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101.
Cook N Nichols RA Wilkinson N Paton CA Barker K Smith HV 《Applied and environmental microbiology》2007,73(22):7388-7391
We report a method for detecting Giardia duodenalis cysts on lettuce, which we subsequently use to examine salad products for the presence of Giardia cysts and Cryptosporidium oocysts. The method is based on four basic steps: extraction of cysts from the foodstuffs, concentration of the extract and separation of the cysts from food materials, staining of the cysts to allow their visualization, and identification of cysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Cyst staining is also performed using proprietary reagents. The method recovered 46.0% +/- 19.0% (n = 30) of artificially contaminating cysts in 30 g of lettuce. We tested the method on a variety of commercially available natural foods, which we also seeded with a commercially available internal control, immediately prior to concentration of the extract. Recoveries of the Texas Red-stained Giardia cyst and Cryptosporidium oocyst internal controls were 36.5% +/- 14.3% and 36.2% +/- 19.7% (n = 20), respectively. One natural food sample of organic watercress, spinach, and rocket salad contained one Giardia cyst 50 g(-1) of sample as an indigenous surface contaminant. 相似文献
102.
Judy K. Morona Renato Morona David C. Miller James C. Paton 《Journal of bacteriology》2002,184(2):577-583
The first four genes of the capsule locus (cps) of Streptococcus pneumoniae (cpsA to cpsD) are common to most serotypes. We have previously determined that CpsD is an autophosphorylating protein-tyrosine kinase, demonstrated that CpsC is required for CpsD tyrosine-phosphorylation, and shown that CpsB is required for dephosphorylation of CpsD. In the present study we show that CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase that belongs to the PHP (polymerase and histidinol phosphatase) family of phosphoesterases. We also show that an S. pneumoniae strain with point mutations in cpsB, affecting one of the conserved motifs of CpsB, is unencapsulated and appears to be morphologically identical to a strain in which the cpsB gene had been deleted. 相似文献
103.
Nicholas Johnson Sarah Ha?denteufel Melanie Theis Adrienne W. Paton James C. Paton Richard Zimmermann Stephen High 《PloS one》2013,8(10)
The metazoan Sec61 translocon transports polypeptides into and across the membrane of the endoplasmic reticulum via two major routes, a well-established co-translational pathway and a post-translational alternative. We have used two model substrates to explore the elements of a secretory protein precursor that preferentially direct it towards a co- or post-translational pathway for ER translocation. Having first determined the capacity of precursors to enter ER derived microsomes post-translationally, we then exploited semi-permeabilized mammalian cells specifically depleted of key membrane components using siRNA to address their contribution to the membrane translocation process. These studies suggest precursor chain length is a key factor in the post-translational translocation at the mammalian ER, and identify Sec62 and Sec63 as important components acting on this route. This role for Sec62 and Sec63 is independent of the signal sequence that delivers the precursor to the ER. However, the signal sequence can influence the subsequent membrane translocation process, conferring sensitivity to a small molecule inhibitor and dictating reliance on the molecular chaperone BiP. Our data support a model where secretory protein precursors that fail to engage the signal recognition particle, for example because they are short, are delivered to the ER membrane via a distinct route that is dependent upon both Sec62 and Sec63. Although this requirement for Sec62 and Sec63 is unaffected by the specific signal sequence that delivers a precursor to the ER, this region can influence subsequent events, including both Sec61 mediated transport and the importance of BiP for membrane translocation. Taken together, our data suggest that an ER signal sequence can regulate specific aspects of Sec61 mediated membrane translocation at a stage following Sec62/Sec63 dependent ER delivery. 相似文献
104.
Genomic sequence duplication is an important mechanism for genome evolution, often resulting in large sequence variations with implications for disease progression. Although paired-end sequencing technologies are commonly used for structural variation discovery, the discovery of novel duplicated sequences remains an unmet challenge. We analyze duplicons starting from identified high-copy number variants. Given paired-end mapped reads, and a candidate high-copy region, our tool, Reprever, identifies (a) the insertion breakpoints where the extra duplicons inserted into the donor genome and (b) the actual sequence of the duplicon. Reprever resolves ambiguous mapping signatures from existing homologs, repetitive elements and sequencing errors to identify breakpoint. At each breakpoint, Reprever reconstructs the inserted sequence using profile hidden Markov model (PHMM)-based guided assembly. In a test on 1000 artificial genomes with simulated duplication, Reprever could identify novel duplicates up to 97% of genomes within 3 bp positional and 1% sequence errors. Validation on 680 fosmid sequences identified and reconstructed eight duplicated sequences with high accuracy. We applied Reprever to reanalyzing a re-sequenced data set from the African individual NA18507 to identify >800 novel duplicates, including insertions in genes and insertions with additional variation. polymerase chain reaction followed by capillary sequencing validated both the insertion locations of the strongest predictions and their predicted sequence. 相似文献
105.
Smith J Sadeyen JR Paton IR Hocking PM Salmon N Fife M Nair V Burt DW Kaiser P 《Journal of virology》2011,85(21):11146-11158
106.
