前沿讲座作业--黄仕英

合集下载

巧用乡土资源,激活初中地理课堂

巧用乡土资源,激活初中地理课堂

巧用乡土资源,激活初中地理课堂
刘明飞
【期刊名称】《地理教学》
【年(卷),期】2012(000)024
【摘要】地理新课程标准要求地理教学要紧密联系生活实践,在初中地理课堂中融入乡土地理就显得尤为重要.对学生进行乡土地理教育,可以提高学生学习地理的积极性和主动性,增强他们综合分析问题的能力,激发学生热爱家乡、热爱祖国的情感.【总页数】3页(P47-49)
【作者】刘明飞
【作者单位】南京市中华中学 210006
【正文语种】中文
【相关文献】
1.巧用乡土资源激活美术课堂 [J], 魏永平
2.巧用思维导图激活高中地理课堂教学研究 [J], 叶保
3.巧用乡情打动学生激活课堂——乡土资源在初中思想品德课教学中的运用 [J], 施林英
4.利用乡土资源激活地理课堂 [J], 朱敏
5.巧用教学智慧激活地理课堂 [J], 陈燕谋
因版权原因,仅展示原文概要,查看原文内容请购买。

在地出新知:国际传播精准化实现路径

在地出新知:国际传播精准化实现路径

在地出新知:国际传播精准化实现路径目录一、内容概述 (2)1.1 背景与意义 (2)1.2 国际传播精准化的概念与内涵 (3)二、国际传播精准化的理论基础 (4)2.1 精准传播理论 (6)2.2 文化传播理论 (7)2.3 情感传播理论 (8)三、国际传播精准化的实践路径 (9)3.1 媒体融合与创新 (10)3.2 数据驱动的传播策略 (12)3.3 跨文化传播的优化 (13)3.4 本土化的内容生产 (14)四、国际传播精准化的挑战与应对 (15)4.1 技术瓶颈与突破 (16)4.2 法律法规与伦理问题 (17)4.3 文化差异与冲突 (18)五、案例分析 (19)六、结论与展望 (20)6.1 主要结论 (21)6.2 未来展望 (22)一、内容概述背景分析:首先介绍全球化对国际传播的影响,以及当前国际传播面临的挑战,包括文化差异、信息过载和舆论多样性等问题。

在地新知的重要性:阐述将地域性知识与文化融入国际传播中的必要性,包括推广多元文化、促进国际交流与合作等方面的价值。

精准化国际传播策略:分析如何实现国际传播的精准化,包括目标受众定位、传播渠道选择、内容定制与优化等方面的策略探讨。

地缘因素与国际传播的结合:讨论地理位置、文化差异等因素在国际传播中的重要性,以及如何结合地域特色制定有效的传播策略。

案例分析与实证研究:通过具体案例分析和实证研究,展示国际传播精准化的实际效果,以及面临的挑战和解决方案。

未来展望与建议:对国际传播精准化的未来发展趋势进行预测,提出针对性的建议和措施,以推动国际传播事业的长远发展。

本文旨在通过对国际传播精准化的系统性研究,为相关领域的实践者提供理论支持和实践指导,以期实现地域性知识与文化的有效传播与交流,推动多元文化的共同发展。

1.1 背景与意义随着全球化的深入发展和信息技术的飞速进步,文化传播和交流日益频繁,国际传播在塑造国家形象、促进国际合作、推动经济社会发展等方面发挥着越来越重要的作用。

母爱的芳香

母爱的芳香

母爱的芳香
王凤英
【期刊名称】《中华家教(上半月)》
【年(卷),期】2015(000)005
【摘要】在我家的客厅和卧室里,总是摆放着几盆盛开的鲜花,那金色的黄、玫瑰的红、纯色的白,点缀得家里春风骀荡,四季放香。

每每有邻居来我家串门,都会对那些花儿赞不绝口!其实,这全是母爱的功劳——那些花儿全都是母亲精心培育的。

【总页数】1页(P59)
【作者】王凤英
【作者单位】
【正文语种】中文
【相关文献】
1.斯霞名人档案浸透母爱芳香
2.提升我国芳香产业水平,促进我国芳香产业发展--2015中国国际芳香产业(洛阳)展览会
3.碘催化芳香酮、芳香醛和芳香胺的Mannich反应:三组分"一锅法"
4.芳香醛、芳香酮和芳香胺的Mannich反应研究概况
5.什么是“芳香性”?芳香性的定义、判据和无机芳香性化合物
因版权原因,仅展示原文概要,查看原文内容请购买。

外国教育史研究与当代中国教育改革

外国教育史研究与当代中国教育改革

外国教育史研究与当代中国教育改革作者:黄凯婷来源:《少年科普报(科教论坛)》2020年第28期摘要:教育是古今中外都在探讨的重要命题,是基础性的社会工作。

研究外国教育史、分析教育共性问题能为中国当代的教育改革提供借鉴。

本文研究了外国教育史的发展特点与态势,着重从教师与学生两个方面进行简要分析。

关键词:外国教育史,当代中国教育,改革中图分类号:G4; 文献标识码:A; 文章编号:(2020)-28-212引言提到当代教育,很多人会想到古代孔子的有教无类等教学主张,但对于外国教育的借鉴则相对较少,特别是对于外国教育史的关注更是寥寥无几。

现阶段我国小学语文教育以古代诗词和现代文阅读作为教学的重点内容,所选范文也多是中国所写的优秀作品,一些学生会对此表示困惑,外国教育史和今天的中国教育有什么关系?实际上最近教育学院的选修课中也很少有人选择外国史的相关课程,一些教师都对此抱有疑问。

随着社会主义市场经济不断发展,中国当代教育与外国教育的相似度不断提高,从外国史的研究中可以窥视到中国今天发展的影子,这一点在教育领域表现得尤为突出。

一、教师方面(一)当代中国的教师状况教师是教育教学活动中的主导者,教师的素质决定了教育的质量,教师教育成为教育工作的源头和重点工程,但当代中国的教师教育状况并不乐观。

二十世纪五十年代至七十年代,我国对教育的重视程度不高,此时的教师在部分人心中与资本主义划着等号,教师在政治上沦落为批判和改造的对象,在经济上愈发贫乏。

与民国时期知识分子获得优厚待遇不同,新中国成立前期的教师工资并没有随着经济发展而增加,部分新人教师的工资水平反而有了一定程度的下降,此时,师范专业的报考率一直不高。

为保证教师的数量和质量,国家出台了一系列措施,在八十年代对师范院校采取“零批录取”的政策,同时,规定在职中小学教师进入高校学习时只能报考师范,限制师范毕业生只能从教,但仍有部分师范生拒绝当教师,投身教育领域的其他行业。

胡主席提出几项重大教育的时代背景是什么?

