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:   被引次数:   他引次数: 提示:输入*表示无穷大
  收费全文   346067篇
  免费   37753篇
  国内免费   210篇
  2021年   3766篇
  2019年   3743篇
  2015年   4897篇
  2014年   5534篇
  2013年   7822篇
  2012年   8743篇
  2011年   8655篇
  2010年   6116篇
  2009年   5921篇
  2008年   7868篇
  2007年   8118篇
  2006年   7687篇
  2005年   7665篇
  2004年   8841篇
  2003年   9371篇
  2002年   9183篇
  2001年   13762篇
  2000年   12840篇
  1999年   10673篇
  1998年   4944篇
  1997年   5345篇
  1996年   4397篇
  1995年   4338篇
  1994年   4641篇
  1993年   4564篇
  1992年   8932篇
  1991年   8633篇
  1990年   8570篇
  1989年   8273篇
  1988年   7946篇
  1987年   7862篇
  1986年   7223篇
  1985年   7486篇
  1984年   6357篇
  1983年   5656篇
  1982年   4522篇
  1981年   4360篇
  1980年   3916篇
  1979年   6422篇
  1978年   5227篇
  1977年   4958篇
  1976年   4691篇
  1975年   5001篇
  1974年   5460篇
  1973年   5320篇
  1972年   5059篇
  1971年   4637篇
  1970年   4264篇
  1969年   3996篇
  1968年   3599篇
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
1.
2.
In alpine regions worldwide, climate change is dramatically altering ecosystems and affecting biodiversity in many ways. For streams, receding alpine glaciers and snowfields, paired with altered precipitation regimes, are driving shifts in hydrology, species distributions, basal resources, and threatening the very existence of some habitats and biota. Alpine streams harbour substantial species and genetic diversity due to significant habitat insularity and environmental heterogeneity. Climate change is expected to affect alpine stream biodiversity across many levels of biological resolution from micro‐ to macroscopic organisms and genes to communities. Herein, we describe the current state of alpine stream biology from an organism‐focused perspective. We begin by reviewing seven standard and emerging approaches that combine to form the current state of the discipline. We follow with a call for increased synthesis across existing approaches to improve understanding of how these imperiled ecosystems are responding to rapid environmental change. We then take a forward‐looking viewpoint on how alpine stream biologists can make better use of existing data sets through temporal comparisons, integrate remote sensing and geographic information system (GIS) technologies, and apply genomic tools to refine knowledge of underlying evolutionary processes. We conclude with comments about the future of biodiversity conservation in alpine streams to confront the daunting challenge of mitigating the effects of rapid environmental change in these sentinel ecosystems.  相似文献   
3.
The functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.  相似文献   
4.
We combine total internal reflection fluorescence structured illumination microscopy with spatiotemporal image correlation spectroscopy to quantify the flow velocities and directionality of filamentous-actin at the T cell immunological synapse. These techniques demonstrate it is possible to image retrograde flow of filamentous-actin at superresolution and provide flow quantification in the form of velocity histograms and flow vector maps. The flow was found to be retrograde and radially directed throughout the periphery of T-cells during synapse formation.Many biological processes are now being visualized with the use of superresolution fluorescence microscopy techniques. However, localization-based techniques primarily rely on fixed or slow moving samples to permit the collection of structural information. The 10-fold gains in resolution afforded by these superresolution techniques are usually possible through sacrificing the factors that originally made microscopy such a powerful tool: the ability to image live cells. In the case of stimulated emission depletion imaging, the scanning approach associated with this technique may fail to detect faster molecular events when imaging whole cellular regions.Structured illumination microscopy (SIM) is an alternative to these methods (1). It increases the resolution of conventional fluorescence microscopy twofold; it has the advantage of using a wide-field system, providing fast acquisition speeds of whole cells with relatively low laser powers; and it is compatible with standard fluorophores. By using a physical grating to produce interference patterns from a laser, periodic illumination is created. This patterned illumination causes information from higher spatial frequencies to be downmodulated (i.e., shifted) into the optical transfer function (support region) of the lens, resulting in higher-resolution spatial information being captured than is ordinarily obtainable.To quantify the directional motion of intracellular molecules, spatiotemporal image correlation spectroscopy (STICS (2)) was applied. Using spatial image correlation in time, STICS measures the similarity of pixels with those surrounding in lagging frames via a correlation function. The