F. Amijee E.J. Allans R.N. Waterhouse L.A. Glover A.M. Paton 《Biocontrol Science and Technology》1992,2(3):203-214
L‐forms of the halo blight pathogen, Pseudomonas syringae phaseolicola, were maintained in a medium which suppressed cell wall synthesis. These L‐forms, unlike revertants (walled forms derived from unstable L‐forms) and cell walled (parent) organisms, did not elicit a hypersensitive response in tobacco leaves. Association of L‐forms with Phaseolus vulgaris was established by seed imbibition in L‐form suspensions compared with appropriate control treatments (5% mannitol or heat‐killed cells). Seedling emergence and plant growth was not affected by L‐form imbibition. The association was detected by agglutination assays using polyclonal antibody. The L‐form association was localized to the lower shoot tissue and was progressively lost with age of plants. Plants with associated L‐forms had vigour and shoot weights equivalent to controls and showed no disease symptoms. The cell walled form could not be isolated from plants showing positive agglutination. On challenge with the pathogen, plants associated with L‐forms showed significantly less disease symptoms than controls. Stem extracts, from associated plants, were inhibitory to in vitro cultures of both L‐forms and parent forms of Ps. syr. phaseolicola. These results indicate that L‐form associations confer induced systemic resistance to bean plants and might be developed as novel biocontrol systems. 相似文献
107.
108.
Distinguishing low frequency mutations from RT-PCR and sequence errors in viral deep sequencing data
Richard J Orton Caroline F Wright Marco J Morelli David J King David J Paton Donald P King Daniel T Haydon 《BMC genomics》2015,16(1)
Background
RNA viruses have high mutation rates and exist within their hosts as large, complex and heterogeneous populations, comprising a spectrum of related but non-identical genome sequences. Next generation sequencing is revolutionising the study of viral populations by enabling the ultra deep sequencing of their genomes, and the subsequent identification of the full spectrum of variants within the population. Identification of low frequency variants is important for our understanding of mutational dynamics, disease progression, immune pressure, and for the detection of drug resistant or pathogenic mutations. However, the current challenge is to accurately model the errors in the sequence data and distinguish real viral variants, particularly those that exist at low frequency, from errors introduced during sequencing and sample processing, which can both be substantial.Results
We have created a novel set of laboratory control samples that are derived from a plasmid containing a full-length viral genome with extremely limited diversity in the starting population. One sample was sequenced without PCR amplification whilst the other samples were subjected to increasing amounts of RT and PCR amplification prior to ultra-deep sequencing. This enabled the level of error introduced by the RT and PCR processes to be assessed and minimum frequency thresholds to be set for true viral variant identification. We developed a genome-scale computational model of the sample processing and NGS calling process to gain a detailed understanding of the errors at each step, which predicted that RT and PCR errors are more likely to occur at some genomic sites than others. The model can also be used to investigate whether the number of observed mutations at a given site of interest is greater than would be expected from processing errors alone in any NGS data set. After providing basic sample processing information and the site’s coverage and quality scores, the model utilises the fitted RT-PCR error distributions to simulate the number of mutations that would be observed from processing errors alone.Conclusions
These data sets and models provide an effective means of separating true viral mutations from those erroneously introduced during sample processing and sequencing.Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1456-x) contains supplementary material, which is available to authorized users. 相似文献109.
Kenneth J. Ferguson Sarah Cleaveland Daniel Thomas Haydon Alexandre Caron Richard A. Kock Tiziana Lembo J. Grant C. Hopcraft Bertrand Chardonnet Thomas Nyariki Julius Keyyu David James Paton Fredrick Mathias Kivaria 《EcoHealth》2013,10(3):314-322
Strategies to control transboundary diseases have in the past generated unintended negative consequences for both the environment and local human populations. Integrating perspectives from across disciplines, including livestock, veterinary and conservation sectors, is necessary for identifying disease control strategies that optimise environmental goods and services at the wildlife-livestock interface. Prompted by the recent development of a global strategy for the control and elimination of foot-and-mouth disease (FMD), this paper seeks insight into the consequences of, and rational options for potential FMD control measures in relation to environmental, conservation and human poverty considerations in Africa. We suggest a more environmentally nuanced process of FMD control that safe-guards the integrity of wild populations and the ecosystem dynamics on which human livelihoods depend while simultaneously improving socio-economic conditions of rural people. In particular, we outline five major issues that need to be considered: 1) improved understanding of the different FMD viral strains and how they circulate between domestic and wildlife populations; 2) an appreciation for the economic value of wildlife for many African countries whose presence might preclude the country from ever achieving an FMD-free status; 3) exploring ways in which livestock production can be improved without compromising wildlife such as implementing commodity-based trading schemes; 4) introducing a participatory approach involving local farmers and the national veterinary services in the control of FMD; and 5) finally the possibility that transfrontier conservation might offer new hope of integrating decision-making at the wildlife-livestock interface. 相似文献
110.
Bradley Clark Vitor P. Costa Brendan J. O'Brien Luiz G. Guglielmo Carl D. Paton 《PloS one》2014,9(12)