全文获取类型
收费全文 | 5258篇 |
免费 | 508篇 |
出版年
2023年 | 18篇 |
2022年 | 46篇 |
2021年 | 89篇 |
2020年 | 65篇 |
2019年 | 63篇 |
2018年 | 90篇 |
2017年 | 91篇 |
2016年 | 128篇 |
2015年 | 229篇 |
2014年 | 286篇 |
2013年 | 352篇 |
2012年 | 440篇 |
2011年 | 438篇 |
2010年 | 304篇 |
2009年 | 271篇 |
2008年 | 361篇 |
2007年 | 375篇 |
2006年 | 345篇 |
2005年 | 315篇 |
2004年 | 297篇 |
2003年 | 274篇 |
2002年 | 249篇 |
2001年 | 42篇 |
2000年 | 40篇 |
1999年 | 53篇 |
1998年 | 87篇 |
1997年 | 35篇 |
1996年 | 53篇 |
1995年 | 45篇 |
1994年 | 51篇 |
1993年 | 34篇 |
1992年 | 22篇 |
1991年 | 13篇 |
1990年 | 18篇 |
1989年 | 18篇 |
1988年 | 11篇 |
1987年 | 13篇 |
1986年 | 6篇 |
1985年 | 5篇 |
1984年 | 10篇 |
1983年 | 11篇 |
1982年 | 6篇 |
1981年 | 10篇 |
1980年 | 5篇 |
1979年 | 8篇 |
1978年 | 7篇 |
1977年 | 13篇 |
1976年 | 9篇 |
1973年 | 3篇 |
1970年 | 3篇 |
排序方式: 共有5766条查询结果,搜索用时 140 毫秒
101.
Peter Schierack Stefan R?diger Christoph Kuhl Rico Hiemann Dirk Roggenbuck Ganwu Li J?rg Weinreich Enrico Berger Lisa K. Nolan Bryon Nicholson Antje R?mer Ulrike Fr?mmel Lothar H. Wieler Christian Schr?der 《PloS one》2013,8(4)
We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars.Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation.In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars. 相似文献
102.
Thomas Vercruysse Eline Boons Tom Venken Els Vanstreels Arnout Voet Jan Steyaert Marc De Maeyer Dirk Daelemans 《PloS one》2013,8(4)
HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb190) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb190-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb190 and the Rev multimerization domains were evaluated in different assays measuring Nb190-Rev interaction or viral production. Seven residues within Nb190 and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb190-Rev structural interface. This Nb190-Rev interaction model can guide further studies of the Nb190 effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb190 epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain. 相似文献
103.
Taylor J. Jensen Tricia Zwiefelhofer Roger C. Tim ?eljko D?akula Sung K. Kim Amin R. Mazloom Zhanyang Zhu John Tynan Tim Lu Graham McLennan Glenn E. Palomaki Jacob A. Canick Paul Oeth Cosmin Deciu Dirk van den Boom Mathias Ehrich 《PloS one》2013,8(3)
Background
Circulating cell-free (ccf) fetal DNA comprises 3–20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.Methods
Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.Results
Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.Conclusions
These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy. 相似文献104.
Yuki Ito Jose Luis Vela Fumiko Matsumura Hitomi Hoshino Aaron Tyznik Heeseob Lee Enrico Girardi Dirk M. Zajonc Robert Liddington Motohiro Kobayashi Xingfeng Bao Jeanna Bugaytsova Thomas Borén Rongsheng Jin Yinong Zong Peter H. Seeberger Jun Nakayama Mitchell Kronenberg Minoru Fukuda 《PloS one》2013,8(12)
Approximately 10–15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl α-glucosides in H. pylori cell wall by α1,4-GlcNAc-capped mucin O-glycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl α-glucosyltransferase (αCgT), which forms cholesteryl α-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of αCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl α-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of αCgT showing a range of enzymatic activity. However, cholesteryl α-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active αCgT into iNKT cell-deficient (Jα18−/−) or wild-type mice, bacterial recovery significantly increased in Jα18−/− compared to wild-type mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Jα18−/− compared to wild-type mice. These findings demonstrate that cholesteryl α-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis. 相似文献
105.