胡主席提出几项重大教育的时代背景是什么?

胡主席提出几项重大教育的时代背景是什么?
王长存
【期刊名称】《解放军理论学习》
【年(卷),期】2006(000)007
【摘要】第一。

当前。

发展是我们党、国家和军队的第一要务。

当前我国发展面
临着难得的战略机遇期。

但战略机遇期也往往是“战略风险期”,国际国内存在诸多复杂的矛盾和不稳定因素。

高科技发展和陆地资源逐渐减少,国家利益的内涵和外延都发生了变化,不仅传统领土、领海和领空范围攸关国家利益,连海洋、太空、电磁空间也与国家利益密切相关,非传统安全因素明显增加。

所有这一切,都要求我军必须肩负起新的使命,对官兵加强新的历史使命教育,增强官兵的使命意识,自觉履行我军历史使命。

【总页数】1页(P59)
【作者】王长存
【作者单位】国防大学军建政工教研部
【正文语种】中文
【中图分类】D2
【相关文献】
1.十六大以来党中央提出一系列重大战略思想的时代背景和重大意义 [J], 季明
2.怎样正确把握几项重大教育在思想政治建设中的地位和作用? [J], 方昱钢;林宪

3.从几项重大改革看三十年来我国高等教育的发展 [J], 肖汉银
4.全国职业教育工作会议提出“十一五”期间我国职业教育发展的目标任务和重大措施 [J], 无
5.农村教育是什么?——基于时代背景的考察 [J], 陈俊山
因版权原因,仅展示原文概要,查看原文内容请购买。

让思想品德教材富有生命力

让思想品德教材富有生命力

让思想品德教材富有生命力
黄伟
【期刊名称】《江苏教育研究》
【年(卷),期】2009(000)012
【摘要】叶圣陶先生曾说:“教材无非是个例子。

”新的课程观把教材定义为“一种育人的媒体和手段”.教材是给教师完成教学目标提供的范例。

既然是“范例”,教师就有对其进行选择、补充、调整的权利。

教师和学生不是“材料员”而是“建筑师”.他们是材料的主人,更是新材料和新教学智慧实施、开发、创生的主体。

【总页数】3页(P50-52)
【作者】黄伟
【作者单位】南京东山外国语学校,211100
【正文语种】中文
【中图分类】G623.152
【相关文献】
1.富有新意才富有生命力——谈学校管理中的几点创新性做法 [J], 张少华
2.富有生命力的中职德育课教学策略解析 [J], 何李媛
3.怎样使"腌笃鲜"
富有生命力? [J], 蔡育发
4.营造富有生命力的家庭“气候”——单亲家庭子女的教育之道 [J], 田国秀;陈盈
5.创造富有生命力的班级文化——新时期学校班级文化建设新路径剖释 [J], 罗志敏
因版权原因,仅展示原文概要,查看原文内容请购买。

