it has been noted that the Glu-Ala repeats are not necessary for cleavage by Kex2, but cleavage after Glu-Lys-Arg may be more efficient when followed by Glu-Ala repeats. A number of amino acids are tolerated at site X instead of Glu in the sequence Glu-Lys-Arg-X. These amino acids include the aromatic amino acids, small amino acids, and histidine. Proline, however, will inhibit Kex2 cleavage. For more information on Kex2 cleavage, see Brake, et al., 1984.There are some cases where Ste13 cleavage of Glu-Ala repeats is not efficient, and Glu-Ala repeats are left on the N-terminus of the expressed protein of interest. This is generally dependent on the protein of interest.Bacterial Transformation Once you have decided on a cloning strategy, you will need to prepare competent E. coli cells for transformation before setting up your ligation reactions. See Current Protocols in Molecular Biology (Ausubel, et al., 1990) or Molecular Biology: A Laboratory Manual (Sambrook, et al., 1989) for preparation of electrocompetent or chemically competent E. coli or use your laboratory's procedure.continued on next pageP AOX1 and Multiple Cloning Site of pPIC9K The sequence below shows the detail of the multiple cloning site and surrounding sequences.Sna BI AOX1 mRNA 3' end (1418)AGGCTTCATT TTTGATACTT TTTTATTTGT AACCTATATA GTATAGGATTSpecial Considerations • The fragment containing the gene of interest must be cloned in frame with the secretion signal open reading frame.• An initiating ATG is provided by the signal sequence. Translation will initiate at the ATG closest to the 5´ end of the mRNA.• If your insert has a Bgl II site, see page 12 for alternate restriction sites to linearize your plasmid for Pichia transformation.Analysis of E. coli TransformantsIntroduction At this point you should have ligation reactions that you will transform by chemicalmeans or electroporation into competent E. coli cells (TOP10 or equivalent) using yourmethod of choice.Analysis of Transformants 1. After transformation, plate 10 µl and 100 µl of the transformation mix onto LBplates with 50-100 µg/ml ampicillin (see Recipes, page 18) and select ampicillin resistant colonies.2. Pick 10 ampicillin resistant transformants and inoculate into 2 ml LB medium with50-100 µg/ml ampicillin. Grow overnight at 37°C with shaking.3. Isolate plasmid DNA by miniprep for restriction analysis and sequencing (seebelow). To sequence your construct in pPIC9K, use the α-factor and the 3´ AOX1 primer sequences (see below).4. Make a glycerol stock of your desired clone for safekeeping by combining 0.85 mlof a overnight bacterial culture with 0.15 ml of sterile glycerol. Mix by vortexing and transfer to a labeled storage tube. Freeze the tube in liquid nitrogen or a dry ice/ethanol bath and store at -70°C.Sequencing Recombinant Clones We strongly recommend that you sequence your construct before transforming into Pichia to confirm that your gene is in frame with the α-factor secretion signal ATG. We suggest using the α-factor and 3´ AOX1 primer sequences to sequence your construct. Refer to the diagram on the previous page for the sequence and location of these primer binding sites.For sequencing protocols, refer to Unit 7 in Current Protocols in Molecular Biology (Ausubel, et al., 1990) or Chapter 13 in Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989).After Sequencing Once you have cloned and sequenced your insert, proceed to Transformation intoPichia, page 12. You will need to generate enough plasmid DNA to transform Pichia (5-10 µg of each plasmid per each transformation).Transformation into PichiaIntroduction At this point you will have your gene cloned in pPIC9K. You should also have about 5-10 µg of each construct for each transformation into Pichia. For methods to transformPichia and select His+ transformants, refer to the Pichia Expression System manual. Tolinearize your construct prior to transformation into Pichia, see below.Linearization of Plasmid DNA It is recommended that you linearize your vector in such a manner to generate both Mut+ and Mut S recombinants. It is possible that one phenotype will express your multicopy integrant better than the other. To linearize pPIC9K containing one copy of your gene: • Bgl II for replacement at AOX1 (GS115, Mut S)• Sac I for insertion at AOX1 (GS115, Mut+ or KM71, Mut S)• Sal I for insertion at HIS4 (GS115, Mut+ or KM71, Mut S)Use strain KM71 if you only want Mut S recombinants. If your insert contains any of the above restriction sites, see the table below for alternate sites.Alternate Restriction SitesThe table below describes alternate restriction sites for linearizing your construct before transformation into Pichia.pPIC9K. Note that an additional Stu I site was added with the inclusion of the kan gene, eliminating the unique Stu I site in HIS4.RestrictionEnzyme5´ AOX1 3´AOX1 Vectorbackbone HIS4 geneSac I 209 -- -- --Pme I 414 -- -- --Bpu 1102 I 589 -- -- --Xcm I 699 -- -- --Aat II* (9102) -- -- --Tth III I* -- (7034) -- --Bgl II† 2 6875 -- -- Dra I†414 6713 6855, 8046, 8065,8757--Sal I -- -- -- 3178Bsp E I-- -- -- 3845 *Restriction sites are outside the AOX1 sequences in the vector backbone, but they are closeenough for efficient recombination to occur.†Restriction sites are used to generate gene replacements at AOX1 in GS115 only.continued on next page。
褪黑素受体Mel 1b基因mRNA和蛋白在鸭不同组织中的表达与分布
浙江农业学报！#!&()(&*! 2018，％0(5): 711 -716 http://ww 刘文举，王淑娟，刘晓丽，等.褪黑素受体D*1B基因mRNA和蛋白在鸭不同组织中的表达与分布％ J].浙江农业学报，2018，％0(5): 711 -716.DOI: 10.3969// issn. 1004-1524. 2018. 05. 06褪黑素受体10基因mRNA和蛋白在鸭不同组织中的表达与分布刘文举1，王淑娟1!!，刘晓丽2,庞训胜1，王立克1(1安徽科技学院动物科学学院，安徽凤阳233100 ;2•华中农业大学动物医学院，湖北武汉430070#摘要:褪黑素广泛的生理作用是通过与其膜受体（Mel 1a、Mel 1b, Mel 16结合介导的。
褪黑素受体在机体内广泛分布，但因受体亚型、物种、组织不同而异。
采用RT-PCR、Nal-time PC R和免疫组化技术探究了 D*1B在鸭不同组织（包括鸭脾脏、心脏、肾脏、大脑、胸肌、卵巢、肺脏、肝脏、胰脏组织）中的表达分布及相对表达量。
结果表明，鸭D*1B mRNA在脾脏、心脏、肾脏、大脑、胸肌、卵巢、肺脏、肝脏、胰脏中均有表达，且M' 1b受体蛋白均存在于大脑、肺脏、肝脏、胸肌、肾脏、心脏、胰脏中（脾脏、卵巢未成功测试）。
R eal-m e PC R结果表明，各组织中D*1BmRNA表达量存在差异，在卵巢中的表达量最高，其次为肺脏，在脾脏、心脏、肾脏和肝脏中的表达量也明显高于大脑中的表达，但在胸肌和胰脏中的表达量要稍低于大脑中的表达量。
M' 1b在鸭各组织中普遍表达分布，进一步说明了其介导褪黑素广泛的生理功能，特别是对生殖功能的调节。
关键词：褪黑素受体;Mel 1b;鸭;real-time PCR中图分类号:S834 文献标志码:A 文章编号:1004-1524(2018)05-0711-06Expression and distribution of Mel 10 mRNA and protein in various tissues of duckLlUW enju1，WANGShujuan1，！，LlUXiaoli2，PANGXunBeng1，WANGLike1(1. College o f A nimal Science, Anhui Science and Technology University，Fengyang 233100, China; 2. College ofAnimal Science，Huazhong Agricultural University，Wuhan 430070，China)Abstract ：A broad p hysiological function of melatonin is mediated by binding to melatonin receptors ( Mel 1 a，Mel1 b and Mel 1c) . Melatonin receptors are widely distributed in the body，but vary accotissues. The study investigated the expression，distribution of DeZ 1B in different tissues of duck spleen，brain，kidney，liver，lung，pancreas and breast muscle) using RT-PCR，real time PCR and immunohisto-chemistry. The results s howed that Del1 b mRNA was extensively expressed in heart，spleen，brain，kidney，liver，lung，pancreas a nd breast muscle of ducks，and Mel 1b protein was distributed in brain，lung，liver，skeletal mus-cle，kidney，heart a nd pancreas. The expression levels of Del1b mRNA were diferent among various tissues. Thehighest expression of Del 1b mRNA was found in ovary，followed by the lung. Compared with the expression level ofMel 1b in brain，the expression levels in spleen，heart kidney and liver were higher，but those in breast muscle andpancreas were lower. Thus，Mel 1b was widely distributed in duck tissues，which f urther confirm收稿日期:2017-09-25基金项目：国家自然科学基金(31301972);安徽省自然科学基金（1308085QC66);安徽省优秀青年人才支持计划（gxyq2018049);安徽省 教育厅自然科学基金（kj20136077)作者简介:刘文举（1981 —)，男，河南驻马店人，硕士，从事动物生殖生理研究。
usefulexpression
usefulexpression1. flagrant: (of an action) shocking because it is done in a very obvious way and shows no respect for people, laws, etc. 骇人听闻的；公然的T: The most flagrant scofflaw of them all is the red-light runner.M: He cheated on the final examination, showing a flagrant disregard for examination rules.2. brazen:(disapproving) open and without shame, usually about sth that people find shocking 厚颜无耻的T: The red-light runner, however, shows no respect whatever for the social rules, and society cannot help being harmed by any repetitious and brazen display of contempt for the fundamentals of order.M: She had become brazen about destroying other’s family.3. hypocrisy: behavior in which sb pretends to have moral standards or opinions that they do not actually have 伪善；虚伪T: If hypocrisy is the tribute that vice pays to virtue, then furtiveness is the true outlaw's salute to the force of law-and-order.M: He condemned the hypocrisy of those politicians who do one thing and say another.4. undermine:to make sth, especially sb’s confidence or authority, gradually weaker or less effective 逐渐削弱（信心，权威等）；使逐步减少效力T: People wiped out Prohibition at last not only because of the alcohol issue but because scofflawry was seriously undermining the authority and legitimacy of government.M: Our confidence in the team has been seriouslyundermined by their recent defeats.1. interrogate:~sb to ask sb a lot of questions over a long period of time, especially in an aggressive way 讯问；审问；盘问T：I am interrogating, I am cross-examining, I am prying and probing for the meaning of a student’s paper.M: Peter was regarded as a suspect of the murder and he was interrogated by the police for over four hours.2. affliction: pain and suffering or sth that cause it 折磨；痛苦T: Taking hi cue from years of higher education, years of reading the textbook and professional journals that are the major sources of his affliction, he writes: “The focus of concentration must rest upon objectives centered around the knowledge of customer areas so that a sophisticated awareness of those areas can serves as an entrepreneurial filter to screen what is relevant from what is irrelevant to future commitments.”M: We sympathize with people in affliction.3. plight: a difficult and sad situation 苦难；困境T：Despite all the current fuss and bother about the extraordinary number of ordinary illiterates who overpopulate our schools, small attention has been given to another kind of illiterate, an illiterate whose plight is, in many ways, more