Wenjing Li Piotr Łączyński Hermes E. Escalona Jonas Eberle Lizhi Huo Xiaosheng Chen Weidong Huang Bingxu Chen Dirk Ahrens Adam Ślipiński Wioletta Tomaszewska Xingmin Wang 《Systematic Entomology》2020,45(2):447-463
Ladybirds of the cosmopolitan tribe Chilocorini prey mainly on coccids and include several important biocontrol agents. The phylogenetic relationships of Chilocorini are poorly known. In this paper, we provide a phylogenetic reconstruction of Chilocorini containing all 27 genera based on five molecular markers and 86 adult morphological characters. Morphological character states were mapped on the combined data tree from Bayesian inference to analyse morphological traits of each genus. Sixteen morphological characters were selected to reconstruct the ancestral states using maximum parsimony and maximum likelihood methods. Divergence times were estimated based on the relaxed molecular clock approach. Our results indicate that Chilocorini, excluding Chilocorellus Miyatake, is monophyletic and closely related to Plotinini. The crown group Chilocorini was estimated to date back to the Middle Cretaceous. Anisorcus Crotch, Egius Mulsant, Phaenochilus Weise and Simmondsius Ahmad & Ghani are synonymized here with Chilocorus Leach ( syn.n. ). The genus Chilocorellus is excluded from Chilocorini. The split of current genera was estimated to have occurred during the Middle Paleogene to Late Paleogene. 相似文献
106.
Kai Hilpert Dirk FH Winkler Robert EW Hancock 《Biotechnology & genetic engineering reviews》2013,29(1):31-106
Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery. 相似文献
107.
Kathleen B?rner Dominik Niopek Gabriella Cotugno Michaela Kaldenbach Teresa Pankert Joschka Willemsen Xian Zhang Nina Schürmann Stefan Mockenhaupt Andrius Serva Marie-Sophie Hiet Ellen Wiedtke Mirco Castoldi Vytaute Starkuviene Holger Erfle Daniel F. Gilbert Ralf Bartenschlager Michael Boutros Marco Binder Konrad Streetz Hans-Georg Kr?usslich Dirk Grimm 《Nucleic acids research》2013,41(21):e199
As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. 相似文献
108.
109.
The palaeoecological visibility of historical human impact on natural ecosystems in tropical East Africa is strongly impeded by an overriding dominant signature of climate change at decadal‐to‐millennial time scales. Better knowledge of the relative magnitude and timing of present and past human impact and climate variability is, however, instrumental to properly assess the resilience, and recovery potential, of East Africa's natural ecosystems. Here, we briefly review comprehensive previous attempts to assess past ecosystem responses to climate change and human impact. We further discuss some key issues of climate‐human‐ecosystem relationships in a multidisciplinary framework and address some future challenges and outcomes, which may pave the way to a better understanding of past climate‐human‐ecosystem interaction‐ in tropical Africa. 相似文献
110.
The pollen–ovule ratio (P/O) is commonly used to estimate the mode of sexual reproduction in flowering plants. In previous studies, a clear correspondence has been detected between this character and the degree of autogamy. We here investigate variation in this character and its expected correlates in the genus Veronica (Plantaginaceae). Pollen–ovule ratios of 45 species representing eleven percent of all the species in the genus were investigated and compared with results from crossing experiments from previous studies. In addition, multiple populations of 17 of the 45 studied species were sampled and a controlled‐environment experiment was conducted to evaluate the extent of intraspecific variation. Moreover, relationships between P/O and other primary and secondary reproductive characters of the Veronica flower were investigated in relation to a phylogenetic hypothesis in order to determine the phylogenetic constraints on reproductive characters. The differences in P/O among species correspond well to the diversity of mating systems in Veronica and correlate well with other floral characters such as corolla size. These characters together seem to allow a powerful and fast tool to infer mating systems. However, causes for intraspecific variation of P/O, such as different cytotypes, ecotypes or different growth conditions, need to be considered. 相似文献