体外分析microRNA功能

体外分析microRNA功能

Analysis of microRNA effector functions in vitroBingbing Wang a ,John G.Doench b ,Carl D.Novinaa,c,*aCancer Immunology and AIDS,Dana-Farber Cancer Institute,and Department of Pathology,Harvard Medical School,Boston,MA 02115,USAbHarvard Institute of Proteomics,Harvard Medical School,Cambridge,MA 02141,USAcBroad Institute of MIT and Harvard,Cambridge,MA 02142,USAAccepted 13April 2007AbstractAnalyses of gene functions in vitro provide the means to dissect biological phenomena.Development of cell-free assays for transcrip-tion,splicing,and translation has yielded great insights into macromolecular interactions and functions required for these processes.This article discusses development of cell-free assays to test macromolecular interactions and activities required for microRNA effector func-tions.This chapter also compares in vitro analyses to complementary studies in cells and the technical considerations that permit molec-ular analysis of microRNA function in cell-free conditions.Ó2007Elsevier Inc.All rights reserved.Keywords:MicroRNA;siRNA;RNA interference;Translation;Cell-free;Gene silencing1.IntroductionmicroRNAs (miRNAs)are an evolutionarily ancient,endogenous class of non-coding RNAs implicated in numerous biological processes [1,2].In mammals,miR-NA-regulated gene expression has been implicated in chro-mosome segregation [3,4],limb morphogenesis [5],and differentiation [6,7].The potential importance of natural gene regulation by miRNAs is highlighted by the identifica-tion of dysregulated miRNA expression in numerous dis-eases,including several cancers (reviewed in [8]).Moreover,bioinformatic methods predict that approxi-mately one-third of all human genes may be targets of miRNAs [9,10]suggesting that miRNAs may govern vast gene regulatory networks.Despite their profound influence over gene expression,the mechanisms used by miRNAs to silence target gene expression are poorly understood.Though there are rare exceptions in mammals [11,12],miRNAs typically bind to the 30-UTR of target mRNAs with imperfectly complementary base pairing and lead totranslational repression of target mRNAs.miRNAs are loaded into an effector complex called the microribonucle-oprotein complex (miRNP),originally discovered by its association with multiple miRNAs and further character-ized to mediate gene silencing [13,14].miRNAs are funda-mentally related to a class of small RNAs,the short interfering RNAs (siRNAs),which are used in another gene silencing pathway,RNA interference (RNAi).siR-NAs,however,are incorporated into the RNA-induced silencing complex (RISC),typically bind to target mRNAs with perfectly complementary base pairing,and lead to cleavage and degradation of target mRNAs [15–17].Importantly,a miRNA can act as an siRNA to induce mRNA cleavage (i.e.RNAi),and an siRNA can act as a miRNA to induce translational gene silencing,simply by manipulating the degree of sequence complementarity between the small RNA and the mRNA target [14,18].Thus,an individual small RNA can lead to biochemically distinct mechanisms of gene silencing:reporter mRNAs with mismatches to the central nucleotides of the small RNA promote translational repression whereas mRNAs with exact sequence complementarity undergo mRNA cleavage.Several cell culture models have used chemically1046-2023/$-see front matter Ó2007Elsevier Inc.All rights reserved.doi:10.1016/j.ymeth.2007.04.003*Corresponding author.E-mail address:carl_novina@ (C.D.Novina)./locate/ymethMethods 43(2007)91–104synthesized,exogenously introduced siRNAs to interrogate the functions of miRNAs([18–22],and others).We have recently adapted one such cell-based system,utilizing the CXCR4siRNA,to an in vitro assay for miRNA-dependent translational repression[23].2.The factors involved in gene silencingA common feature of all characterized silencing effector complexes,such as the miRNP and RISC,is the presence of at least one member of the Argonaute(Ago)family of proteins[13,14,24–27].Ago proteins are defined by the presence of a PAZ(Piwi–Argonaute–Zwille)domain and a PIWI domain[28].The human genome encodes eight Ago proteins,which are further separated into the Ago and PIWI sub-families.Ago1,2,3,and4are closely related and have each been shown to bind to miRNAs[29].Fur-thermore,Ago2has been shown genetically[22]and bio-chemically[29]to be responsible for siRNA-directed mRNA cleavage.Structural studies of the Ago proteins indicate that Agos-1and-4lack the histidine residue required to coordinate Mg++[30]and consequently are dis-pensable for the mRNA cleavage reaction[22].Interest-ingly,Ago3contains the active site histidine residue but is still dispensable for the mRNA cleavage reaction[22].While Ago2is clearly the only Argonaute capable of performing RNAi,it is dispensable for translational repres-sion.Knockout mouse embryonicfibroblasts lacking Ago2 can no longer cleave mRNA targets perfectly complemen-tary to the CXCR4siRNA but can still translationally repress mRNA with imperfectly complementary CXCR4 target sites[22]suggesting that any or all of Agos-1,-3, and-4are responsible for miRNA-directed translational gene silencing.Indeed,it may be that any individual Ago is capable of translational repression,but that the activity is governed by other trans-associated factors in the miRNP complex.A unified view of the various proteins and associated activities that comprise either RISC or the miRNP has not yet emerged.For example,RISC activity has been found associated with complexes ranging from140kDa to500kDa,and as large as80S(reviewed in[31]).The Dro-sophila80S‘‘holo-RISC’’identified by Sontheimer and col-leagues demonstrates two levels of assembly and contains the RNAi proteins Dicer2,Ago2,VIG,TSN,and dFXR [32],as well as ribosomal components L5,L11and5S rRNA[33].Similarly,the miRNP was isolated as a550-kDa complex containing Gemin3and Gemin4[13,14]. For both the miRNP and RISC,many associated factors remain to be identified.With the exception of Ago2in RISC,the functional requirement for any of these proteins in gene silencing is not yet understood.3.Biochemical reconstitution of miRNA functionRecent data have presented conflicting views of the pre-cise mechanism of translational repression by miRNAs.Some reports suggest that miRNAs target actively translat-ing mRNAs[34,35]either by blocking translation initiation [20,23,36]or translation post-initiation[21,37,38].Other reports suggest that miRNAs target mRNAs in P-bodies [39,40]or stress granules[41],sites that do not support active translation.By selectively removing and reconstitut-ing different proteins in a controlled setting,cell-free reac-tions permit a precise molecular understanding of the mechanisms used by miRNAs to repress translation.Addi-tionally,cell-free reactions can clarify the molecular func-tions of the factors implicated in gene silencing.Biochemical reconstitution of miRNA-directed gene silencing in vitro is a prerequisite for determining functional requirements for miRNP-associated factors.In thefirst in vitro RNAi reaction performed in Drosophila embryo lysates,Tuschl,Zamore,Bartel and Sharp demonstrated dsRNA-mediated cleavage of the mRNA target comple-mentary to the input long dsRNA[16]in an ATP-depen-dent fashion[17].Other cell-free RNAi reactions that have been described include Drosophila[42]and human [43,44]Dicer cleavage of dsRNA and Drosha cleavage of primary miRNA transcripts[45–47].Thus,biochemical requirements are becoming well-defined for these processes but the requirements for miRNP-directed translational repression remain unknown.Recently,we described the first cell-free translational repression reaction that recapit-ulates known molecular hallmarks of miRNA function in cells[23].These assays permit analysis of the precise molec-ular mechanisms of miRNA function in translational repression in comparison to mRNA cleavage in vitro.Sev-eral assays are presented that permit molecular analysis of gene silencing in vitro.4.Preparation of reporter mRNAsThe reporter system used for in vitro analyses utilizes the same constructsfirst described in cell-based analyses of miRNA functions that elucidated and extended some of the early principles of miRNA targeting([18,19,21], Fig.1).One such hallmark of miRNA targeting recapitu-lated in vitro,discovered both experimentally and compu-tationally,is the requirement for perfect base pairing through the seed region of the miRNA:mRNA duplex, defined as nucleotides2–7from the50-end of the miRNA [19,48,49].While our original in vitro studies were per-formed by delivering the small RNA as a siRNA duplex, experiments using endogenous miRNAs are possible.This method has the possible advantage of more accurately rep-resenting genuine miRNA-directed repression without the effects of miRNA processing,per se,as the miRNA biogen-esis machinery may also participate in small RNA effector functions[32,50–52].Considerations for the use of endog-enous miRNAs to repress natural target mRNAs are dis-cussed below.For purposes of clarity in this report,we are defining small RNAs as siRNAs when they have perfect comple-mentarity to their mRNA target and as miRNAs when92 B.Wang et al./Methods43(2007)91–104they have