important, because he is more influential.M: After the earthquake, the people in this area is in a desperate plight.4. grapple with sth: to try hard to find a solution to a problem 努力设法解决T: If he is majoring in sociology, he must grapple with such journals as the American Sociological Review, journals bulging with barbarous jargon, such as “ego-integrative action orientation”a nd “orientation toward improvement of the gratificational-deprivation balance of the ac tor” (the latterof which monstrous phrases represents, to quot e Malcolm Cowley, the sociologist’s way of saying “the pleasure principle”).M: The new government has yet to grappling with the problem of air pollution.Unit 71. Deprive sb/sth of sth:to prevent sb from having or doing sth, especially sth important 剥夺；使丧失；使不能享有T: It was principally the influence of Christianity that deprived beauty of the central place it had in classical ideals of human excellence.M: They are imprisoned and deprived of their basic rights.2. Interminable: lasting a very long time and therefore boring or annoying 冗长的；没完没了的T: One could hardly ask for more important evidence of the dangers of considering persons as split between what is "inside" and what is "outside" than that interminable half-comic half-tragic tale, the oppression of women.M: Their quarrels about the trivia are always interminable.3. Reinforce:① to make a feeling, an idea, etc. stronger 加强；充实；使更强烈T: It does not take someone in the throes of advanced feminist awareness to perceive that the way women are taught to be involved with beauty encourages narcissism, reinforces dependence and immaturity.M: Your positive response will reinforce her actions.② to make a structure or material stronger, especially b y adding another material to it 加固；使更结实M：All buildings are now reinforced to withstandearthquakes.③ to send more people or equipment in order to make an army, etc. stronger 给……加强力量（或装备）；使更强大M: The UN has undertaken to reinforce its military presence along the borders.4. Enchantment: a feeling of great pleasure 狂喜；陶醉T: By limiting excellence (virtus in Latin) to moral virtue only, Christianity set beauty adrift ---- as an alienated, arbitrary, superficial enchantment.M: The beauty of the scene filled us with enchantment.Unit 81. Imperceptible: very small and therefore unable to be seen or felt 无法察觉的；感觉不到的T: Besides, the whole toffeeness of toffees was imperceptibly diminished by the gross act of having eaten it.M: Lily used a kind of whitening product for a month, but the change is imperceptible.2. Inexhaustible:that cannot be entirely consumed or used up 用之不竭的；无穷无尽的T: No, the best was in wanting it, in sitting and looking at it, when one tasted an inexhaustible treasure - house of flavours.M: She has worked for nearly a month without rest. It seems that her energy is inexhaustible.3. Flawless: without flaw and therefore perfect 完美的；无暇的T: For in this condition, of course, I know that the object of desire is always at its most flawlessly perfect.M: He has prepared for a long time, so his performance was almost flawless.4. Homage: something that is said or done to show respect for sb 敬辞；表示敬意的举动T: Fasting is an act of homage to the majesty of appetite.M: He shows his homage to the king.Unit 91. Pronounced: very noticeable, obvious or strongly expressed 显著的；很明显的；表达明确的T: In such a land Lee stood for the feeling that it was somehow of advantage to human society to have a pronounced inequality in the social structure.M: He has very pronounced views on art.2. Tenacious:continuing to exist, have influence, etc. for longer than you might expect 顽强的；坚韧不拔的T: The Westerner, on the other hand, would fight with an equal tenacity for the broader concept of society.M: She is very tenacious and will work hard and long to achieve objectives.3. Indomitable: not willing to accept defeat, even in a difficult situation; very brave and determined 不屈不挠的；勇敢坚定的T: In each man there was an indomitable quality ... the born fighter's refusal to give up as long as he can still remain on his feet and lift his two fists.M: It'll require indomitable will to accomplish the task.。
Unit 3 Words and Expressions高中英语(人教版2019选择性必修第三册)
最新的进展情况已向大众传播媒介发布。
【练透】 单句语法填空
①The factory keeps releasing
（release） smoke， making the air dirty.
②The kind girl decided to release the bird from
the cage.
1. The problem was how to get enough food to sustain life. 维持
问题在于如何找到足够的食物维持生命。 2. The company sustained losses of millions of dollars. 遭受
公司遭受了数百万美元的巨大损失。 3. Arches sustain the weight of the roof. 承受 拱形结构承受着屋顶的重量。 sustain damage/an injury/a defeat/heavy losses
The mountain climber could not find many footholds on the _m__e_lt_in_g_ (melt) ice.
溶解成；消散在……中 melt into
消失，消散；逐渐散去 melt away
使某人的心变软
melt one's heart
熔点
regions
.
令我们震惊的是，每年都有许多e欠ith发er达di地e f区ro的m 孩the子co死ld于or饥st饿arve to death
⑥If the situation goes on, they’ll
.
【写美】 完成句子 许多人渴望成功，但他们往往因为缺乏决心而失败。
was exhibited higher levels of expression -回复
was exhibited higher levels of expression -回复"Was exhibited higher levels of expression" refers to a situation where someone or something displayed increased or elevated levels of expression. In this article, we will explore the meaning of expression, the factors that contribute to higher levels of expression, and the significance of expressing oneself authentically.Expression is a fundamental aspect of human communication and creativity. It allows individuals to convey their thoughts, emotions, and perspectives. Whether it is through art, music, writing, or body language, expression serves as a powerful tool that connects people on a deeper level. When someone exhibits higher levels of expression, it signifies a greater intensity or richness in their communication.There are several factors that can contribute to higher levels of expression. First and foremost is self-awareness. Being aware of one's own thoughts and emotions is essential to articulate oneself effectively. When individuals have a deeper understanding of themselves, they can express their innermost feelings and ideas more authentically. This is why self-reflection and introspection play a significant role in nurturing higher levels of expression.Another factor is practice. Like any skill, expression requirespractice and effort to improve. Whether it is practicing an instrument, honing writing skills, or refining public speaking abilities, regular practice enables individuals to refine their expressions and develop their unique style. Through practice, individuals can find their voice and discover innovative ways to communicate effectively.Moreover, inspiration plays a vital role in increasing expression. Inspiration can come from various sources such as nature, people, experiences, or art. When individuals feel inspired, their creative energy is heightened, enabling them to express themselves with more enthusiasm and passion. This influx of inspiration often leads to higher levels of expression.Furthermore, a supportive environment fosters higher levels of expression. When individuals feel encouraged and appreciated, they are more likely to express themselves freely. On the contrary, judgment or criticism can dampen one's expression. Creating a safe space where individuals feel safe to express themselves without fear of judgment or rejection is crucial for promoting higher levels of expression.Expressing oneself authentically is of great significance in personal development and fulfillment. When individuals express themselves honestly, it allows them to explore their own beliefs,values, and passions. This process of self-discovery leads to a deeper understanding of oneself and enables individuals to live more fulfilling lives. Authentic expression also allows individuals to connect with others on a genuine level, fostering meaningful relationships and establishing a sense of belonging.Higher levels of expression can also contribute to personal growth and resilience. It provides a healthy outlet for emotions, allowing individuals to manage stress, anxiety, or any other psychological challenges. Expression serves as a form ofself-expression therapy, allowing individuals to cope with their emotions in a constructive manner. By expressing themselves, individuals can process their feelings and experiences, fostering personal growth and resilience.In conclusion, "was exhibited higher levels of expression" refers to an increased or elevated display of communication intensity or richness. Factors such as self-awareness, practice, inspiration, and a supportive environment contribute to higher levels of expression. Expressing oneself authentically holds great significance in personal development, fulfillment, and resilience. It allows individuals to connect with others genuinely and fosters personalgrowth. Embracing and nurturing higher levels of expression is crucial for self-discovery and leading a more meaningful and fulfilling life.。
factorization the following expressions 选择题
