全文获取类型
收费全文 | 2937篇 |
免费 | 338篇 |
国内免费 | 425篇 |
专业分类
3700篇 |
出版年
2023年 | 91篇 |
2022年 | 65篇 |
2021年 | 91篇 |
2020年 | 126篇 |
2019年 | 166篇 |
2018年 | 110篇 |
2017年 | 125篇 |
2016年 | 141篇 |
2015年 | 114篇 |
2014年 | 121篇 |
2013年 | 188篇 |
2012年 | 123篇 |
2011年 | 168篇 |
2010年 | 138篇 |
2009年 | 163篇 |
2008年 | 154篇 |
2007年 | 168篇 |
2006年 | 149篇 |
2005年 | 136篇 |
2004年 | 106篇 |
2003年 | 116篇 |
2002年 | 102篇 |
2001年 | 88篇 |
2000年 | 68篇 |
1999年 | 45篇 |
1998年 | 55篇 |
1997年 | 44篇 |
1996年 | 39篇 |
1995年 | 36篇 |
1994年 | 36篇 |
1993年 | 41篇 |
1992年 | 32篇 |
1991年 | 35篇 |
1990年 | 15篇 |
1989年 | 29篇 |
1988年 | 18篇 |
1987年 | 29篇 |
1986年 | 17篇 |
1985年 | 16篇 |
1984年 | 20篇 |
1983年 | 10篇 |
1982年 | 26篇 |
1981年 | 19篇 |
1980年 | 13篇 |
1979年 | 17篇 |
1978年 | 15篇 |
1977年 | 13篇 |
1976年 | 14篇 |
1975年 | 11篇 |
1974年 | 11篇 |
排序方式: 共有3700条查询结果,搜索用时 15 毫秒
21.
Matthias Rüdt Philipp Vormittag Nils Hillebrandt Jürgen Hubbuch 《Biotechnology and bioengineering》2019,116(6):1366-1379
Virus-like particles (VLPs) have shown great potential as biopharmaceuticals in the market and in clinics. Nonenveloped, in vivo assembled VLPs are typically disassembled and reassembled in vitro to improve particle stability, homogeneity, and immunogenicity. At the industrial scale, cross-flow filtration (CFF) is the method of choice for performing reassembly by diafiltration. Here, we developed an experimental CFF setup with an on-line measurement loop for the implementation of process analytical technology (PAT). The measurement loop included an ultraviolet and visible (UV/Vis) spectrometer as well as a light scattering photometer. These sensors allowed for monitoring protein concentration, protein tertiary structure, and protein quaternary structure. The experimental setup was tested with three Hepatitis B core Antigen (HBcAg) variants. With each variant, three reassembly processes were performed at different transmembrane pressures (TMPs). While light scattering provided information on the assembly progress, UV/Vis allowed for monitoring the protein concentration and the rate of VLP assembly based on the microenvironment of Tyrosine-132. VLP formation was verified by off-line dynamic light scattering (DLS) and transmission electron microscopy (TEM). Furthermore, the experimental results provided evidence of aggregate-related assembly inhibition and showed that off-line size-exclusion chromatography does not provide a complete picture of the particle content. Finally, a Partial-Least Squares (PLS) model was calibrated to predict VLP concentrations in the process solution. values of 0.947–0.984 were reached for the three HBcAg variants. In summary, the proposed experimental setup provides a powerful platform for developing and monitoring VLP reassembly steps by CFF. 相似文献
22.
23.
Xiaoyu Yang Wei Xu Svetlana Dukleska Sabrina Benchaar Selina Mengisen Valentyn Antochshuk Jason Cheung Leslie Mann Zulfia Babadjanova Jason Rowand Rico Gunawan Alexander McCampbell Maribel Beaumont David Meininger Daisy Richardson Alexandre Ambrogelly 《MABS-AUSTIN》2013,5(5):787-794
Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2−8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources. 相似文献
24.