imperfect complementarity to their mRNA tar-get.Small RNA can either be introduced as a duplex(i.e. as a canonical siRNA)or as a single strand(i.e.a canonical mature miRNA);the degree of complementarity to the tar-get,not the method of introduction,governs silencing activity in vitro.The CXCR4small RNA serves as the test small RNA while a GFP small RNA serves as the control. Test mRNAs contain binding sites for the CXCR4small RNA while control mRNAs lack binding sites,with the canonical‘‘wild-type’’site shown in Fig.1.One important consideration in choosing control small RNAs is that con-trol RNAs should demonstrate activity on other target mRNAs with sites for that control RNA.Controls that are important especially for analysis of miRNA activity at endogenous mRNA targets include the use of antagomirs.Antagomirs are antisense RNAs that bind to miRNAs to represses their natural function [53–56].Usually,antagomirs have one or more modifica-tions that increase the affinity of the antisense RNAs for their miRNAs.These modifications may be20O-methyl-ated RNA backbones,locked nucleic acid backbones,or other modifications available through several vendors (Dharmacon,Exiqon Gene linkä,Integrated DNA Tech-nologies,Sigma–Aldrich,and Qiagen).Often,antagomirs possess one of several substitutions that permit visualiza-tion in cells or promote transduction across membranes. These may be used for in vitro analyses,though the latter substitutions are considered unnecessary and may actually inhibit the efficiency of the antagomir in vitro.4.1.In vitro transcription of reporter plasmidsAlthough it is theoretically possible to perform coupled transcription-translation reactions to measure miRNA functions in vitro,the major advantage of performing these reactions separately is that the reactions may be uncoupled. Stated another way,performing in vitro transcription reac-tions and purifying the transcripts enables more controlled analyses of the translation step.Purified transcripts may be modified in a variety of ways and may also be added in dif-ferent amounts and at different times to gain insights into the thermodynamic and kinetic parameters governing miRNA function.Linearizing the plasmid reporters permits analysis of translational repression from defined mRNAs containing different ing mRNAs lacking30UTRs also tests the dependence of miRNA function upon cognate miRNA binding sites.1.Linearize pcDNA3.1-based(Invitrogen)plasmidswith EcoRV to generate zero,two,four or six miC-XCR4binding site reporters.2.Linearize pRL-TK-based(Promega)plasmidswith XhoI to generate the one siCXCR4binding site reporter and the RL0X internal control reporter.3.The restriction digestion reactions proceed at37°Cfor2h.4.Extract the linearized plasmids with phenol:chloro-form:isoamyl alcohol(25:24:1,pH8.0).5.Add DEPC-treated H20to the supernatant to afinalvolume of100l L.6.Add2.5volumes of100%ethanol and1/10volumesof3M NaOAC(pH5.5).7.Precipitate RNA pellets by storing the mixture atÀ80°C for1h.8.Resuspend RNA pellets in afinal volume of20l L ofDEPC-treated H2O.RNase-free conditions are important during this and all subsequent steps involv-ing mRNAs and miRNAs.9.Perform in vitro transcription with5l g of linearizedplasmids using the RiboMax T7Large-Scale RNA Production kit according to the manufacturer’s pro-tocols(Promega).10.Purify the RNAs using the RNeasy mini kit accord-ing to the manufacturer’s protocols(Qiagen).4.2.Capping in vitro transcriptsUnlike siRNA-directed mRNA cleavage,miRNA-direc-ted translational repression requires an mRNA cap for function in vitro[23].The issue of whether miRNAs repress translation initiation or post-initiation in cells hingespartly B.Wang et al./Methods43(2007)91–10493upon whether translation driven by viral internal ribosome entry sites(IRESes)could be repressed by miRNAs.It is difficult to determine whether uncapped mRNAs are capped after transfection of mRNAs into cells.One princi-ple advantage of testing mRNA cap-dependency of miRNA functions in vitro is that either capped or uncapped mRNAs may be added to translation repression reactions directly,circumventing the issue of whether viral IRESes accurately represent cellular translation.Indeed,the mRNA cap structure may be important for translational repression by miRNAs.Similar to results observed in cells [20],physiological mRNA caps are required for transla-tional repression by miRNAs in vitro[23].Cap analogs available in some translation kits do not promote transla-tional repression by miRNAs[23].The exact molecular mechanism of the mRNA cap dependency for miRNA function is still unknown.The recombinant version of the mRNA capping enzyme has been reported[57–59]and may be obtained commercially(Ambion).1.The capping reaction buffer is composed of75mMTris(pH7.9),9mM KCl,3.75mM DTT,1.88mM MgCl2,0.15mg/mL BSA,1mM GTP,and1mM S-adenosyl Methionine(Ambion).2.Add20pmol mRNA reporter per7-methyl G cap-ping reaction.3.Add capping enzyme to afinal volume of15l L.4.Incubate the capping reaction at37°C for1h.5.Terminate the reaction by placing the mixture on ice.6.Extract the capped mRNAs by adding phenol:chloro-form:isoamyl alcohol(25:24:1,pH6.0).7.Add2.5volumes of100%ethanol and1/10volumesof3M NaOAC(pH5.5).8.Precipitate RNAs by storing the mixture atÀ80°Cfor1h.9.Resuspend RNA pellets in DEPC-treated H2O to afinal volume of20l L.10.Quantitate RNAs by UV spectrophotometry.4.3.Polyadenylating in vitro transcriptsmiRNA-directed translational repression requires a poly(A)tail for function in vitro[23].Thefirst report of miRNA-directed translational repression in vitro described polyadenylation of transcripts to200adenosines because mRNAs in vivo are on average between200and250nucle-otides in length[60]and because200adenosines promote robust translational repression while shorter poly(A)tails demonstrate less activity.Transcripts are polyadenylated in vitro using recombinant,purified poly(A)polymerase (PAP;[61])which is commercially available(Ambion and USB).Calibrating activity of the enzyme will determine reaction conditions and duration of the polyadenylation reaction.The approximate length of the poly(A)tail and yield of the reaction can be assessed by preparing serial dilutions of RNAs of known lengths.In fact,it may even be advisable to reassess length of the mRNA after the in vitro translational repression assay.Though,it has not been tested in vitro,miRNAs have been reported to pro-mote deadenylation of target mRNAs in cells[62].Alternatively,mRNAs containing poly(A)tails may be generated through use of plasmids containing adenosine tracts.This method has the advantage that an accurate and reproducible number of adenosines may be consis-tently incorporated into target mRNAs without the need to continuously monitor and calibrate the polyadenylation reaction.However,one limitation to this method is the instability and poor expression of plasmids containing long tracts of adenosines.For this reason,the poly(A)polymer-ase method of polyadenylating mRNAs is recommended.1.Polyadenylation reaction buffer is composed of20mMTris–HCl(pH7.0),50mM KCl,0.7mM MnCl2,0.2mM EDTA,100l g/mL acetylated BSA,10%glyc-erol,0.5mM ATP.2.Add10pmol of7-methyl G capped-mRNA.3.Add600U PAP to afinal reaction volume of25l L.4.Incubate at37°C for5–30min.5.Terminate polyadenylation reactions using1l L of5mM EDTA.6.Monitor the polyadenylation reaction on1%agarose–formaldehyde e a0.28–6.58kb RNA marker (Promega)to assess migration of RNAs and thus extent of polyadenylation of reporter RNAs.4.4.Biotinylating the poly(A)tails of in vitro transcriptsOne very important variation on polyadenylation of transcripts in vitro is biotinylation of poly(A)tails[23].This method enables several different downstream analyses of miRNA functions(discussed below).Streptavidin precipi-tation of biotinylated poly(A)tails permits highly efficient recovery of transcripts.Thus,factors strongly associated with target RNAs may be separated from factors more transiently associated with target RNAs.Most impor-tantly,the presence of biotins in the poly(A)tail does not inhibit miRNA repression in the ratios described below [23].The ratio of biotinyl-adenosine to adenosine used in the following reactions may be modified to incorporate more or less biotin-A as required for each experiment.It is recommended that miRNA-dependent translational repression be reassessed at significantly higher levels of bio-tin in the poly(A)tail.1.Perform biotinylation reactions as described above using2.5nM biotin–ATP(Perkin–Elmer)added into reactionscontaining0.5mM ATP,20mM Tris–HCl(pH7.0), 50mM KCl,0.7mM MnCl2,0.2mM EDTA,100l g/ mL BSA,10%glycerol,with0.5mM ATP,5nM bio-tin–ATP(Perkin–Elmer),and600U PAP.Thus,on average,every poly(A)tail has one biotinyl-adenosine for every99adenosines.94 B.Wang et al./Methods43(2007)91–1042.Incubate reactions at37°C for30min.3.Terminate and monitor the progress of these reactions asdescribed above.5.In vitro mRNA translation assayOurfirst attempts to develop a miRNA-dependent translational repression reaction were performed in rabbit reticulocyte lysate(RRL)for several reasons.First,RRL presents a robust source of highly active extract for in vitro analysis.RRL demonstrates