Factorization of the Following Expressions - Multiple ChoiceQuestionsIn this document, we will explore multiple choice questions related to the factorization of various expressions. Factorization is an important concept in algebra and is a powerful technique used to simplify and solve equations. Let’s dive into the questions and test our understanding!Question 1:Factorize the expression: x^2 - 4.a.(x - 2)(x - 2)b.(x + 2)(x - 2)c.(x + 4)(x - 4)d.(x^2 - 4)Correct Answer: b. (x + 2)(x - 2)Explanation: To factorize the given expression x^2 - 4, we can use the difference of squares formula, which states that a^2 - b^2 can be factorized as (a - b)(a + b). Comparing this formula with the given expression, we can see that a is x and b is 2. Thus, the factorization of x^2 - 4 is (x + 2)(x - 2).Question 2:Factorize the expression: 4x^2 - 9y^2.a.(2x - 3y)(2x + 3y)b.(2x + 3y)(2x + 3y)c.(2x - 3y)(2x - 3y)d.(4x^2 - 9y^2)Correct Answer: a. (2x - 3y)(2x + 3y)Explanation: To factorize the given expression 4x^2 - 9y^2, we can use the difference of squares formula again. Comparing 4x^2 with a^2 and 9y^2 with b^2, we can see that a is 2x and b is 3y. Therefore, the factorization of 4x^2 - 9y^2 is (2x - 3y)(2x + 3y).Question 3:Factorize the expression: x^3 - 8y^3.a.(x - 2y)(x^2 + 2xy + 4y^2)b.(x + 2y)(x^2 - 2xy + 4y^2)c.(x - 2y)(x^2 - 2xy + 4y^2)d.(x^3 - 8y^3)Correct Answer: a. (x - 2y)(x^2 + 2xy + 4y^2)Explanation: To factorize the given expression x^3 - 8y^3, we can use the formula for the difference of cubes: a^3 - b^3 = (a - b)(a^2 + ab + b^2). In this case, a is x and b is 2y. Therefore, the factorization of x^3 - 8y^3 is (x - 2y)(x^2 +2xy + 4y^2).Question 4:Factorize the expression: 6x^2 + 11xy - 10y^2.a.(3x + 5y)(2x - 2y)b.(2x - 5y)(3x + 2y)c.(2x + 5y)(3x - 2y)d.(6x^2 + 11xy - 10y^2)Correct Answer: c. (2x + 5y)(3x - 2y)Explanation: Factoring the given expression 6x^2 + 11xy - 10y^2 can be done by splitting the middle term method. We are looking for two numbers whose product is -60 (the product of 6 and -10) and whose sum is 11 (the coefficient of thexy term). The numbers are 15 and -4. Using these numbers, we can rewrite the expression as (2x + 5y)(3x - 2y).Question 5:Factorize the expression: x^2 + 7x + 12.a.(x + 3)(x + 4)b.(x + 6)(x + 2)c.(x + 7)(x + 5)d.(x^2 + 7x + 12)Correct Answer: a. (x + 3)(x + 4)Explanation: To factorize the given expression x^2 + 7x + 12, we need to find two numbers whose sum is 7 (the coefficient of the x term) and whose product is 12. The numbers are 3 and 4. Therefore, the factorization of x^2 + 7x + 12 is (x + 3)(x + 4).Conclusion:Factorization is a valuable technique in algebra that allows us to simplify and solve equations. By understanding the formulas and methods involved, we can factorize various expressions and make complex problems more manageable. Remember to practice these concepts and explore more examples to strengthen your understanding of factorization.。
Common Expressions in Nursing English 4
Expressions for cardiac surgery
"Your heart rate and blood pressure are normal. We need to monitor your condition closure
Expressions related to die
'A balanced die is important for maintaining good health' What is your manual die like? ' Do you eat a variety of foods or stick to a few favorite dishes
Common Expressions in Nursing English 4
汇报人： 日期:
目 录
• Daily care expression • Disease Care Expression • Health education expression • Expression of numbering operation
Dietary Care Expression
Breakfast is ready
used to notify the patient that breakfast is ready
Eat slowly
used to remind the patient to eat slowly
Please drink more water
2. Please take your time and don't sit to let me know if you need anything- This presence is used to resource the patient to move slowly and not rush through the exam, while also reminding them that they can ask for assistance if needed
小学上册第八次英语第5单元期末试卷
小学上册英语第5单元期末试卷英语试题一、综合题(本题有100小题，每小题1分，共100分.每小题不选、错误，均不给分)1. A __________ (多样性) in chemical compounds contributes to the complexity of life.2.The _____ (老虎) is a fierce hunter in the jungle.3.I love to eat ______ in the summer.4.The phone is _____ (ringing/silent).5.What is the freezing point of water?A. 0 degrees CelsiusB. 100 degrees CelsiusC. 32 degrees FahrenheitD. Both A and CD6.What is 5 x 2?A. 8B. 10C. 12D. 14B7.What is the capital of the Philippines?A. ManilaB. CebuC. DavaoD. Zamboanga8.What is the opposite of happy?A. SadB. JoyfulC. ExcitedD. AngryA9.What is 20 - 8?A. 10B. 12C. 14D. 1610.The frog catches insects with its sticky ______ (舌头).11.The chemical symbol for uranium is ______.12. A cheetah is the fastest _______ in the animal kingdom.13.What do we call the process of forming clouds?A. EvaporationB. CondensationC. PrecipitationD. SublimationB14. A _______ (小剑鱼) swims swiftly in the ocean.15.How many wheels does a bicycle have?A. 2B. 3C. 4D. 5A16.The Sun is primarily composed of hydrogen and ______.17.What is the longest river in the world?A. AmazonB. NileC. MississippiD. Yangtze答案:B18.I want to _______ (买) a toy.19.The invention of the computer has changed modern _____.20.The _____ (虫子) play a role in pollinating plants.21.What do we call a person who studies the implementation of laws?A. LawyerB. JuristC. SolicitorD. AttorneyA22.My favorite animal is a ________ (小狗).23.The _____ (叶片) are essential for photosynthesis.24.What is the name of the famous American singer known for his hit song "Thriller"?A. Michael JacksonB. PrinceC. Elvis PresleyD. MadonnaA25.What is the process of keeping food cold to preserve it?A. CookingB. RefrigerationC. FreezingD. DehydrationB Refrigeration26.The ________ is a famous structure built in Paris.27.The chemical symbol for argon is ______.28. A _______ is a change that involves the formation of new substances. (化学反应)29. A physical change alters a substance without changing its ______.30.What is the name of the famous beach in Rio de Janeiro?A. CopacabanaB. IpanemaC. BondiD. Waikiki31.The ________ was a crucial battle in the campaign for independence.32.What do we call a person who studies ancient civilizations?A. ArchaeologistB. HistorianC. AnthropologistD. Paleontologist33.Gardening tools like shovels and rakes make planting ______. (铲子和耙子等园艺工具使种植变得容易。
小学上册第十二次英语第五单元测验试卷
小学上册英语第五单元测验试卷英语试题一、综合题(本题有100小题，每小题1分，共100分.每小题不选、错误，均不给分)1._____ (自然界) is full of fascinating plants.2.The ________ is the imaginary line that goes around the middle of the Earth.3.The __________ is the longest mountain range in the world. (安第斯山脉)4.The __________ (奴隶制) was abolished in many countries in the 19th century.5.The ________ (register) is open for sign-ups.6.My friend is always __________ (乐于助人的) when I need help.7.We have a ______ (小) family.8.The kitten sleeps on the _______ (小猫睡在_______上).9.ts grow best in ______ (潮湿) conditions. Some pla10.________ (植物多样性保护措施) are enacted.11.The __________ (历史的挑战者) question established narratives.12. A saturated solution can reach its limit when no more solute can be ______.13.Planetary rings are made of ice and rock ______.14.What is the capital of Libya?A. TripoliB. BenghaziC. MisrataD. SirteA15. A volcano can create new land when it erupts ______.16.The sun shines _____ (brightly/dimly).17.I like to pretend I'm a scientist with my toy ________ (玩具名称).18.The bridge is very ___ (strong).19.What is a solar system?A. A collection of starsB. A group of planets orbiting a starC. A type of galaxyD. A cloud of gas20.How many Earth years does it take for Neptune to orbit the sun?A. 15B. 84C. 165D. 25021.What is a common pet that purrs?A. DogB. CatC. BirdD. FishB22.The bird can _______ (唱歌) beautifully.23.What do you call a group of birds?A. SwarmB. FlockC. PackD. GaggleB24.There are ten _____ (students) in the class.25.What do we call a sweet drink made from tea and milk?A. ChaiB. Bubble TeaC. Iced TeaD. All of the above26.We have a ______ (有趣的) day planned for next month.27.My dog has a shiny ______ (毛发).28.The _____ (sun/cloud) is shining.29.We visit the ______ (自然博物馆) to learn about ecosystems.30.What is a common beverage made from leaves?A. CoffeeB. SodaC. TeaD. JuiceC Tea31.What do we call a scientist who studies insects?A. EntomologistB. BiologistC. ChemistD. EcologistA32.The moon is ___. (bright)33.The average distance from the Earth to the Sun is million ______.34.The bicycle has ______ (two) wheels.35.The peacock dances to attract a _________. (伴侣)36.My brother is a __________ (审计师).37.The antelope runs swiftly from danger in the ____.38.I like to race my ________ (玩具名称) with my brother.39.What do you call an animal that lives both in water and on land?A. MammalB. ReptileC. AmphibianD. FishC40.The moon affects the ______ of the oceans.41.The Milky Way is just one of billions of _____ in the universe.42.The ancient Greeks studied ________ to learn about the universe.43.Which of these is a vegetable?A. AppleB. CarrotC. BananaD. GrapeB44. f Reason emphasized logic and ________ (理性). The Alam45.What do we call a story that is made up?A. FactB. FictionC. HistoryD. Biography46.What do you call a person who studies the environment?A. EcologistB. BiologistC. GeologistD. All of the aboveD47.I have a ________ (地球仪) in my classroom.48.What do you call the person who flies a plane?A. PilotB. DriverC. CaptainD. SailorA49.The ancient Romans built _______ to entertain the public. (竞技场)50.How many states are in the USA?A. 50B. 52C. 48D. 5151.The ________ (植物演替) changes ecosystems.52.__________ can be found in everyday products like soap and shampoo.53.The __________ is an important tool for environmental management.54.My teacher is a ______. She loves to inspire students.55.The bird has bright _______ (鸟有鲜艳的_______).56.My favorite game is _______ (乒乓球).57.I enjoy visiting ______ during summer break.58.The library is _______ and quiet.59.The country with the kangaroo is __________. (澳大利亚)60.The _____ (wind) is blowing.61.This is my best . (这是我最好的。
小学上册第6次英语第一单元全练全测(含答案)
小学上册英语第一单元全练全测(含答案)英语试题一、综合题(本题有100小题，每小题1分，共100分.每小题不选、错误，均不给分)1.She is helping her mom in the ___. (kitchen)2.The ________ (海洋) is home to many animals.3.I like to ________ (experiment) with ideas.4.My dad takes care of the ________ in our backyard.5.Which of these is a natural resource?A. PlasticB. WoodC. GlassD. Metal答案:B6.The chemical symbol for potassium is ________.7.What is the name of the famous palace in France?A. Buckingham PalaceB. Palace of VersaillesC. Neuschwanstein CastleD. Schönbrunn Palace答案:B8. A meteor shower happens when many meteors enter the _____.9.What is the color of an orange?A. BlueB. GreenC. OrangeD. Purple答案:C10.What do you call a young dog?A. PuppyB. KittenC. CalfD. Chick答案:A11.They are _____ (going/come) to the picnic.12.The ______ is always smiling.13.ts grow in ______ (水中). Some pla14.An endothermic reaction absorbs ______.15.What is the main language spoken in the USA?A. SpanishB. FrenchC. EnglishD. German答案:C16.My sister is a ______. She enjoys helping at the shelter.17.Some plants are _______ and can climb walls.18.What do you call a baby turtle?A. HatchlingB. PupC. KitD. Calf19.The garden is ________ (郁郁葱葱).20.I like to draw _____ in my sketchbook.21.What is the name of the famous bridge in San Francisco?A. Golden Gate BridgeB. Brooklyn BridgeC. London BridgeD. Sydney Harbour Bridge答案:A22.The sun is shining brightly on the ______ (草地). It's a perfect day for a ______ (野餐).23.The city of Pompeii was buried under _______. (火山灰)24.The ________ is a tiny animal that lives in the forest.25.We are visiting the ___. (art gallery)26.What is the opposite of 'thin'?A. SlimB. FatC. NarrowD. Lean答案:B27.What is the capital of Egypt?A. CairoB. AlexandriaC. LuxorD. Aswan28.My friend has a lovely _______ (动物). 