全文获取类型
收费全文 | 10371篇 |
免费 | 783篇 |
国内免费 | 2篇 |
专业分类
11156篇 |
出版年
2023年 | 68篇 |
2022年 | 120篇 |
2021年 | 243篇 |
2020年 | 189篇 |
2019年 | 211篇 |
2018年 | 273篇 |
2017年 | 259篇 |
2016年 | 378篇 |
2015年 | 585篇 |
2014年 | 580篇 |
2013年 | 757篇 |
2012年 | 889篇 |
2011年 | 776篇 |
2010年 | 534篇 |
2009年 | 478篇 |
2008年 | 621篇 |
2007年 | 614篇 |
2006年 | 561篇 |
2005年 | 490篇 |
2004年 | 473篇 |
2003年 | 394篇 |
2002年 | 394篇 |
2001年 | 115篇 |
2000年 | 93篇 |
1999年 | 90篇 |
1998年 | 106篇 |
1997年 | 80篇 |
1996年 | 70篇 |
1995年 | 78篇 |
1994年 | 67篇 |
1993年 | 57篇 |
1992年 | 54篇 |
1991年 | 58篇 |
1990年 | 37篇 |
1989年 | 43篇 |
1988年 | 41篇 |
1987年 | 31篇 |
1986年 | 31篇 |
1985年 | 26篇 |
1984年 | 25篇 |
1983年 | 25篇 |
1982年 | 23篇 |
1981年 | 22篇 |
1980年 | 13篇 |
1979年 | 11篇 |
1978年 | 13篇 |
1975年 | 6篇 |
1974年 | 9篇 |
1973年 | 5篇 |
1966年 | 4篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
101.
Angelo Porciuncula Natalia Zapata Elizabeth Guruceaga Xabier Agirre Miguel Barajas Felipe Prosper 《Gene expression patterns : GEP》2013,13(1-2):12-20
MicroRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression, play multiple roles in the cell, including cell fate specification. We have analyzed the differential expression of miRNAs during fibroblast reprogramming into induced pluripotent stem cells (iPSCs) and endoderm induction from iPSCs upon treatment with high concentrations of Activin-A. The reprogrammed iPSCs assumed an embryonic stem cell (ESC)-like miRNA signature, marked by the induction of pluripotency clusters miR-290–295 and miR-302/367 and conversely the downregulation of the let-7 family. On the other hand, endoderm induction in iPSCs resulted in the upregulation of 13 miRNAs. Given that the liver and the pancreas are common derivatives of the endoderm, analysis of the expression of these 13 upregulated miRNAs in hepatocytes and pancreatic islets revealed a tendency for these miRNAs to be expressed more in pancreatic islets than in hepatocytes. These observations provide insights into how differentiation may be guided more efficiently towards the endoderm and further into the liver or pancreas. Moreover, we also report novel miRNAs enriched for each of the cell types analyzed. 相似文献
102.
Migla Miskinyte Ana Sousa Ricardo S. Ramiro Jorge A. Moura de Sousa Jerzy Kotlinowski Iris Caramalho Sara Magalh?es Miguel P. Soares Isabel Gordo 《PLoS pathogens》2013,9(12)
Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity. 相似文献
103.
Olivia Twu Natalia de Miguel Gila Lustig Grant C. Stevens Ajay A. Vashisht James A. Wohlschlegel Patricia J. Johnson 《PLoS pathogens》2013,9(7)
Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization. 相似文献
104.
Dominik Thimm Melanie Knospe Aliaa Abdelrahman Miguel Moutinho Bernt B. A. Alsdorf Ivar von Kügelgen Anke C. Schiedel Christa E. Müller 《Purinergic signalling》2013,9(3):415-426
The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation “P0-receptors” has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [3H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K D values of 286 nM (mAde1R, B max 1.18 pmol/mg protein) and 301 nM (cAdeR, B max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure–activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC50 9.77 nM) and the cAdeR (EC50 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC50 6.24 nM) and was found to be additionally coupled to Gq proteins. 相似文献
105.
106.
Ana Aranda Paloma Campo Arantxa Palacin Inmaculada Do?a Cristina Gomez-Casado Luisa Galindo Araceli Díaz-Perales Miguel Blanca 《PloS one》2013,8(1)
Background
Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated.Objective
To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthmaMaterials and methods
An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies.Results
Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA−), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases.Conclusions
Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens. 相似文献107.
Aurélie Bedel Jean- Max Pasquet éric Lippert Miguel Taillepierre Valérie Lagarde Sandrine Dabernat Pierre Dubus Lucie Charaf Fran?ois Beliveau Hubert de Verneuil Emmanuel Richard Fran?ois-Xavier Mahon Fran?ois Moreau-Gaudry 《PloS one》2013,8(8)
Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34+ blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents. 相似文献
108.
Francisco Campos Tomás Sobrino Alba Vieites-Prado María Pérez-Mato Manuel Rodríguez-Yá?ez Miguel Blanco José Castillo 《PloS one》2013,8(11)
Hyperthermia is a predictor of poor outcome in ischemic (IS) and intracerebral hemorrhagic (ICH) stroke. Our aim was to study the plausible mechanisms involved in the poor outcome associated to hyperthermia in stroke. We conducted a case-control study including patients with IS (n = 100) and ICH (n = 100) within the first 12 hours from symptom onset. Specifically, IS and ICH patients were consecutively included into 2 subgroups, according to the highest body temperature within the first 24 hours: Tmax <37.5°C and Tmax ≥37.5°C, up to reach 50 patients per subgroup of temperature for both IS and ICH patients. Body temperature was determined at admission and every 4 hours during the first 48 hours. Main outcome variable was poor functional outcome (modified Rankin scale score >2) at 3 months. Serum levels of glutamate and active MMP-9 were measured at admission. Our results showed that Tmax ≥37.5°C within the first 24 hours was independently associated with poor outcome in both IS (OR, 12.43; 95% CI, 3.73–41.48; p<0.0001) and ICH (OR, 4.29; 95% CI, 1.32–13.91; p = 0.015) after adjusting for variables with a proven biological relevance for outcome. However, when molecular markers levels were included in the logistic regression model, we observed that glutamate (OR, 1.01; 95% CI, 1.00–1.02; p = 0.001) and infarct volume (OR, 1.06; 95% CI, 1.01–1.10; p = 0.015) were the only variables independently associated to poor outcome in IS, and active MMP-9 (OR, 1.04; 95% CI, 1.00–1.08; p = 0.002) and National Institute of Health Stroke Scale (NIHSS) at admission (OR, 1.29; 95% CI, 1.13–1.49; p<0.0001) in ICH. In conclusion, these results suggest that although the outcome associated to hyperthermia is similar in human IS and ICH, the underlying mechanisms may be different. 相似文献
109.
Jo?o Miguel Freire Ana Salomé Veiga Thaís M. Concei??o Wioleta Kowalczyk Ronaldo Mohana-Borges David Andreu Nuno C. Santos Andrea T. Da Poian Miguel A. R. B. Castanho 《PloS one》2013,8(12)
Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins. 相似文献
110.
Cristina Virto Carlos A. Zárate Miguel López-Ferber Rosa Murillo Primitivo Caballero Trevor Williams 《PloS one》2013,8(8)
With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected insects: i) healthy males (H♂)×healthy females (H♀); ii) infected males (I♂)×healthy females (H♀); iii) healthy males (H♂)×infected females (I♀) and iv) infected males (I♂)×infected females (I♀). Experimental adults and their offspring were analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Male-mediated venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virus-based insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between bioinsecticide applications. 相似文献