very low lot-to-lot var-iability,as consistency between preparations is critical dur-ing in vitro assay development.Second,RRL demonstrates high specific translation activity.In other words,even small amounts of input RNA yield high levels of encoded pro-tein.Indeed,one major insight towards the development of in vitro miRNA-dependent translation repression was reducing input mRNA levels far below the generally rec-ommended levels used in general application of RRL to produce proteins.Third,RRL demonstrates mRNA cap-independent translation.Because the cap-dependency of miRNA-dependent translational repression was not known and RRL permits both cap-dependent and cap-indepen-dent translation,RRL permitted the greatest chance of identifying miRNA activity in extracts.The essential properties of this miRNA-directed transla-tional silencing in vitro are that(1)miRNA-directed trans-lational gene silencing occurs during translation,not by simply reducing steady-state mRNA levels;(2)transla-tional gene silencing requires50-seed region complementar-ity between miRNAs and target mRNAs[9,19,48,49];(3) miRNAs require50-phosphates[63]for translational gene silencing activity;and(4)miRNA-targeted mRNAs require both a7-methyl G cap[20,36]and a poly(A)tail[20]for translational gene silencing,whereas siRNA-targeted mRNAs may be uncapped and unpolyadenylated and still be cleaved.While the use of unphosphorylated siRNAs is common in transfection of cells,our studies indicate that the kinase activity that phosphorylates siRNAs in cells is either absent or weak in RRL and thus phosphorylated small RNAs should be used.The sequences of the CXCR4(test)siRNA are50-P-GUUUUCACUCCAGCUAACACA-30(sense strand)and50-P-UGUUAGCUGGAGUGAAAACUU-30 (antisense strand).The sequences of the GFP(control) siRNA are50P-GGCUACGUCCAGGAGCGCACC-30 (sense strand)and50-P-UGCGCUCCUGGACGUAGC CUU-30(antisense strand).The20O-methylated RNAs used to inhibit small RNAs have identical base sequence to the antisense strand of the CXCR4siRNA with a20car-bon O-methyl substituent on every nucleotide,though other modifications to the inhibitory RNA backbones are possible.One important consideration for robust repression is the amount of starting mRNA that is added and,related to this,the stoichiometry of small RNA to mRNA.Another important consideration is the order of addition ofreagents.The observation that small RNAs and targetRNAs should be mixed and heated togetherfirst was anindication that a RISC loading complex(RLC)activityand a miRNP loading activity(miRLC)was absent fromthe RRL[23].One consequence of the lack of RLC andmiRLC activities is that the two strands must be separatedprior to beginning the gene silencing reactions.In the caseof the translation repression assay,the best repressionoccurred when the miRNA was pre-incubated with thereporter mRNA[23].miRNA function in vitro is optimalwhen the stoichiometry of the small RNAs matches thestoichiometry of the miRNA-binding sites.The fact thatthe miRNA appears to bind the target before the miRNPloads may be the explanation for the strict stoichiometricrequirements for miRNA function in vitro.However,theexact reason for this stoichiometric relationship is stillunknown.Another important consideration is the time atwhich the reaction is terminated and repression is mea-sured.Our studies indicate that repression is mostpotent at early time points,indicating that the miRNPor other trans-acting factors have a limited half-lifein vitro.Further underscoring this idea,we have foundthat the number of freeze-thaw cycles for the lysate iscritical,as each additional cycle dramatically reducesactivity.There are two general methods to visualize the resultsof miRNA-dependent gene silencing reactions,either gelelectrophoresis followed by transfer and autoradiographyor dual luciferase assay.Whereas autoradiography istime-consuming and more accurate,luciferase assays aremore rapid and cost effective.The method of autoradiog-raphy is more accurate because it permits direct visualiza-tion of full-length products offirefly and Renillaluciferases.Non-specific and/or prematurely terminatedproducts offirefly and Renilla luciferases during transla-tion can be excluded from quantitation.In addition,amore precise calculation can be obtained if the back-ground is subtracted from mature bands scanned fromphosphorimager analysis.Nonetheless,measuring genesilencing by luciferase assay has the advantage of immedi-ate results and is suitable for screening large numbers ofreactions.Importantly,luciferase assays require very littlereporter RNAs.Thus,luciferase assays are more compat-ible to test gene silencing of reporter mRNAs in parallelwith testing other assays such as ribosome binding assay(described below).In addition,autoradiography of 35S-incorporated protein production is not possible in assays using50-end-labeled reporter mRNAs(see below).In these cases,luciferase assays are the preferred methodof measuring gene silencing.5.1.Gene silencing assays1.Obtain nuclease-treated RRL from Promega(Cat.#L4960).B.Wang et al./Methods43(2007)91–104952.Mix0.025pmol test or control reporter mRNAs with0.15pmol test or control siRNAs in a total reaction vol-ume of2.2l L.In this example using an mRNA reporter with six binding sites,the ratio of siRNA to mRNA is 6:1.3.Heat the reaction mixture to75°C for3min and cooledto room temperature for5min.4.Place the reaction mixtures on ice.5.Prepare translation master mixes by assembling7l LRRL,0.2l L of4–8U RNasin(Promega),0.2l L 20l M amino acid mixture minus methionine and cys-teine(Promega),and0.4l L(5.7l Ci)Promix L-[35S] in vitro cell labeling mix(Amersham Biosciences).6.Add7.8l L of master mix to each siRNA–mRNAreaction mixture for a total reaction volume of 10l L.7.Incubate the reaction at30°C for10to60min.8.Stop the reactions by transferring the reaction mixturesto ice.5.2.Gel electrophoresis1.Resolve reaction products by12%SDS–PAGE in1·SDS–PAGE buffer(25mM Tris base,200mM glycine,and0.1%SDS)at120V at room temperature for1–2h.2.Transfer proteins from SDS–PAGE to PVDF mem-branes(Bio-Rad)in chilled1·Western buffer composed of20mM Tris base and200mM glycine at80V for1h.3.Quantitate gene silencing by PhosphorImager(Molecu-lar Dynamics).e ImageQuant software.Measure the intensity of eachvisible band and the background.To obtain the absolute volume of each band,subtract the background from each band intensity.5.3.Dual luciferase activity assay1.Perform dual luciferase activity assay according tomanufacturer’s protocol(Promega)using mRNA labeled with[a-32P]ATP in the poly(A)tail in transla-tional gene silencing reactions.2.Measurefirefly luciferase activity in96-well reactiontrays.This method keeps luciferase reaction volumes low and minimizes reaction volume losses.a.Add2l L of each reaction to one well of a96-wellplate.b.Immediately add20l L LAR II(Promega)to eachwell.c.Countfirefly luciferase activity in a luminometer(Victor3V,Perkin–Elmer)for5s.3.Measure Renilla luciferase activity.a.Add20l L Stop&Glo(Promega)to each well andmix.b.Count Renilla luciferase activity for another5s.5.4.Calculate gene silencing1.Normalize gene silencing by measuring the ratio offire-fly luciferase activity to Renilla luciferase activity in each reaction.2.Calculate gene silencing using the following formula:1-[(+siRNA FL6X/RL0)/(ÀsiRNA FL6X/RL0)].5.5.Northern blotting analysisOne method to assess the effect of gene silencing on reporter mRNAs is to perform Northern blotting reac-tions.Even though the reactions are labeled with35S-methionine and35S-cysteine,the degree of radioactivity emitted form this source is minimal relative to the b emis-sion from32P used in traditional Northern blotting.More-over,most of the35S should be extracted away from the RNAs in thefirst step of this procedure.Thus,reactions assessing amino acid incorporation do not substantially affect the signal measured in Northern blotting.This method has the advantage of directly assessing the effect of gene silencing on mRNAs directly without enzymatic amplification required by real-time,reverse transcription PCR.1.Extract RNAs from gene silencing reactions by phe-nol:choloroform:isoamyl alcohol(25:24:1,pH6.0,Ambion).2.Precipitate RNA in2.5volumes of100%ethanoland1/10volumes of3M NaOAc(pH5.5)at–80°C for1h.3.Resuspend the precipitate in10l L of DEPC-trea-ted H2O and quantitate RNAs by UVspectrophotometry.4.Resolve RNAs on a1%agarose–formaldehyde gel.5.Transfer resolved RNAs to Hybond N+mem-branes(Amersham Biosciences).6.Hybond N+membranes are auto UV-crosslinkedat1200l F.7.Generate the probe tofirefly luciferase mRNA bydouble digesting the pGL3vector with ScaI andSphI(New England Biolabs).8.Gel-purify the498bp band.9.Generate the probe to Renilla luciferase RNA byamplifying a200-bp PCR product using thepRL-TK vector as template.Primer sequencesare50-GCTTATCTACGTGCAAGTGATGATTTACC-30and50-TTGAGAACTCGCTCAACGAACG-30.10.Random-label probes using the DECA-Prime II kit(Ambion)in the presence of[a-32P]ATP accordingto manufacturer’s protocols.11.Remove unincorporated isotopes by MicroSpinG-50columns(Amersham Biosciences).12.Hybridize membranes with the labeled probes at42°C overnight.96 B.Wang et al./Methods43(2007)91–104。