它的性格很 _______ (形容词).29.The capital of the Philippines is __________.30.The Earth's crust is composed of various types of ______ rocks.31.I like to write ______ (故事) and share them with my friends. It’s fun to come up with new characters and adventures.32.What is 15 - 7?A. 6B. 7C. 8D. 933.Galileo was one of the first to use a ______.34.I want to _______ a great leader.35.When it rains, I like to jump in ______ (水坑).36.We are going to ___ a trip. (take)37.The first man to fly solo across the Atlantic was ________ (林白).38.What do we call a baby elephant?A. CalfB. FoalC. KitD. Pup39.My favorite game is ________ (拼图).40.I can create a world of imagination with my toy ________ (玩具名称).41.What do you call a place where animals are kept for public display?A. ZooB. AquariumC. FarmD. Sanctuary答案:A42. A ______ (蜥蜴) can change colors to hide from predators.43. A _______ (小狸猫) is known for its playful nature.44.What is the capital of Australia?A. SydneyB. MelbourneC. CanberraD. Brisbane45._____ (植物特色) attract people to gardens.46.Penguins cannot _________ (飞).47.What is the largest organ in the human body?A. HeartB. LiverC. SkinD. Brain答案:C48.We have a ______ (快乐的) family gathering every month.49.How many players are there on a basketball team?A. 5B. 7C. 9D. 11答案:A50.The smallest unit of a compound is a _______.51.Stellar evolution describes the life cycle of a _______.52. A __________ (溶胶) is a colloidal mixture with solid particles dispersed in a liquid.53.The ______ loves to sing songs.54.The Earth's surface is covered with about ______ percent water.55.The process of ______ can lead to the breakdown of rocks.56.What do you call the leader of a country?A. PresidentB. MayorC. GovernorD. Senator57.My brother has a _____ collection of toys. (large)58.Planting _____ (本地树种) contributes to ecological stability.59.Which fruit is yellow and curved?A. OrangeB. BananaC. AppleD. Grape答案:B60.How many hearts does an octopus have?A. 1B. 2C. 3D. 4答案:C61. A sound that is loud has a high ______ (amplitude).62.The capital city of Canada is _______.63.The chemical formula for -methylbutanoic acid is ______.64.What do you call a doctor for teeth?A. SurgeonB. DentistC. PediatricianD. Pharmacist答案:B65.What do you call a scientist who studies space?A. BiologistB. ChemistC. AstronomerD. Physicist答案:C66.How many letters are in the English alphabet?A. 24B. 25C. 26D. 2767. A flamingo stands on one ________________ (脚).68.The ____ has a distinctive call and can be loud.69.I like to collect ________ (邮票) from all over the world. Each one tells a different ________ (故事).70.What do you call a large mammal that lives in the ocean?A. SharkB. WhaleC. DolphinD. Seal答案:B71.I have a toy _____ that can roar.72.What is the name of the famous monument in Washington, D. C.?A. Lincoln MemorialB. Washington MonumentC. Jefferson MemorialD. White House73.The _____ (植物观察) helps understand biodiversity.74.The __________ is soft and white after it snows. (雪)75.The sun is _______ (非常耀眼)。
Beyond Undecidable
a rX iv:mat h /66713v1[mat h.GM ]28J un206BEYOND UNDECIDABLEPAOLA CATTABRIGA Abstract.The predicate complementary to the well-known G¨o del’s provabil-ity predicate is defined.From its recursiveness new consequences concerning the incompleteness argumentation are drawn and extended to new results of consistency,completeness and decidability with regard to Peano Arithmetic and the first order predicate calculus.Keywords:decision problem,provability predicate,G¨o del numbering.Introduction Of all the remarkable logical achievements of the twentieth century perhaps the most outstanding is the celebrated G¨o del incompleteness argumentation of 1931[1,2].In contrast to Hilbert’s program called for embodying classical mathematics in a formal system and proving that system consistent by finitary methods [4],G¨o del paper showed that not even the first step could be carried out fully,any formal system suitable for the arithmetic of integers was incomplete.The present article,in the most absolute respect for the extraordinary contribu-tion given by G¨o del to the logical inquiry,brings G¨o del’s achievement into question by the definition of the refutability predicate.As it is well-known self-reference plays a crucial role in G¨o del’s incompleteness argumentation and the methods of achiev-ing self-referential statements is the so-called “diagonalization”.The refutability predicate,defined by arithmetization as a number-theoretic statement,gives rise to new consequences properly regarding G¨o del’s incompleteness argumentation and the method of diagonalization.This article proposes a revision based on the logical investigation of the interactive links between provability and refutability predicates.Originally devised by G¨o del in order to arithmetize metamathematical notions,G¨o del numbering turns out to be the key of the problem in defining refutability with the same recursive status as provability.The inquiry comes up with a final solution for finitary methods and the related decision problem [3].The paper is organized as follows.Firstly,in the following of this section,we introduce diagonalization and the famous incompleteness argumentation of G¨o del.Section 1presents two new primitive recursive predicates for refutability and the enucleation of some of their consequences,which represent the first main result of this paper:G¨o del’s incompleteness argumentation is not a theorem in Peano Arithmetic.Section 2shows that any formula of Peano Arithmetic is proved if andonly if it is not refuted,and extends this result to the accomplishment of consistency and completeness for Peano Arithmetic and then to the achievement of decidability forfirst order predicate calculus.Basic Setup.We shall assume afirst order theory which adequately formalizes Peano Arithmetic(see for example the system S,with all the necessary assumptions, in[5]116-175).Let us call it PA.As is well known by means of the G¨o del numbering, each expression in PA can refer to itself.Numerals,as usual,are defined recursively,n+1is(n,and this name can be substituted back intoφ(v).This self-reference procedure is admitted by the so-called diagonalization lemma as follows.Diagonalization.For any formulaφwith only the variable v free there is a sen-tenceδsuch that⊢PAδ⇐⇒φ(n)= φ(m).We shall show thatδis the sentence we were looking for.To this purpose we notice that in PA they hold the following equivalences⊢δ⇐⇒β(m,β(v) ,β(δ )by definition.G¨o del’s Incompleteness.We present the version of the so-called G¨o del’sfirst incompleteness Theorem as it is given in([5]161-162),to which the reader can refers for the definition of the concepts which are involved.Letφ(v)be the formula∀x¬P f(x,v),hence by diagonalization lemma we attain⊢P Aδ⇐⇒∀x¬P f(x,r,r,δ ).By Biconditional Elimination,⊢P A∀x¬P f(x,r,δ ), Biconditional Elimination yields⊢P A¬∀x¬P f(x,δ ).On the other hand,since PA isω-consistent,PA is consistent.But,⊢P A¬δ.Hence,not⊢P Aδ;that is,there is no proofin PA ofδ.So Pf(n,q)is false for every natural number n and,therefore,⊢P A¬P f( δ )for every natural number n.(Remember that q.)Byω-consistency,not⊢P A∃x P f(x,1We shall not reproduce entirely this long list of definitions which is already well-known(see also[1]162-176).∃u u<x∃v v<x∃z z<x∃w w<x([x=2w∧Ax(w)]∨[Prf(u)∧Fml((u)w)∧x=u∗2v∧Gen((u)w,v)]∨[Prf(u)∧Fml((u)z)∧Fml((u)w)∧x=u∗2v∧MP((u)z,(u)w,v)]∨[Prf(u)∧x=u∗2v∧Ax(v)].Pf(x,v):x is the G¨o del number of a proof in PA of the formula with G¨o del number v:Prf(x)∧v=(x)lh(x) –1.By means of such definitions,we shall define two new predicates,Rf and Ref.Rf(x,v):x is the G¨o del number of a proof in PA of the negation of the formula with G¨o del number v:Pf(x,z)∧z=Neg(v).In other terms Rf(x,v)states x is the G¨o del number of a refutation in PA of the formula with G¨o del number v2.Rf is primitive recursive,as the relations obtained from primitive recursive rela-tions by means of propositional connectives are also primitive recursive([5]137). For its recursiveness Rf(x,v)is expressible in PA by a formula Rf(x,v).Ref(x):x is the G¨o del number of a refutation in PA:Prf(v)∧v=Neg(x)In other terms Ref(x)states x is the G¨o del number of a proof in PA of its negation.Ref(x)is primitive recursive,as the relations obtained from primitive re-cursive relations by means of propositional connectives are also primitive recursive. For its recursiveness Ref(x)is expressible in PA by a formula Ref(x).Lemma1.For any natural number n and for any formulaαnot both Rf(n, α ) and Pf(n, α ).Proof.Let us suppose to have both Rf(n, α )and Pf(n, α ).We should have thenPrf(n)∧ α =(n)lh(n) –1and Pf(n,z)∧z=Neg( α ),i.e.Prf(n)∧ α =(n)lh(n) –1and Prf(n)∧Neg( α )=(n)lh(n) –1.By the definition of Prf(x)this would mean to have∃u u<n∃v v<n∃z z<n∃w w<n([n=2w∧Ax(w)]∨[Prf(u)∧Fml((u)w)∧n=u∗2v∧Gen((u)w,v)]∨[Prf(u)∧Fml((u)z)∧Fml((u)w)∧n=u∗2v∧MP((u)z,(u)w,v)]∨[Prf(u)∧n=u∗2v∧Ax(v)]and bothα =(n)lh(n) –1and Neg( α )=(n)lh(n) –1and hence the four cases(1)[n=2 α ∧Ax( α )]and[n=2Neg( α )∧Ax(Neg( α ))](2)[Prf(u)∧Fml((u)w)∧n=u∗2 α ∧Gen((u)w, α )]and[Prf(u)∧Fml((u)w)∧n=u∗2Neg( α )∧Gen((u)w,Neg( α ))](3)[Prf(u)∧Fml((u)z)∧Fml((u)w)∧n=u∗2 α ∧MP((u)z,(u)w, α )]and[Prf(u)∧Fml((u)z)∧Fml((u)w)∧n=u∗2Neg( α )∧MP((u)z,(u)w,Neg( α ))](4)[Prf(u)∧n=u∗2 α ∧Ax( α )]and[Prf(u)∧n=u∗2Neg( α )∧Ax(Neg( α ))]which are all immediately impossible by the definitions of Ax(y),Gen(x,y)and MP(x,y,z)and thence by the definitions of the axioms of PA,the Generalization Rule and Modus Ponens,because no axiom belongs to PA together with its negation and the two inference rules preserve logical validity.We now recall the definition of characteristic function.If R is a relation of n arguments,then the characteristic function C R is defined as followsC R(x1,...,x n)= 0if R(x1,...,x n)is true,1if R(x1,...,x n)is false.Let us call the characteristic functions of Pf(x,v),Prf(x),Rf(x,v)and Ref(x) respectively C Pf,C Prf,C Rf,and C Ref.A relation R(x1,...,x n)is said to be primitive recursive(recursive)if and only if its characteristic function C R(x1,...,x n)is primitive recursive(recursive)([5] 137).As Pf(x,v),Prf(x),Rf(x,v)and Ref(x)are primitive recursive then also C Pf,C Prf,C Rf and C Ref are primitive recursive.Every recursive function is representable in PA([5]143),thence C Pf,C Prf,C Rf and C Ref,are representable in PA.We shall assume C P f,C P rf,C Rf and C Ref to represent respectively C Pf,C Prf,C Rf and C Ref in PA.Lemma2.For any formulaα,and n as the G¨o del number of a proof in PA ofα⊢P A C P f( α )=n,1Proof.One can easily see that the two conjuncts are true:as n is the G¨o del number of a proof in PA ofαC P f( α )=n,0∧C P f( α )=n,0is true.By Lemma(1)Pf(n, α )does not hold,therefore it is true that n is not the G¨o del number of a proof in PA of α. Lemma4.For any formulaα(i)not both⊢P A P f( α )⊢P A Rf( α ),(ii)for n as the G¨o del number of a refutation in PA ofα⊢P A Rf( α )⇐⇒¬P f( α ),(iii)for n as the G¨o del number of a proof in PA ofα⊢P A P f( α )⇐⇒¬Rf( α ).Proof.