Mathieu E Loup P Dellamonica P Daures JP 《Biometrical journal. Biometrische Zeitschrift》2005,47(6):834-846
The purpose of this study was to evaluate the evolution of HIV infected patients and to bring out some significant factors associated with this pathology. The main criteria revealing the State of illness is viral load measurement (VL). However the CD4 lymphocytes also represent an important marker as these reflect the State of the immune reservoir. Many studies have been carried out in this field and different models have been proposed with a view to a better understanding of this disease. Multi State Markov models defined in terms of CD4 counts, or in terms of viral load, have proved to be very useful tools for modelling HIV disease progression. The model we have developed in this study is based on both the CD4 lymphocytes counts and VL. Markov models are characterized by transition intensities. In this paper we explored several structures in succession. First, we used a homogeneous continuous time Markov process with four states defined by crossed values of CD4 and VL in a given patient at a given time. Then, the effect of certain covariates on the infection process was introduced into the model via the transition intensity functions, as with a Cox regression model. Since the hypothesis of homogeneity may be unrealistic in certain cases, we also considered piecewise homogeneous Markov models. Finally, the effects of covariates and time were combined in a piecewise homogeneous model with a covariate. We applied these methods to data from 1313 HIV-infected patients included in the NADIS cohort. 相似文献
25.
Hanania EG Fieck A Stevens J Bodzin LJ Palsson BØ Koller MR 《Biotechnology and bioengineering》2005,91(7):872-876
Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened. 相似文献
26.
Xun Wang Nora Fung-Yee Tam Shi Fu Aray Ametkhan Yun Ouyang Zhihong Ye 《Annals of botany》2014,114(2):271-278
Background and Aims
Mercury (Hg) is an extremely toxic pollutant, especially in the form of methylmercury (MeHg), whereas selenium (Se) is an essential trace element in the human diet. This study aimed to ascertain whether addition of Se can produce rice with enriched Se and lowered Hg content when growing in Hg-contaminated paddy fields and, if so, to determine the possible mechanisms behind these effects.Methods
Two cultivars of rice (Oryza sativa, japonica and indica) were grown in either hydroponic solutions or soil rhizobags with different Se and Hg treatments. Concentrations of total Hg, MeHg and Se were determined in the roots, shoots and brown rice, together with Hg uptake kinetics and Hg bioavailability in the soil. Root anatonmy was also studied.Key Results
The high Se treatment (5 μg g–1) significantly increased brown rice yield by 48 % and total Se content by 2·8-fold, and decreased total Hg and MeHg by 47 and 55 %, respectively, compared with the control treatments. The high Se treatment also markedly reduced ‘water-soluble’ Hg and MeHg concentrations in the rhizosphere soil, decreased the uptake capacity of Hg by roots and enhanced the development of apoplastic barriers in the root endodermis.Conclusions
Addition of Se to Hg-contaminated soil can help produce brown rice that is simultaneously enriched in Se and contains less total Hg and MeHg. The lowered accumulation of total Hg and MeHg appears to be the result of reduced bioavailability of Hg and production of MeHg in the rhizosphere, suppression of uptake of Hg into the root cells and an enhancement of the development of apoplastic barriers in the endodermis of the roots. 相似文献27.
28.
29.
Elke Thnnes Marie-Colette van Lieshout 《Biometrical journal. Biometrische Zeitschrift》1999,41(6):721-734
Summary functions such as the empty space function F and the nearest neighbour distance distribution function G are often used as test statistics for point patterns. Van Lieshout and Baddeley recently proposed an alternative statistic, the J-function, which is defined as J = (1 — G)/(1 — F). Theoretical advantages of the J-function over the F- and G-statistics are that it measures the type, strength and range of interaction, and that it can be evaluated explicitly for a larger class of models. In this simulation study we investigate empirically how the power of tests based on J compares to that of tests based on F and G. 相似文献
30.
An attempt to use phylogenetic invariants for tree reconstruction was made at the end of the 80s and the beginning of the 90s by several researchers (the initial idea due to Lake [1987] and Cavender and Felsenstein [1987]). However, the efficiency of methods based on invariants is still in doubt (Huelsenbeck 1995; Jin and Nei 1990). Probably because these methods only used few generators of the set of phylogenetic invariants. The method studied in this paper was first introduced in Casanellas et al. (2005) and it is the first method based on invariants that uses the "whole" set of generators for DNA data. The simulation studies performed in this paper prove that it is a very competitive and highly efficient phylogenetic reconstruction method, especially for nonhomogeneous models on phylogenetic trees. 相似文献