全学科“研读评”一体化,赋能提质

全学科“研读评”一体化,赋能提质

全学科“研读评”一体化,赋能提质发布时间:2022-04-25T00:52:20.985Z 来源:《中小学教育》2022年1期作者:王淑英刘玉玲[导读] “双减”背景下的“核心素养导向”的课改新时期,如何真正走向以学生为中心的教育?王淑英刘玉玲保定师范附属学校“双减”背景下的“核心素养导向”的课改新时期,如何真正走向以学生为中心的教育?保师附校推进了全学科阅读,以“研读评一体化”助力双减提质。

以中小学全部学科所关联的各类符号信息为阅读客体,通过拓宽阅读材料、提升阅读技巧,达到促进学科理解、培育核心素养的目的。

旨在引领教师和学生阅读在“研读评一体化”的全学科阅读中,从“学习阅读”走向“阅读学习”,从量变到阅读的质变,培育和提升师生的阅读素养。

一、共创“研读评”一体化模式,教师专业阅读领航。

“研”就是规划教师阅读素养为核心的专业专长路径,结合教师梯队,将教师成长分为六个阶段,即“新任期、发展期、熟练期、示范型、专家型、教育家型”。

每位教师结合自身的教龄和业务发展现状,设定阅读目标,采取专业阅读+生活阅读路径,撰写《个人愿景发展规划阅读规划》,教研组撰写《学科建设发展规划》,教师工作站、工作坊撰写《工作站(坊)发展规划》。