(i)Immediately by Lemma(1)and the definition of being expressible which holds for both Pf(x,v)and Rf(x,v)([5]130).(ii)Let us assume⊢P A Rf( α ),then Lemma(3)yields⊢P A C P f( α )=n,n,n,n,n,n,n,1.Hence by definition Rf( α )is false,consequently⊢P A¬Rf( α ).Con-versely let us assume⊢P A¬Rf( α )then Rf( α )is false and by Lemma(2) we attain⊢P A P f( α ).All preceding lemmas were carried out constructively,needlessly to assume con-sistency.We are now able to consider the consequences yielded by such lemmas to the G¨o del’s argumentation.(a′)Assume⊢P Aδ.Let r be the G¨o del number of a proof in PA ofδ.Then Pf(r,q).Hence,⊢P A P f(q),that is⊢P A P f( δ ).Hence by Lemma(4)(i)⊢P A Rf( δ )is not admitted,which means that r cannot be the G¨o delnumber of a refutation ofδ(indeed Lemma(2)yields⊢P A C Rf( δ )=r,r,r,0).(b′)Assume⊢P A¬δ.Let r be the G¨o del number of a proof in PA of¬δ.Then Rf(r,q).Hence⊢P A Rf(q)that is⊢P A Rf( δ ).Hence by Lemma(4)(i)⊢P A P f( δ )is not admitted.This means that r cannot be theG¨o del number of a proof ofδ(in fact,r is the G¨o del number of a refutation ofδ,Lemma(3)yields⊢P A C P f( δ )=r,r,r,0).We have thus shown that previous Lemmas prevent any accomplishment of(a) and(b)within G¨o del’s argumentation3.We have then established the following theorem.Theorem5.By the arithmetization of the refutability predicate G¨o del’s incom-pleteness does not hold as a theorem of PA.2.Consistency,Completeness and DecidabilityA recursive predicate defines a decidable set,by reason that its characteristic function is considered to be effectively computable([5]165,249).Let us call T P A the set of G¨o del numbers of theorems of PA and R P A the set of G¨o del numbers of refutations of PA.By the recursiveness of Pf(x,v),C Pf(x,v)=0if v∈T P A and C Pf(x,v)=1if v/∈T P A.By the recursiveness of Rf(x,v),C Rf(x,v)=0if v∈R P A and C Rf(x,v)= 1if v/∈R P A.We can than state the following theorem.Theorem6.T P A and R P A are decidable sets.r,It is furthermore well-known that if we have a computable function f(x1,...,x n) such thatf(x1,...,x n)= 0if<x1,...,x n>∈S1if<x1,...,x n>/∈S(where S is a set of natural number which turns out to be decidable just by this definition),then the function g(x1,...,x n)defined byg(x1,...,x n)= 1if f(x1,...,x n)=00if f(x1,...,x n)=1is effectively computable too.Accordingly the complement of S is decidable.One can easily see that for f(x1,...,x n)primitive recursive,g(x1,...,x n)is primitive recursive too.Consequently we haveC¬Prf(x)= 1if C Prf(x)=00if C Prf(x)=1C¬Ref(x)= 1if C Ref(x)=00if C Ref(x)=1C¬Pf(x,v)= 1if C Pf(x,v)=00if C Pf(x,v)=1C¬Rf(x,v)= 1if C Rf(x,v)=00if C Rf(x,v)=1where¬Prf,¬Pf,¬Ref and¬Rf are respectively complementary of Prf,Pf,Ref and Rf.Let us summarize,Prf,Pf,Ref and Rf are primitive recursive,then C Prf,C Pf, C Ref and C Rf are primitive recursive too.But C¬Prf(x)=1−C Prf(x),C¬Pf(x,v)= 1−C Prf(x,v),C¬Ref(x)=1−C Ref(x),and C¬Rf(x,v)=1−C Rf(x,v),thence¬Prf,¬Pf,¬Ref and¬Rf are primitive recursive too.We have then the following statements.Lemma7.For every xPrf(x)if and only if¬Ref(x).Proof.Let us assume Prf(x).C Prf(x)=0.Hence C Prf(Neg(x))=1,by the effective computability of C Prf.Prf(Neg(x))is false,then Ref(x)is false.Accordingly, C Ref(x)=1.Thus C¬Ref(x)=0and¬Ref(x).Conversely,let us assume¬Ref(x).Then C¬Ref(x)=0and C Ref(x)=1.If Ref(x)is false by its definition Prf(Neg(x))is false.Thus C Prf(Neg(x))=1and C¬Prf(Neg(x))=0.Consequently C¬Prf(x)=1,and C Prf(x)=0.Hence Prf(x).If we convent to formalize“a proof in PA ofθ”withθ1...θr⊢P Aθthen we have ⊢P A(θ1⇒(θ2⇒...(θr⇒θ)...))(Herbrand,1930).Indeed Lemma(7)could be read as follows:forθ1,...,θr,θformulas in PA Prf( (θ1⇒(θ2⇒...(θr⇒θ)...)) )if and only if¬Ref( (θ1⇒(θ2⇒...(θr⇒θ)...)) ).Furthermore,by the recursiveness of Prf(x),C Prf(x)=0if x∈T P A and C Prf(x)=1if x/∈T P A.By the recursiveness of Ref(x)C Ref(x)=0if x∈R P A and C Ref(x)=1if x/∈R P A.Lemma8.For every<x,v>Pf(x,v)if and only if¬Rf(x,v).Proof.Let us assume Pf(x,v).C Pf(x,v)=0.Hence C Pf(x,Neg(v))=1.Accord-ingly C¬Pf(x,Neg(v))=0.Thence C¬Rf(x,v)=0and¬Rf(x,v).Conversely,let us assume¬Rf(x,v).We have then¬Pf(x,Neg(v))and C¬Pf(x,Neg(v))=0.Therefore C¬Pf(x,v)=1,by the effective computability of C¬Pf.Accordingly C Pf(x,v)=0 and Pf(x,v).Indeed Lemma(8)could be read as follows:forθ1,...,θr,αformulas in PA Pf( (θ1⇒(θ2⇒...(θr⇒α)...) , α )if and only if¬Rf( (θ1⇒(θ2⇒...(θr⇒α)...) , α ).Lemma9.For m= α and n= (θ1⇒(θ2⇒...(θr⇒θ)...))(i)m∈T P A iffm/∈R P A,(ii)n∈T P A iffn/∈R P A.Proof.Immediately(i)by Lemma(8),(ii)by Lemma(7). Theorem10.PA is consistent;that is,there is no formulaαsuch that bothαand ¬αare theorems of PA.Proof.Let us assume m to be the G¨o del number of a proof of a formulaαof PA and n to be the G¨o del number of a proof of¬α.Then n,m∈T P A.But since n is the G¨o del number of a proof of¬αwe have also n∈R P A,accordingly n belongs to both T P A and R P A,which is impossible by Lemma(9). Theorem11.PA is complete;that is for any well formed formulaαof PA either ⊢P Aαor⊢P A¬α.Proof.Letαbe a well formed formula of PA,we can then yield by G¨o del numbering m= α .By Lemma(9)either m∈T P A or m∈R P A.Therefore either⊢P Aαor ⊢P A¬α.Let us call PF the fullfirst-order predicate calculus([5]172).Let T P F be then the set of G¨o del number of theorems of PF.Theorem12.T P F is decidable.Proof.By G¨o del Completeness Theorem,a formulaαof PA is provable in PA if and only ifαis logically valid,andαis provable in PF if and only ifαis logically valid.Hence⊢P Aαif and only if⊢P Fα.Accordingly,for n as the G¨o del number of a proof ofαin PA,n∈T P A iffn∈T P F.Hence,by theorem(6),T P F is decidable.Calling our attention to the diagonalization lemma we note that it holds for any formulaφwith only the variable v free.In other termsφcan be replaced by any formula with only one free variable.Let us suppose now that a sentenceδis a theorem of PA,i.e.⊢P Aδ.For n as the G¨o del number of a proof in PA ofδwe have⊢P A P f( δ ).But forφ(v)as∀x Rf(x,v)diagonalization lemma could have already yielded⊢PAδ⇐⇒∀x Rf(x,we have⊢PA∀x Rf(x,n,n,n,n,n,n,n,。
平顶山2024年09版小学5年级第七次英语第三单元暑期作业
平顶山2024年09版小学5年级英语第三单元暑期作业考试时间：80分钟（总分:100）B卷考试人：_________题号一二三四五总分得分一、综合题(共计100题)1、填空题：My parents often take me to __________. (旅行)2、 Wall was built over many _______. (世纪) 填空题：The Grea3、听力题：A zebra is known for its black and ______ stripes.4、填空题：In summer, the grass grows __________ (茂盛).5、What do you call a baby dog?A. PuppyB. KittenC. CalfD. Chick答案: A6、听力题：A chemical reaction can occur in _____ conditions.7、What do you call the main character in a story?A. ProtagonistB. AntagonistC. HeroD. Villain答案:AThe seahorse is unique because it is one of the few species of ________________ (鱼) that exhibits reverse sexual roles.9、听力题：We celebrate Christmas in _____ (December/June).10、填空题：The __________ (古迹) tells stories of the past.11、What do we call a young hawk?A. EyasB. ChickC. KitD. Calf答案:A. Eyas12、填空题：A ________ (湿地) helps filter water.13、听力题：The baby is _____ in its crib. (sleeping)14、听力题：A telescope helps us see distant ______.15、选择题：Which month comes after January?A. MarchB. FebruaryC. AprilD. May16、填空题：The air is cool and fresh in the ______ (早晨).17、听力填空题：I enjoy watching movies, especially __________ because __________.18、Which season comes after summer?A. WinterB. SpringC. AutumnD. Fall答案: CThe chemical symbol for mercury is __________.20、听力题：A black hole cannot be observed directly because its gravity prevents light from ______.21、听力题：A _______ is a chemical reaction where energy is absorbed.22、填空题：I enjoy drawing ______ (动物) in my sketchbook. They bring me ______ (快乐).23、填空题：My favorite form of exercise is _______ (游泳).24、听力题：The ______ teaches us about sports.25、填空题：The ________ was an important document in the founding of the United States.26、填空题：A ladybug is ______ (小) and often red with spots.27、填空题：The classroom is ________ (安静的).28、听力题：The sun sets in the _____ (west).29、听力题：A pendulum swings back and ______.30、填空题：The ancient Romans used ________ as a form of entertainment.31、What animal is known for having a long neck?A. RhinoB. GiraffeC. ZebraD. Hippo答案: B32、填空题：The invention of the radio revolutionized _____ communication.Metamorphism occurs when rocks are subjected to high ______ and temperature.34、填空题：A _____ is an area with many different plants and animals.35、What do you call a person who repairs watches?A. WatchmakerB. JewelerC. ClockmakerD. Craftsman答案:A36、填空题：We _______ (喜欢) to watch movies together.37、听力题：Combustion is a chemical reaction involving _______.38、Which of these is a holiday in December?A. ThanksgivingB. ChristmasC. Independence DayD. Labor Day答案:B39、填空题：When it rains, I like to wear my __________ shoes. (防水的)40、Which fruit is red and often associated with teachers?A. BananaB. AppleC. OrangeD. Grape41、What is the capital of Serbia?A. BelgradeB. Novi SadC. NisD. Kragujevac答案: A42、填空题：I call my friend's sister __________. (她的名字)43、In which direction does the sun rise?a. Northb. Southc. Eastd. West答案:c44、填空题：A lion is a mighty _______ that rules its kingdom.45、What is the name of the largest moon of Saturn?A. EuropaB. TitanC. GanymedeD. Callisto46、听力题：The ______ is known for her impactful writing.47、填空题：The _____ (兔子) nibbles on carrots.48、What do we call the practice of keeping animals for