correlation function provides information on both flow velocities and directionality, while discounting static structures through the immobile object filter, achieved by subtracting a moving average of pixel intensities.The formation of an immunological synapse between T cells and antigen-presenting cells is a process requiring many dynamic (3) and subdiffraction-limited clustering events (4–6) to take place. The polymerization of actin is important for the spreading of cells over their target antigen-presenting cells (7), as well as cell mobility and migration (8). Retrograde flow of densely meshed cortical actin is observed at the basal membrane of synapse-forming T cells, where it may have a role in the corralling and clustering of signaling molecules at the plasma membrane (9), as well as at the leading edge of migrating cells (10). Filamentous actin is an extremely dynamic (7), densely packed, and thin (7-nm) structure (11,12).Here, we perform STICS on SIM data acquired on a total internal reflection fluorescence (TIRF) microscope system, which generated an evanescent field of 75-nm depth for excitation. To our knowledge, this is the first demonstration of an image correlation approach to quantify molecular dynamics on subresolution length scales using wide-field microscopy. To demonstrate the technique, we analyze two-dimensional actin flows in CD4+ T cells during immunological synapse formation, performed after cross-linking of antigen T cell receptors on a coverslip coated with specific antibodies.Fig. 1 a shows a schematic of the TIRF SIM setup. Excitation light (488 nm) passes through a polarizing module and then a phase-grating block, producing diffracted beams. These are then passed through a diffraction filter module to isolate the −1 and +1 order laser beams. These first-order laser beams are angled through the objective to produce total internal reflection conditions at the glass-water interface. The two evanescent waves interfere at the sample, producing structured illumination. The setup then produces lateral and rotational shifts through three orientations, producing nine raw images containing higher spatial frequencies than can normally be acquired by an objective using standard light microscopy. Fig. 1 b demonstrates the increased resolution obtained from TIRF SIM. Shown are the collected Fourier frequencies compared to those of a conventional microscope (dotted red line). Resolution was also measured using sparse 100-nm diameter fluorescent beads. Fig. 1 c shows a magnified image of these beads from which a line profile was obtained (yellow arrow). The full width at half-maximum of this profile (Fig. 1 d) gives a lateral resolution for the system of 120 nm.Open in a separate windowFigure 1(a) Schematic of the TIRF SIM setup. (b) Demonstration of the doubling of spatial resolution of collected frequencies through a Fourier transform (superimposed red circle demonstrating regular spatial frequency limits). (c) SIM reconstructed image of 100-nm bead (scale bar 0.5 μm). (d) (Plotted line) Bead showing full width at half-maximum of 120 nm.We then applied STICS analysis to quantify actin flow in T cell synapses acquired using TIRF SIM (Fig. 2). Fig. 2 a shows a schematic of the STICS analysis. From the raw data, immobile objects are first filtered by subtracting a moving average of the pixel values. Vector maps were obtained from correlation analysis of the time-series as previously published in Hebert et al. (2) and Brown et al. (13). Fig. 2 b shows a reconstructed TIRF SIM image of a mature T cell immunological synapse, representative of a time-point derived from the time series acquired at 1.28 fps (see Movie S1 in the Supporting Material). From this reconstructed image, two representative regions have been selected. In these regions, pseudo-colored actin flow vectors are overlaid onto the fluorescence intensity image. These range in magnitude from 0.01 μm/min (blue) to 5.61 μm/min (red). It can be observed that all flow vectors are directed radially toward the synapse center. A histogram of this flow is shown in Fig. 2 c. The histogram shows a peak retrograde flow velocity of 1.91 ± 1.27 μm/min. These data are representative of n = 7 T-cell synapses imaged by TIRF SIM.Open in a separate windowFigure 2(a) STICS analysis, performed by isolating mobile from immobile structures through a moving average filter (i) and binning a subset of pixels into blocks of superpixels (ii); the STICS software correlates spatial fluorescence fluctuations through time (iii). The code then outputs vector maps showing directionality and flow velocities. (b) TIRF SIM image of actin flow in a T cell 5 min after contact with a stimulatory coverslip. (Zoomed regions) Retrograde actin flow at the synapse periphery. (c) Histograms showing flow speed statistics of vectors from T-cell synapses (n = 7).  相似文献   
5.
Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore-forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating.  相似文献   
6.
We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR_0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed.  相似文献   
7.
Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.  相似文献   
8.
Lipid-mimetic metallosurfactant based luminophores are promising candidates for labeling phospholipid membranes without altering their biophysical characteristics. The metallosurfactants studied exhibit high structural and physicochemical similarity to phospholipid molecules, designed to incorporate into the membrane structure without the need for covalent attachment to a lipid molecule. In this work, two lipid-mimetic phosphorescent metal complexes are described: [Ru(bpy)2(dn-bpy)]2 + and [Ir(ppy)2(dn-bpy)]+ where bpy is 2,2′-bipyridine, dn-bpy is 4,4′-dinonyl-2,2′-bipyridine and ppy is 2-phenylpyridine. Apart from being lipid-mimetic in size, shape and physical properties, both complexes exhibit intense photoluminescence and enhanced photostability compared with conventional organic fluorophores, allowing for prolonged observation. Moreover, the large Stokes shift and long luminescence lifetime associated with these complexes make them more suitable for spectroscopic studies. The complexes are easily incorporated into dimyristoil-phosphatidyl-choline (DMPC) liposomes by mixing in the organic solvent phase. DLS reveals the labeled membranes form liposomes of similar size to that of neat DMPC membrane. Synchrotron Small-Angle X-ray Scattering (SAXS) measurements confirmed that up to 5% of either complex could be incorporated into DMPC membranes without producing any structural changes in the membrane. Fluorescence microscopy reveals that 0.5% label content is sufficient for imaging. Atomic Force Microscopic imaging confirms that liposomes of the labeled bilayers on a mica surface can fuse into a flat lamellar membrane that is morphologically identical to neat lipid membranes. These results demonstrate the potential of such lipid-mimetic luminescent metal complexes as a new class of labels for imaging lipid membranes.  相似文献   
9.
Cranial/intracranial abscess disease is an emerging source of significant mortality for male white-tailed deer (Odocoileus virginianus). Most cases of cranial/intracranial abscess disease are associated with infection by the opportunistic pathogen Trueperella pyogenes although the relationship between the prevalence of the bacteria and occurrence of disease is speculative. We examined 5,612 hunter-harvested deer from 29 sites across all physiographic provinces in Georgia for evidence of cranial abscess disease and sampled the forehead, lingual, and nasal surfaces from 692 deer. We used polymerase chain reaction (PCR) to determine presence of T. pyogenes from these samples. We found T. pyogenes prevalence at a site was a predictor for the occurrence of cranial abscess disease. Prevalence of T. pyogenes did not differ between samples from the nose or tongue although prevalence along the forehead was greater for males than females (p = 0.04), particularly at sites with high occurrence of this disease. Socio-sexual behaviors, bacterial prevalence, or physiological characteristics may predispose male deer to intracranial/cranial abscess disease. Determination of factors that affect T. pyogenes prevalence among sites may help explain the occurrence of this disease among populations.  相似文献   
10.
Fiber tractography plays an important role in exploring the architectural organization of fiber trajectories, both in fundamental neuroscience and in clinical applications. With the advent of diffusion MRI (dMRI) approaches that can also model “crossing fibers”, the complexity of the fiber network as reconstructed with tractography has increased tremendously. Many pathways interdigitate and overlap, which hampers an unequivocal 3D visualization of the network and impedes an efficient study of its organization. We propose a novel fiber tractography visualization approach that interactively and selectively adapts the transparency rendering of fiber trajectories as a function of their orientation to enhance the visibility of the spatial context. More specifically, pathways that are oriented (locally or globally) along a user-specified opacity axis can be made more transparent or opaque. This substantially improves the 3D visualization of the fiber network and the exploration of tissue configurations that would otherwise be largely covered by other pathways. We present examples of fiber bundle extraction and neurosurgical planning cases where the added benefit of our new visualization scheme is demonstrated over conventional fiber visualization approaches.  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