形成专业阅读+学科思想+破解教学难题+工作站(坊)示范引领。

构建学科阅读共同体,打造学科领袖。

进行全学科阅读模式的研究、阅读教学成果交流研讨会。

“读”就是创新了以学科阅读为项目,以阅读教学研究为突破,以指导学生阅读提质为目的,以评价为导向进行的“研读评一体化”教学研究模式。

基于学生视角、基于阅读素养、基于学生成长,形成一种 “读—学—讲—展—评”阅读项目学习模式。

一跟,就是推广“声音工作室老师读课文”公众平台。

二学,就是课内学读活动,借助声音工作室定期推出朗读讲座视频课程,推送给家长和学生观看。

通过课程学习应该怎样读?三研,是开展研学活动,开展“我是志愿讲解员、讲好保定故事”活动(博物馆等地志愿讲解员选拨活动),激发学生爱祖国爱家乡的情感,增强文化自信,为品质之城助力,为儿童友好城市。

  1. 1、下载文档前请自行甄别文档内容的完整性,平台不提供额外的编辑、内容补充、找答案等附加服务。
  2. 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
  3. 3、如文档侵犯您的权益,请联系客服反馈,我们会尽快为您处理(人工客服工作时间:9:00-18:30)。

黄仕英社会责任会计的信息披露制度引言从过去2008年的“三聚氰胺奶粉事件”到今年的老酸奶和毒胶囊事件,这一系列引发的食品安全问题再次成为举国关注的焦点,而且在国际市场也掀起了不小的波澜。

最近几年企业社会责任缺失的案例愈演愈烈,提高企业社会责任的呼声也日益高涨。

本文在这一背景下,从会计视角透视我国社会责任信息披露现状,通过分析发现当前我国社会责任会计信息披露存在的主要问题是国内尚未建立有关“社会责任会计”的规范体系和企业的社会责任意识淡薄,针对这些问题,然后从国家宏观经济发展的需要和企业自身发展的需要两个角度阐述了加快构建我国社会责任会计信息披露制度的必要性,最后在借鉴西方国家较为成熟的社会责任会计信息披露制度基础上,提出构建符合我国国情的社会责任会计信息披露制度设想,即以政府为主导,按照企业规模进行分层次披露,并配套建立社会责任财务评价体系的初步设想。

一、社会责任会计的内涵界定20世纪六十年代,西方发达国家的企业在追求高额利润同时,暴露出工业化的弊端,高发的环境污染事故和职工伤亡事故把企业社会责任缺失推上风口浪尖。

各国政府和社会公众开始关注企业履行社会责任问题,如何将企业社会责任信息收集、汇总、整理、加工、输出和评价成为传统会计的一大挑战,一个新的会计分支——社会责任会计应运而生。

1963年斯坦福研究所提出利益相关者理论,强调利益相关者与企业之间的依存性。

1984年 Freeman在《战略管理:利益相关者分析方法》里,把利益相关者定义为影响企业的经营活动或受企业经营活动影响的个人或团体,包括股东、债权人、消费者、雇员、社区、环境等。

利益相关者理论从利益依存的角度说明企业须关注除股东之外的其他社会成员的利益,即企业应当主动承担社会责任。

阿基–B·卡罗(ArchieB.Caxxol1)认为,企业社会责任是社会在一定时期对企业提出的经济、法律、道德和慈善期望。

1968年,美国会计学家戴维·F·林诺维斯在《会计杂志》发表了题为《社会经济会计》一文,首创“社会责任会计”一词。

他指出“社会责任会计是会计在社会学、政治学和经济学等社会学科中的应用”。

戴维还进一步提出了“社会责任会计是衡量和分析政府及企业行为的社会公共部门所产生的经济和社会效果”的基本观点。

日本《新版会计学大典》中认为:社会责任会计是把企业与社会之间的相互关系当作社会责任,并以它为中心而展开的会计。

陈讯等 (2005)根据社会责任与企业关系的紧密程度把企业社会责任分成三个层次:一是基本企业社会责任,包括对股东的责任和善待员工;二是中级企业社会责任,包括对消费者的责任和服从政府的领导、搞好与社区的关系和保护环境;三是高级企业社会责任,包括积极慈善捐助和热心公益事业。

笔者认为,社会责任会计就是以货币或非货币形式,将企业在环境保护、解决就业、雇员培训、医疗劳保、产品质量等方面的贡献和损害通过一定的会计方法加以衡量和报告,对企业的社会责任及其履行情况的活动进行反映和监督。

二、加快建立我国社会责任会计信息披露制度的必要性(一)是国家宏观经济发展的需要1.是规范市场经济的需要会计不仅具有反映的只能,而且具有监督、控制、评价与考核的职能。

建立我国社会责任会计信息披露制度,既建立了一个监督约束机制。

通过揭示和公布企业责任的履行情况,把企业纳入整个社会的监督之下,使企业的不良行为透明化。

对我国市场机制健康有序的运转起到积极的作用。

2.是解决当前我国一系列社会问题的需要随着我国经济继续保持高速的增长,逐利、好利的企业有时为了自身短期的利益而置社会整体利益于不顾,一系列社会问题接踵而至。

(二)是企业自身追求发展的需要1.企业通过披露社会责任履行情况树立良好形象企业通过披露自身履行社会责任的良好业绩,体现企业对社会的关爱,对环境的爱护,对职工的关怀,可以充分展示自身良好的企业文化,是向社会公众宣传展示自身形象的最佳机会。

社会责任会计信息披露制度的建立,为企业的利益相关者和社会公众提供一个了解企业的窗口,企业通过披露产品质量、售后服务、公益活动、环境治理等信息扩大企业的知名度和美誉度。

2.是企业参与国际竞争的需要随着社会责任标准SA8000的推广,国际上超过200家跨国公司已经制定并推行了企业的社会责任。

随着SA8000未来逐步在我国的实施,社会责任会计最终将取代传统企业会计,为此建立一套适合我国外贸企业并且最终适合所有企业的社会责任评价指标体系和报告模式成为提高我国企业竞争力的关键。