food, work, or companionship?A. AgricultureB. FarmingC. DomesticationD. Husbandry答案:C49、What is the largest animal in the ocean?A. SharkB. WhaleC. DolphinD. Octopus答案: B50、听力题：She is _______ (sharing) her toys with others.51、填空题：The ______ (小鸟) builds a nest.52、Which of these is a vegetable?A. AppleB. CarrotC. BananaD. Cherry答案:B53、填空题：A kitten can be very ______ (调皮).54、填空题：The ancient Egyptians built monumental _____ for their leaders.55、What is the term for a baby kangaroo?A. JoeyB. CalfC. KitD. Cub答案:A56、Which one is a type of tree?A. RoseB. OakC. DaisyD. Lily答案: B57、听力题：We visit our __________ on holidays.58、填空题：The ________ was a significant battle in World War II.59、填空题：The rabbit is very ______.60、填空题：The scientist discovered a new _____ (物种).61、What is the capital of South Korea?A. SeoulB. BusanC. IncheonD. Gwangju答案:A62、听力题：I like to ______ my friends on weekends. (meet)63、听力题：A _______ is a mixture where the components are evenly distributed.64、填空题：My _____ (小猫) likes to climb trees.65、听力题：The __________ is known for its unique cultural practices.66、What do we call a place where you can see many different types of animals?A. FarmB. ZooC. ParkD. Aquarium67、What is the capital city of the Seychelles?A. VictoriaB. MaleC. Port LouisD. Antananarivo答案: A68、What is the name of the famous monument in Egypt with a lion's body?A. Great PyramidB. SphinxC. ObeliskD. Temple答案: B69、What do you call an animal that can live both in water and on land?A. FishB. MammalC. AmphibianD. Reptile答案:C70、听力题：A reaction that produces energy is called an ______ reaction.71、听力题：The ______ (teacher) is explaining the lesson.72、How many months are there in a year?A. TenB. TwelveC. ElevenD. Thirteen答案:B73、听力题：There are ______ (five) birds in the tree.74、What is the capital of Germany?A. BerlinB. MunichC. FrankfurtD. Hamburg答案:A. Berlin75、填空题：My dog loves to chase _________ (球).76、听力题：I found a ___. (shell) on the beach.77、听力题：You can find _______ in a garden or park.78、听力题：The boy plays the ________.79、填空题：I like to keep a journal where I write my ______ (梦想) and aspirations. It motivates me to work hard.80、听力题：I have a _____ (gift) for you.81、填空题：World War II began in __________. (1939年)82、听力题：The Great Wall of China can be seen from ______ space.83、听力题：Fossils can provide information about past ______ environments.84、填空题：The ________ (植物经济) is vital for trade.85、填空题：My sister is learning to play the __________ (乐器).I call my mother _____ (妈妈) in Chinese.87、What is the main ingredient in a burrito?A. TortillaB. RiceC. BeansD. All of the above答案:A88、听力题：Acids can donate ______ ions in solution.89、听力题：A galaxy is a collection of ______ and other matter.90、听力题：We can see ________ in the sky.91、What is the primary function of the kidneys?A. To pump bloodB. To filter wasteC. To digest foodD. To produce hormones答案: B. To filter waste92、填空题：My grandmother tells me __________. (故事)93、填空题：The ______ (鲸鱼) is the largest mammal in the ocean.94、填空题：The __________ (社会责任) is important for community well-being.95、填空题：The __________ (历史传说) often blend fact and fiction.96、听力题：The chemical symbol for iron is _______.97、填空题：The elephant is known for its ______ (记忆).A garden needs regular ______ (浇水) to keep plants healthy.99、What is the primary reason we need to drink water?A. To feel fullB. To stay hydratedC. To wash our handsD. To eat food答案:B100、What do we call the area of land that is always covered with ice?A. DesertB. GlacierC. TundraD. Antarctic答案: B。
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Submit a Manuscript: https:// World J Gastroenterol 2019 August 21; 25(31): 4452-4467 DOI: 10.3748/wjg.v25.i31.4452ISSN 1007-9327 (print) ISSN 2219-2840 (online)ORIGINAL ARTICLE Basic StudyHigh expression of APC is an unfavorable prognostic biomarker in T4 gastric cancer patientsWei-Bo Du, Chen-Hong Lin, Wen-Biao ChenORCID number: Weibo Du(0000-0001-5779-6159); Chenhong Lin (0000-0002-9730-5614); Wenbiao Chen (0000-0002-1028-6319).Author contributions: Chen WB designed and supervised the research; Du WB, Lin CH, and Chen WB interpreted the data; Du WB wrote the manuscript; Lin CH constructed the figures and tables; Chen WB revised the manuscript.Supported by the Major National S&T Projects for Infectious Diseases, No. 2018ZX10301401-005; the National Key Research and Development Program of China, No. 2018YFC2000500; the Key Research and Development Plan of Zhejiang Province, No.2019C04005; and the National Natural Science Foundation of China, No. 81571953.Institutional review board statement: The study was approved by the Clinical Research Ethics Committee of College of Medicine, Zhejiang University.Conflict-of-interest statement: The authors declare that they have no competing interests.Data sharing statement: All research data can be downloaded from public databases.ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.Open-Access: This article is an open-access article which was Wei-Bo Du, Chen-Hong Lin, Wen-Biao Chen, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, ChinaCorresponding author: Wen-Biao Chen, PhD, Doctor, Postdoctoral Fellow, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University. No. 79, Qingchun Road, Hangzhou 310003, Zhejiang Province, China. chenwenbiao@Telephone: +86-183********Fax: +86-571-87236114AbstractBACKGROUNDAdenoma polyposis coli (APC) mutation is associated with tumorigenesis via the Wnt signaling pathway.AIMTo investigate the clinical features and mechanism of APC expression in gastric cancer (GC).METHODSBased on APC expression profile, the related genome-wide mRNA expression, microRNA (miRNA) expression, and methylation profile in GC, the relationship between APC and GC, as well as the prognostic significance of APC were systematically analyzed by multi-dimensional methods.RESULTSWe found that high expression of APC (APC high) was significantly associated with adverse outcomes of T4 GC patients. Genome-wide gene expression analysis revealed that varying APC expression levels in GC were associated with some important oncogenes, and corresponding cellular functional pathways. Genome-wide miRNA expression analysis indicated that most of miRNAs associated with high APC expression were downregulated. The mRNA-miRNA regulatory network analysis revealed that down-regulated miRNAs affected their inhibitory effect on tumor genes. Genome-wide methylation profiles associated with APC expression showed that there was differential methylation between the APC high and APC low groups. The number of hypermethylation sites was larger than that of hypomethylation sites, and most of hypermethylation sites were enriched in CpG islands.selected by an in-house editor andfully peer-reviewed by externalreviewers. It is distributed inaccordance with the CreativeCommons Attribution NonCommercial (CC BY-NC 4.0)license, which permits others todistribute, remix, adapt, buildupon this work non-commercially,and license their derivative workson different terms, provided theoriginal work is properly cited andthe use is non-commercial. See:/licenses/by-nc/4.0/Manuscript source : Unsolicited manuscript Received: April 18, 2019Peer-review started: April 18, 2019First decision: June 16, 2019Revised: July 18, 2019Accepted: August 7, 2019Article in press: July 19, 2019Published online: August 21, 2019P-Reviewer: Bordonaro M, TanabeKS-Editor: Ma RYL-Editor: Wang TQE-Editor: Ma YJ CONCLUSION Our research demonstrated that high APC expression is an unfavorable prognostic factor for T4 GC patients and may be used as a novel biomarker for pathogenesis research, diagnosis, and treatment of GC.Key words: Adenoma polyposis coli; Gastric cancer; Prognosis; mRNA; miRNA;Methylation ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.Core tip: We found that high expression of adenoma polyposis coli (APC ) was associated with a poor prognosis in T4 gastric cancer (GC) patients. There was differentialexpression of mRNAs and miRNAs as well as differential DNA methylation between thehigh expression of APC (APC high ) and low expression of APC (APC low ) groups. The linkbetween APC high and differential expression of mRNAs, miRNAs, and DNA methylationmay contribute to the poor prognosis in T4 GC patients, and be involved in thepathogenesis of GC. APC could be used as a novel biomarker for clinical diagnosis,therapy, and assessment of prognosis in T4 GC patients, as well as for further research of the pathogenesis of GC.Citation: Du WB, Lin CH, Chen WB. High expression of APC is an unfavorable prognostic biomarker in T4 gastric cancer patients. World J Gastroenterol 2019; 25(31): 4452-4467URL : https:///1007-9327/full/v25/i31/4452.htm DOI : https:///10.3748/wjg.v25.i31.4452INTRODUCTIONGastric cancer (GC) is one of the most common malignant tumors in China, and itsmorbidity and mortality rank first in digestive tract tumors [1]. As the early detectionrate of GC is low, most patients are initially diagnosed at an advanced stage, whichseriously affects the prognosis and survival rate of the patients. With the developmentof endoscopic technique and the improvement of medical technology, the 5-yearsurvival rate of GC has increased to 90%[2,3]. However, recurrence, metastasis, anddrug resistance limit the therapeutic effect. There are still about 350000 deaths due toGC in China every year [4]. Therefore, the early prevention, diagnosis, and anti-tumorcomprehensive treatment of GC have attracted more and more attention.GC is a complex multi-factorial, multi-step progressive process involving coding ornon-coding genes and epigenetic change [5]. Many kinds of molecular events, such asDNA mutations [6], aberrant expression of mRNAs [7] and microRNAs (miRNAs)[8], andabnormal proteomes [9], have been found to participate in the pathogenesis of GC.Currently, these molecular events have been extensively studied as biomarkers for theprognosis, diagnosis, and therapy of GC. Li et al [10] found that MUC16 gene mutationwas associated with a higher load of tumor mutations and improved prognosis inpatients with GC. These findings may have an impact on prognosis prediction andtreatment guidance for GC. MiR-1265 inhibits the progression and carcinogenicautophagy of GC by reducing the expression