首页 | 本学科首页   官方微博 | 高级检索  
文章检索
  按 检索   检索词:      
出版年份:从到   被引次数:从到   他引次数:从到 提示：输入*表示无穷大
全文获取类型
  收费全文   22448篇
  免费   2164篇
  国内免费   9篇
专业分类
电工技术   1385篇
综合类   409篇
化学工业   9964篇
金属工艺   274篇
机械仪表   452篇
建筑科学   1528篇
矿业工程   172篇
能源动力   221篇
轻工业   2599篇
水利工程   174篇
石油天然气   88篇
无线电   577篇
一般工业技术   3402篇
冶金工业   861篇
原子能技术   54篇
自动化技术   2461篇
出版年
  2023年   795篇
  2022年   649篇
  2021年   1255篇
  2020年   1119篇
  2019年   812篇
  2018年   690篇
  2017年   522篇
  2016年   750篇
  2015年   919篇
  2014年   984篇
  2013年   1665篇
  2012年   774篇
  2011年   720篇
  2010年   982篇
  2009年   1123篇
  2008年   636篇
  2007年   588篇
  2006年   425篇
  2005年   387篇
  2004年   344篇
  2003年   316篇
  2002年   205篇
  2001年   148篇
  1998年   224篇
  1997年   167篇
  1996年   232篇
  1995年   214篇
  1994年   191篇
  1993年   248篇
  1992年   173篇
  1990年   170篇
  1989年   193篇
  1988年   160篇
  1987年   183篇
  1986年   200篇
  1985年   178篇
  1984年   174篇
  1983年   184篇
  1982年   169篇
  1981年   213篇
  1980年   167篇
  1979年   180篇
  1977年   150篇
  1976年   150篇
  1975年   204篇
  1974年   189篇
  1973年   369篇
  1972年   214篇
  1971年   152篇
  1968年   154篇
排序方式： 共有10000条查询结果，搜索用时 15 毫秒
991.
    
Small interfering RNA (siRNA) can effectively silence target genes through Argonate 2 (Ago2)-induced RNA interference (RNAi). It is very important to control siRNA activity in both spatial and temporal modes. Among different masking strategies, photocaging can be used to regulate gene expression through light irradiation with spatiotemporal and dose-dependent resolution. Many different caging strategies and caging groups have been reported for light-activated siRNA gene silencing. Herein, we describe a novel caging strategy that increases the blocking effect of RISC complex formation/process through host/guest (including ligand/receptor) interactions, thereby enhancing the inhibition of caged siRNA activity until light activation. This strategy can be used as a general approach to design caged siRNAs for the photomodulation of gene silencing of exogenous and endogenous genes.  相似文献   
992.
The utilities of an α-methylene-β-lactone (MeLac) moiety as a warhead composed of multiple electrophilic sites are reported. We demonstrate that a MeLac-alkyne not only reacts with diverse proteins as a broadly reactive measurement probe, but also recruits reduced endogenous glutathione (GSH) to assemble a selective chemical probe of GSH-β-lactone (GSH-Lac)-alkyne in live cells. Tandem mass spectrometry reveals that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael or acyl addition. A peptide-centric proteomics platform demonstrates that the proteomic selectivity profiles of orlistat and parthenolide, which have distinct reactivities, are measurable by MeLac-alkyne as a high-coverage probe. The GSH-Lac-alkyne selectively probes the glutathione S-transferase P responsible for multidrug resistance. The assembly of the GSH-Lac probe exemplifies a modular and scalable route to develop selective probes with different recognizing moieties.  相似文献   
993.
    
Biomaterials with dynamically tunable properties are critical for a range of applications in regenerative medicine and basic biology. In this work, we show the reversible control of gelatin methacrylate (GelMA) hydrogel stiffness through the use of DNA crosslinkers. We replaced some of the inter-GelMA crosslinks with double-stranded DNA, allowing for their removal through toehold-mediated strand displacement. The crosslinks could be restored by adding fresh dsDNA with complementary handles to those on the hydrogel. The elastic modulus (G’) of the hydrogels could be tuned between 500 and 1000 Pa, reversibly, over two cycles without degradation of performance. By functionalizing the gels with a second DNA strand, it was possible to control the crosslink density and a model ligand in an orthogonal fashion with two different displacement strands. Our results demonstrate the potential for DNA to reversibly control both stiffness and ligand presentation in a protein-based hydrogel, and will be useful for teasing apart the spatiotemporal behavior of encapsulated cells.  相似文献   
994.
    
There is growing interest in developing intracellular RNA tools. Herein, we describe a strategy for N3-kethoxal (N3K)-based bioorthogonal intracellular RNA functionalization. With N3K labeling followed by an in vivo click reaction with DBCO derivatives, RNA can be modified with fluorescent or phenol groups. This strategy provides a new way of labeling RNA inside cells.  相似文献   
995.
    
Template-guided chemical reactions between nucleic acid strands are an important process in biomedical research. However, almost all of these reactions employ an oligonucleotide-templated approach that is based on the double-helix alignment. The moderate stability of the double helix makes this approach unsuitable for many chemical reactions, so alternative nucleic acid alignment mechanisms, demonstrating higher thermal and chemical stability, are desirable. Earlier, we described a noncovalent coupling mechanism between DNA strands through a quadruplex-and-Mg2+ connection (QMC). QMC is based on G-quadruplexes and allows unusually stable and specific interactions. Herein, a novel catalytic nucleic acid reaction, based on QMC, is described. This approach uses G-tetrads as a structural and recognition element without employing Watson-Crick complementarity rules at any stage of substrate/catalyst formation or interaction between them. Quadruplex-templated ligation can be achieved through the self-ligation of two nucleic acid strands, or through a quadruplex catalyst, which forms a G-triplex and specifically connects the strands. The process is extraordinarily robust and efficient. For instance, the ligation of carbodiimide-activated substrates can proceed in boiling solutions, and complete ligation is demonstrated within a minute. The quadruplex-templated and catalyzed reactions will create new opportunities for chemical reactions requiring harsh experimental conditions.  相似文献   
996.
    
Synthetic vaccines, based on antigenic peptides that comprise MHC−I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3CysSK4. A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.  相似文献   
997.
    
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.  相似文献   
998.
    
Selenium-modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The widespread application of 2-selenopyrimidine nucleosides is currently limited by low yields in established synthetic routes. Herein, we describe the optimization of the synthesis of 2-Se-uridine and 2-Se-thymidine derivatives by thermostable nucleoside phosphorylases in transglycosylation reactions using natural uridine or thymidine as sugar donors. Reactions were performed at 60 or 80 °C and at pH 9 under hypoxic conditions to improve the solubility and stability of the 2-Se-nucleobases in aqueous media. To optimize the conversion, the reaction equilibria in analytical transglycosylation reactions were studied. The equilibrium constants of phosphorolysis of the 2-Se-pyrimidines were between 5 and 10, and therefore differ by an order of magnitude from the equilibrium constants of any other known case. Hence, the thermodynamic properties of the target nucleosides are inherently unfavorable, and this complicates their synthesis significantly. A tenfold excess of sugar donor was needed to achieve 40−48 % conversion to the target nucleoside. Scale-up of the optimized conditions provided four Se-containing nucleosides in 6–40 % isolated yield, which compares favorably to established chemical routes.  相似文献   
999.
    
Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment – typically at a single site or at a few sites per protein – of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l -phenylalanine (pPR), with ∼30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production – using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain – of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling.  相似文献   
1000.
    
  相似文献   
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号