三、我国社会责任会计信息披露的现状社会责任会计从20世纪八十年代开始传入我国,自1990年常勋教授提出“社会责任会计”开始,理论界便开始对社会责任会计理论框架进行探索,但实务界关于社会责任会计信息的披露仍停留在原始的自发阶段,仅有个别行业和部门对此做了相关规定和要求,总体而言,我国至今仍未建立起较为完善的社会责任会计信息披露制度。

1985年,财政部公布了评价企业十大经济效益指标,其中仅有两个指标与社会责任有关:其一,社会贡献率=企业社会贡献总额/平均资产总额;其二,社会积累率=上缴国家财政总额/企业社会贡献总额。

企业社会贡献总额包括工资、劳保退休统筹及其他社会福利支出、利息净支出额、应交增值税、营业税、消费税、所得税等各种税金及附加费和上缴净利润等。

1997年,美国民间组织社会责任国际(Social Accountability International,简称SAI)发表了全球首个社会责任认定标准SA8000(Social Accountability 8000)。

它主要保护企业内部劳工的权利,并规定了企业必须承担的对社会和利益相关者的责任。

该项认定标准的发布对我国出口贸易企业社会责任信息的披露提出了挑战。

如何突破传统会计提供的信息来迎合A8000认证的需求已成为我国出口贸易企业迫在眉睫的问题。

1999年,中国证监会就要求公开发行股票的公司在法律意见书中要说明是否由环境保护、知识产权、产品质量、劳动安全等原因产生侵权之债。

2002年,财政部印发的《2002年企业效绩评价标准》中增设了“综合社会贡献”指标,主要考查企业在履行社会责任、承担社会义务、维护商业信誉、保护环境等方面的情况。

2003年的《公开发行证券的公司信息披露内容与格式准则第1号——招股说明书》中规定发行人应主动披露投资项目因环保因素引致的风险、我国社会责任会计核算体系对环保政策的限制等。

2006年9月25日,深交所发布了《深圳证券交易所上市公司社会责任指引》。

该指引指出,上市公司社会责任是指上市公司对国家和社会的全面发展、自然环境和资源,以及股东、债权人、职工、客户、消费者、供应商、社区等利益相关方所应承担的责任,公司可将社会责任报告与年度报告同时对外披露。

我国中央企业正式发布第一份社会责任报告是国家电网公司2006年发布的《国家电网公司2005社会责任报告》,该报告主要阐述了国家电网公司的价值观、核心业务运作的基本理念、对各方面利益相关者的责任定位和具体行动,以及公司重要的“六个角色定位”和“十个方面的社会责任”,向公众展示了一个真诚、开放、勇于承担责任、不断追求卓越的国家电网公司形象,但其内容也不能全面概括其社会责任。

至2007年底,已有11家中央企业、全国共超过60家企业发布了社会责任报告或可持续发展报告。

2008 年1月4日,国务院国资委又发布了《关于中央企业履行社会责任的指导意见(定稿)》,并将企业社会责任具体界定为切实提高产品质量和服务水平、加强资源节约和环境保护、推进自主创新和技术进步等八个方面。

目前国资委所属的中央企业已经有多家发布社会责任报告。

2008年5月14日,上海证券交易所发布《关于加强上市公司社会责任承担工作的通知》以及《上海证券交易所上市公司环境信息披露指引》,倡导各上市公司积极履行社会责任,重视利益相关者的共同利益,为构建和谐社会、促进社会经济的可持续发展贡献力量。

据《2008年中国上市公司100强公司治理评价报告》显示,百强上市公司中只有10%的公司正式发表了“公司社会责任报告”。

近几年,媒体也越来越关注企业社会责任问题。

2009年,由经济观察报、中国社科院城市发展与环境研究中心共同发起的“2009中国最具社会责任房地产企业G20评选活动”,并发布首份《中国房地产社会责任绿皮书》。

主要关注中国房地产企业在与社会责任相关的十大领域中的表现:建筑质量、消费者满意度、节能环保、合法经营及纳税、就业贡献及善待员工、产业化贡献及善待合作伙伴、慈善捐赠、企业信用、全装修水平、保障房建设。

四、我国社会责任会计信息披露存在的问题1.国内尚未建立有关“社会责任会计”的规范体系纵观社会责任信息披露在我国的发展,缺少一个自上而下、层层牵制“社会责任会计”规范体系。

虽然深交所、上交所都发布了社会责任指引,但并没有对社会责任信息披露的内容、披露方式等做出强制、统一的要求,很多行业的社会责任信息披露只是停留在自发原始阶段,既不规范又不完整。

披露的内容很不全面、随意性强;信息披露的模式单一;缺少定量披露,难以形成综合评价指标体系;2.企业对社会责任信息的披露的意识淡薄“经济人”假设,决定了企业朝着“自利”的方向对社会责任信息披露的相关要求做出反应。

企业把利润最大化或股东财富最大化视为企业的唯一目标,没有将企业作为社会的重要部分自觉履行社会责任,并为社会整体效益做出贡献。

我国企业的社会责任意识尚处于较低层次,相比之下,西方国家的企业已把履行社会责任上升到企业的战略高度,这种认识上的差距是我国企业发布社会责任报告的主动性远不及西方国家的主要原因。

五、西方国家社会责任会计信息披露制度1.美国美国作为企业社会责任会计的发源地,其政府机构和民间组织都对社会责任会计起到了较好的规范作用。

美国政府机构如联邦贸易委员会、环境保护局、职业安全和事故防护局等都要求企业提供有关社会责任履行情况的信息;美国会计学会(AAA)也要求企业在年报中披露社会责任活动业绩、人力资源、社会费用及企业活动对社会的影响等信息;平等就业机会委员会(the Equal Employment Opportunity Commission)要求拥有15名或以上雇员的雇主保存和报告雇员的记录;美国证券交易委员会(SEC)要求企业披露因遵守联邦、州和地方有关环境保护的法律而产生的实质效果、为环境控制设施所花费的资本支出以及因环境事宜导致的法律诉讼所发生的资本支出。

由此看出,美国披露的社会责任信息内容以环境会计信息为主,目前尚未出现完整的社会责任会计报表,主要以社会责任报告为主要披露方式。

2.法国法国是世界上最重视披露社会责任会计信息的国家,在社会责任会计信息披露实践方面处在领先地位。

相关文档
最新文档