of CAB39 and regulating the AMPK-mTOR signal transduction pathway. Therefore, it was speculated that miR-1265 mayrepresent a potential therapeutic target for GC [11]. DNA methylation is common innon-neoplastic gastric mucosa infected with Helicobacter pylori , which is associatedwith a high risk of GC. Therefore, DNA methylation could be used as a usefuldiagnostic tool for GC risk assessment [12]. Despite these, the molecular mechanism ofGC is currently still unclear, and the identification of biomarkers is of greatsignificance for the clinical prognosis, diagnosis, and treatment of GC.Adenoma polyposis coli (APC ) encodes a tumor suppressor protein that can inhibitthe Wnt signal pathway in tumor, and mutation and inactivation of APC are uniquekey and early events in tumorigenesis, especially colorectal cancer. The diagnostic andprognostic value of APC in colorectal cancer is high [13,14]. Compared with colorectalcancer, there have been much fewer studies on APC in GC. However, some successfulstudies have initially shown that APC could be used as a biomarker for GC. Throughresearch and analysis of big data, Bria et al [15] defined a risk classification systemcomprising biomolecular (including APC ) and clinicopathological predictors, whichDu WB et al.APC in period-T4 gastric cancerDu WB et al.APC in period-T4 gastric cancercould divide resected GC patients into three types of risk for guiding treatment. Bystatistically analyzing a large number of GC samples, Merchant et al[16] revealed thatthe incidence of APC-related GC in young Hispanic men has been rising in the UnitedStates. And the high expression of APC was correlated with advanced stage and lowdifferentiation of GC. Therefore, we speculated that APC may have an important rolein the pathogenesis and clinical application of GC.In this study, we demonstrated that APC was a biomarker for poor prognosis in T4GC patient, and identified APC-related mRNA and miRNA expression changes, andDNA methylation profile on a genome-wide scale by multi-dimensional methods.Our findings indicated the value of APC as a new target for the diagnosis andtreatment of T4 GC patients and highlighted the important role of APC in thepathogenesis of GC.MATERIALS AND METHODSResearch data sourceThe research data, including high throughput RNA sequencing (RNA-Seq), miRNA-Seq, Illumina Infinium Human Methylation 450, and clinical follow-up informationdata, which provided all mRNA and miRNA expression and methylation profiles forfollowed GC patients, were downloaded from the Cancer Genome Atlas (TCGA)[17] onOctober 31, 2018. Samples with more than 30 d of follow-up were screened fromclinical follow-up data to further match the RNAseq expression profile, miRNAexpression profile, and methylation profile. We obtained 387 samples and 2173miRNAs, 335 samples and 19754 mRNAs, and 364 samples and 358418 methylationsites, respectively. The study was approved by the Clinical Research EthicsCommittee of College of Medicine, Zhejiang University.Statistical analysisOverall survival (OS) was defined as the time from randomization to death for anyreason, while relapse-free survival rate (RFS) was defined as complete remission(approximately one month after diagnosis) to recurrence or the end of follow-up. Inorder to evaluate the prognostic value of APC in GC samples, we selected theappropriate cut-off value for subdivision. The optimal cut-off was defined by thestatistical distribution of APC expression. APC expression was normally distributed inGC samples, and there was a significantly evident distinction along the median value.Therefore, GC samples were divided into APC high and APC low groups by the medianAPC expression (Supplemental Figure 1A). Next, the T4 GC patients were classified into four groups according to quartiles of APC expression: Q1, < 25%; Q2, 25%–50%; Q3,50%–75%; Q4, > 75%). The results showed that Q4 had the worst prognosis both in OSand RFS. Also, a significant difference was observed between Q2 vs Q4 and Q2 vs Q4in OS and RFS (Supplemental Figure 1B and 1C). Then, the relationship between APCexpression and OS and RFS was evaluated by the Kaplan-Meier (KM) method andfurther examined by log-rank test. To investigate the association between APC andclinical and molecular characteristics, we divided the T4 GC patients into APC high andAPC low groups according to median APC expression value and used the t-test and chi-square tests to compare the clinical and molecular characteristics between the twogroups. The t-test and false discovery rate (FDR) were applied to reveal thedifferential expression of genome-wide genes, miRNAs, and methylation betweenAPC high and APC low groups. The significantly statistical cut-off values were fold-change(FC) > 2 and FDR < 0.05 for genes, FC > 1.5 or < 0.66 and P < 0.05 for miRNAs, and FC> 2 and P < 0.05 for methylation. In order to observe the relationship between APCexpression and cellular functional pathways based on the differential expression ofgenome-wide genes, we screened all GC samples with gene expression profiles, andused Gene Set Enrichment Analysis (GSEA) tools[18] to perform KEGG pathwayenrichment analysis on samples from APC high and APC low groups with a selectionthreshold of P < 0.05. KEGG pathway enrichment of methylation was performedusing R ClusterProfiler with a selection threshold of P < 0.05. To observe therelationship between APC expression and related miRNAs, we screened miRNAswith expression levels greater than 0 and ratios greater than 50% in each sample, andused the Pearson correlation coefficient to estimate the correlation between miRNAexpression profiles and APC expression. The significantly statistical cut-off was P <0.05 and a correlation coefficient > 0.3 or < -0.3 to determine the correlation of APCwith related miRNAs. In additional, we used the miRWalk database to predictmiRNA target genes and constructed mRNA-miRNA regulatory networks inconjunction with the most significantly related miRNAs.Figure 1 Prognostic value of APC expression in gastric cancer patients. A: Overall survival of gastric cancer patients with high APC expression and those with low expression (based on median value); B: Relapse-free survival of gastric cancer patients with high APC expression and those with low expression; C: Distribution of APC expression; D: Relapse-free survival of T4 gastric cancer patients with high APC expression and those with low expression. APC : Adenoma polyposis coli; GC:Gastric cancer.Du WB et al.APC in period-T4 gastric cancerDu WB et al.APC in period-T4 gastric cancerRESULTSAPC is related to an unfavorable prognosis of T4 GCAnalysis of OS and RFS was performed based on the 335 GC samples to investigatethe association between APC expression and prognosis. The results showed that therewas no significant relationship between APC expression and OS (P = 0.58) and RFS (P= 0.98) in the overall population (Figure 1A and 1B). Subsequently, we analyzed theassociation between APC expression and prognosis in GC of different stages andfound that the expression of APC in T4 samples was high in the middle and low onboth sides with a median value of 10.53 (Figure 1C). According to this median value,the 88 T4 GC samples were divided into either an APC high (n = 44) or APC low group (n =44). It was found that patients with APC high GC showed significantly shorter RFS thanthose with APC low GC (log-rank P = 0.037) (Figure 1D). These results suggested thathigh expression of APC was associated with a poor prognosis in T4 GC patients.Association of APC with clinical characteristics and known prognostic genesThe proportions of GC patients with tumor stages III and IV, tumor grades 2 and 3,and lymph node metastases N2 and N3 were significantly higher in the APC high groupthan in the APC low group (P = 0.043, 0.047, 0.039, 0.031, and 0.035, respectively). Inaddition, APC high was associated with a greater chance of high expression of someknown prognostic biomarkers, such as KIT, PIK3CA, KRAS, and MLH1 (P = 0.031, P <0.001, P = 0.038, and P = 0.038, respectively), all of which are associated with anunfavorable prognosis (Table 1). Kit, a cell surface receptor of stem cell factor, isconsidered to be a oncogenic signal and has a acquired functional mutation in humangastrointestinal stromal tumors[19]. The frequency of PIK3CA mutation was low in GC,but the prognosis of GC patients with PIK3CA mutation was poor[20]. Fu et al[21] foundthat KRAS mutation was a poor prognostic factor in Chinese CG patients. MLH1methylation was associated with oxaliplatin resistance in GC patients[22]. Collectively,these results indicated that APC may be a biomarker of poor prognosis in T4 GCpatients.Differentially expressed mRNAs associated with APC expressionTo observe the association between APC expression and other genes, we made acomparison between the APC high and APC low groups by genome-wide microarrayanalysis. Twelve significantly differentially expressed mRNAs were revealed to beassociated with APC expression by the t-test and Wilcoxon rank sum test (FC > 2 andFDR < 0.05) (Figure 2A, Table 2). The heat map of these 12 mRNAs is presented inFigure 2B. Previous studies have revealed that most of these 12 mRNAs are closelyrelated to tumorigenesis. ARID5B plays a role in the growth and differentiation of B-lymphocyte progenitor cells that are associated with acute lymphoblastic leukemia[23].JMY has strong carcinogenic characteristics, whose expression was elevated inprimary colorectal cancer and head and neck cancer in response to DNA damage incells[24]. MAP3K14 is involved in the induction of NF-kappaB signaling in cancer cellinvasion[25]. IL6ST is a signal transduction factor common to many cytokine activators,and it is involved in the STAT3 pathway in sub-groups of primary lung cancer and isassociated with tumor progression and a poor prognosis[26]. Rev3L plays an importantrole in regulating the response of cervical cancer cells to cisplatin[27]. Genetic variationof estrogen receptor PPARGC1B is important in familial breast cancer[28]. PARD3 playsan important role in invasiveness and metastasis of lung squamous cell carcinomabecause of its repeated inactivation of cell polarity regulator[29]. PDE4D acts as apromoter of cell proliferation in prostate cancer[30].In order to further analyze the cell molecular function of APC related genes, weinvestigated the association between cellular functional pathways of APC relatedgenes with APC expression. The results revealed that 18 pathways were associatedwith APC high, including 6 upregulated and 12 downregulated pathways (P < 0.05)(Table 3). The APC related genes were enriched in upregulated pathways such asreceptor signaling (Figure 3A), pancreatic cancer (Figure 3B), and prostate cancer ofupregulated pathway, whereas downregulated pathways such as ascorbate andaldarate metabolism (Figure 3C), PPAR signaling (Figure 3D), drug metabolism otherenzymes, and steroid hormone biosynthesis are closely related to tumor signaling,drug resistance, hormone receptor, and metabolism. These results indicated thatAPC high associated genes are closely related to the occurrence and development of GC.Relationship between APC high expression and genome-wide miRNA sequencingIn order to further understand the relationship between biological diversities of GCand different levels of APC expression, we conducted a full-genome analysis ofmiRNA sequencing data to identify the miRNA profiles with a significant correlationwith APC expression. We found that 674 miRNA expression profiles were correlatedDu WB et al.APC in period-T4 gastric cancer Table 1 Relationship between APC high and clinical characteristic and known prognostic genesAPC: Adenoma polyposis coli.with APC expression. After Pearson correlation coefficient calculation, a total of 35 miRNAs with a P-value < 0.05 and a correlation coefficient > 0.3 or < -0.3 were identified. The relationship between the correlation coefficient and the distribution of P-value is shown in Figure 4A. The heatmap of the 35 miRNAs is presented in Figure 4B, from which we can see that most of miRNA were negatively correlated with APC, and a few were positively correlated with APC. Most of the 35 miRNAs have been previously found to be involved in tumorigenesis mechanisms or be targets for clinical diagnosis and treatment, prognostic markers, and mediators of tumor drug resistance. Hsa-miR-636 may affect the tumorigenesis of hepatocellular carcinoma by downregulating the Ras signaling pathway[31]. The enrichment of hsa-miR-200c-3p secreted in urine was a non-invasive marker for the diagnosis of endometrial carcinoma[32]. The downregulated expression of has-miR-130b-3p was predicted to be associated with cell proliferation, colony formation, anti-apoptosis, and self-renewal in hepatocellular carcinoma[33]. Hsa-miR-339-5p plays a key role in the regulation of lung cancer and may be used as a biomarker to predict the progression of lung cancer[34]. The upregulated expression of miR-451 in papillary thyroid carcinoma with lymph node metastasis is a unique marker for prognosis and progression of thyroid papillary carcinoma[35]. Increasing expression of hsa-miR-103a-3p predicted improved breast cancer survival[36]. Overexpression of miR-940 in breast cancer cells induced extensive osteoblast damage in metastatic bone tumors by promoting osteogenic differentiation of host mesenchymal cells[37]. LncRNA LINC00460 could upregulate IL6 through sponging tumor suppressor gene miR-149-5p to promote the carcinogenesis of nasopharyngeal carcinoma[38]. Ectopic expression of has-let-7c-5p promoted the efficacy of all-trans retinoic acid in the treatment of acute myeloid leukemia cells[39]. Low expression of miR-100-5p endowed lung cancer cells with cisplatin drug resistance[40]. These APC related miRNAs, together with APC, may be associated with the pathogenesis of GC, which further supports APC as a marker for the poor prognosis of T4 GC.Furthermore, we combined the most significantly related miRNAs, using the miRWalk database, to predict the miRNA target genes to construct the mRNA-miRNA regulatory network (Figure 4C). The regulatory network included 6 upregulated miRNAs, 24 downregulated miRNAs, and 11 genes. It can be seen that some downregulated miRNAs may affect the inhibition of oncogenes, thusDu WB et al.APC in period-T4 gastric cancerTable 2 Association between differential mRNAs and APC highAPC: Adenoma polyposis coli.aggravating the result of deterioration. For example, hsa-miR-200c-3p, a member ofmiR-200 family, targets ARID5B and plays an important role in inhibiting tumorepithelial-mesenchymal transition (EMT)[41]. Has-miR-940 targeted PDE4D and ZNF81were downregulated in prostate cancer and can inhibited the migratory and invasivepotential of cancer cell[42]. In the meantime, some upregulated miRNAs decreased thefunction of tumor suppressor genes, thus promoting tumorigenesis. For example,ARID5B is targeted by has-miR-150-5p, and the upregulated expression of has-miR-150-5p increased the response of multiple myeloma to glucocorticoid specifictherapy[43]. Hsa-miR-195-3p, regarded as one of colon cancer metastasis biomarkers,targeted PARD3B and other genes that are associated with protein binding, celladhesion, and cancer metastasis[44]. Taken together, these findings may contribute tounderstanding why APC high could predict a poor prognosis of T4 GC.Relationship between APC high and genome-wide DNA methylation sequencingDNA methylation is a common and important epigenetic change and plays animportant role in the occurrence, development, and prognosis of many malignanttumors. Also, DNA methylation can regulate gene expression and the differentiationof cancer cells to different directions via DNA methyltransferase, including DNMT1,DNMT3A, and DNMT3B[45]. Therefore, we analyzed the differences in transcriptionlevels of the three methyltransferase between APC high and APC low. DNMT1 andDNMT3A were found to be significantly higher in APC high than in APC low (Figure 5),which suggested that there may be different methylation patterns in APC high andAPC low. Five hundred and forty-three differential methylation sites, including 530hypermethylation sites and 13 hypomethylation sites, were identified after making acomparison between APC high and APC low (FC > 2 and P < 0.05) (Figure 6A). Sincehypermethylation of tumor suppressor genes and hypomethylation of oncogeneswere associated with carcinogenesis[46], our finding that there were morehypermethylation sites than hypomethylation sites may suggest that DNAhypermethylation regulated APC expression is involved in T4 GC. In addition, weanalyzed the distribution of these 543 different methylation sites around CPG islands,and found that there was a significant difference in the distribution of differentialDNA methylation sites around CPG islands, i.e., the hypermethylation sites aremainly distributed on the CPG islands (Figure 6B), which is consistent with the viewthat the methylation of DNA methylation-regulated genes involved in tumorigenesisoccurs mainly on CPG islands[47]. Furthermore, we analyzed the distribution oftranscription initiation site (TSS) distance between these differential DNA methylationsites and the nearest genes. Most of the DNA methylation sites are located between600 bp upstream and 1200 bp downstream of TSS (Figure 6C), and thehypermethylation sites tend to be located upstream of TSS, which is related to animportant regulatory area. Moreover, the genes corresponding to the nearest TSS ofthe differential DNA methylation were extracted for KEGG pathway analysis. Therewere ten significant KEGG pathways, including six upregulated and fourdownregulated items. The upregulated DNA methylation-related genes were mainlyenriched in pathways such as basal cell carcinoma, Hippo signaling pathway, mTORsignaling pathway, and signaling pathways regulating pluripotency of stem cells,Figure 2 Differentially expressed mRNAs associated with APC high . A: Volcanic map of differentially expressed mRNAs associated with APC high ; (B) Heatmap of differentially expressed mRNAs associated with APC high . APC : Adenoma polyposis coli.which are associated with the pathogenesis of GC (Figure 6D ). Whereas, thedownregulated DNA methylation-related genes participate in viral carcinogenesis,herpes simplex infection, alcoholism, and systemic lupus erythematosus, which wereregarded as inducers of tumorigenesis (Figure 6E ).DISCUSSIONThe identification of prognostic factors for GC is important for its diagnosis andtreatment as well as the development of new targeted therapy. Especially, for precisetreatment strategy based on the risk stratification, the prognostic factors for GC cannot only provide clinicians with the earliest diagnostic markers and definite prognosisprediction, but also can help them select the best treatment plan. The aim of ourresearch was to identify novel biomarkers for GC prognosis. APC encodes animportant negative regulator that serves as an antagonist of the Wnt signalingpathway, which is closely related to cancer cell proliferation, metastasis, invasion,adhesion, and activation [48]. Here, our results showed that the expression of APC wasan independent and useful factor for poor prognosis of T4 GC. KM analysis showedthat APC high was associated with a shorter RFS in T4 GC patients. In addition, APC highwas found to be associated with advanced tumor stage and high tumor grade in GCand have significant relevance to some known prognostic biomarkers (KIT , PIK3CA ,KRAS , and MLH1). However, previous research revealed that APC is a tumorsuppressor gene, and upregulating APC expression can inactivate the Wnt/β-cateninpathway in tumorigenesis [49,50]. However, APC was found as a poor prognostic markerin patients with T4 GC, although there was no significant association between APCand OS or RFS in the overall GC population. Such discrepancy may be due to acombination of the following reasons. On one hand, APC , as a tumor suppressor gene,was verified by experimental evidence, not by statistical analysis of a large number ofclinical samples. On the other hand, a study showed that Wnt receptor (Fzd7) can beresponsible for mediating Wnt signaling in human GC cells both in the presence andabsence of APC mutations [51]. Therefore, we hypothesize that APC is not the onlyDu WB et al.APC in period-T4 gastric cancer。