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A 6 Å resolution electron density map of pig muscle phosphoglucose isomerase has been obtained. From this map it has been possible to isolate a single molecule and to assign tentative subunit boundaries. The binding of the competitive inhibitor 6-phosphogluconate has been studied. The binding site appears to be close to the subunit interface. 相似文献
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BACKGROUND: Autologous PBSC transplantation is the standard care for patients with multiple myeloma. The most common regimen used to mobilize PBSC consists of CY and G-CSF. METHODS: We retrospectively analyzed the efficacy and toxicity of two regimens of CY for PBSC mobilization: low-dose CY (1-2 g/m(2), LD-CY, n=61) plus G-CSF, and intermediate-dose CY (3-4 g/m(2), ID-CY, n=26) plus G-CSF. RESULTS: In the LD-CY group, 5.17 (0.23-17.3)x10(6) CD34(+) cells/kg, and in the ID-CY group 7.71 (0.08-26.4)x10(6) CD34(+) cells/kg (P=0.018), were collected. Although >/=2x10(6)/kg CD34(+) cells were collected in 89% of the LD-CY group and 92% of the ID-CY group, this was achieved after a single leukapheresis in 54% of the LD-CY group and 92% of the ID-CY group (P=0.0001). Patients who are to have tandem autologous PBSC transplants require >/=4x10(6)/kg CD34(+) cells. This was achieved in only 65% patients in the LD-CY group but 88% in the ID-CY group (P=0.05). Among patients who had not had prior melphalan and/or >12 months of prior treatment, 74% in the LD-CY group and 100% in ID-CY group mobilized >/=4x10(6)/kg CD34(+) cells. Febrile neutropenia was more frequent in the ID-CY group (38% vs. 13%). DISCUSSION: In conclusion, compared with LD-CY, patients receiving ID-CY were more likely to collect a total CD34(+) cell number adequate for tandem autologous PBSC transplantation. The increased toxicity was manageable and considered acceptable. 相似文献
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Contrasting patterns in genetic diversity following multiple invasions of fresh and brackish waters 总被引:1,自引:1,他引:0
Biological invasions may combine the genetic effects of population bottlenecks and selection and thus provide valuable insight into the role of such processes during novel environmental colonizations. However, these processes are also influenced by multiple invasions, the number of individuals introduced and the degree of similarity between source and receiving habitats. The amphipod Gammarus tigrinus provides a useful model to assess these factors, as its invasion history has involved major environmental transitions. This species is native to the northwest Atlantic Ocean, although it invaded both brackish and freshwater habitats in the British Isles after introduction more than 65 years ago. It has also spread to similar habitats in Western Europe and, most recently, to Eastern Europe, the Baltic Sea, and the Laurentian Great Lakes. To examine sources of invasion and patterns of genetic change, we sampled populations from 13 native estuaries and 19 invaded sites and sequenced 542 bp of the mitochondrial COI gene. Strong native phylogeographical structure allowed us to unambiguously identify three allopatrically evolved clades (2.3-3.1% divergent) in invading populations, indicative of multiple introductions. The most divergent clades occurred in the British Isles and mainland Europe and were sourced from the St Lawrence and Chesapeake/Delaware Bay estuaries. A third clade was found in the Great Lakes and sourced to the Hudson River estuary. Despite extensive sampling, G. tigrinus did not occur in freshwater at putative source sites. Some European populations showed reduced genetic diversity consistent with bottlenecks, although selection effects cannot be excluded. The habitat distribution of clades in Europe was congruent with the known invasion history of secondary spread from the British Isles. Differences in salinity tolerance among lineages were suggested by patterns of habitat colonization by different native COI clades. Populations consisting of admixtures of the two invading clades were found principally at recently invaded fresh and brackish water sites in Eastern Europe, and were characterized by higher genetic diversity than putative source populations. Further studies are required to determine if these represent novel genotypes. Our results confirm that biological invasions need not result in diminished genetic diversity, particularly if multiple source populations, each with distinctive genetic composition, contribute to the founding populations. 相似文献
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Delphine Rapp John Waller Gale Brightwell Richard W. Muirhead 《Applied and environmental microbiology》2010,76(5):1686-1688
Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log10 and 6 log10 CFU g−1.Bovines are a reservoir for verotoxigenic Escherichia coli O157:H7 and Campylobacter jejuni, pathogenic microorganisms responsible for severe human gastrointestinal disease (5, 12). Qualitative and quantitative detection of these organisms in bovine feces is essential for evaluating risk to human health. Real-time PCR (quantitative PCR [qPCR]) assays have been developed to detect and quantify both E. coli O157:H7 and C. jejuni bacteria by using DNA directly extracted from animal feces (20, 22). Analysis of DNA extracted from bovine feces can generate a high level of correlation between the actual target cell density and the PCR signal (7, 8). However, the detection of E. coli O157:H7 and C. jejuni by direct DNA extraction is less sensitive and more variable than detection by procedures based on a preliminary enrichment step (e.g., laboratory culture) (7, 9, 16, 20). We explored the potential of lyophilization for improving overall detection by qPCR through increasing the amount of bovine fecal material available for DNA extraction.Four sets of five fresh bovine fecal samples were collected, and each sample was divided into four equal portions. Samples were seeded with either (i) E. coli O157:H7 (strain NZRM 3614) grown for 18 h at 37°C in tryptic soy broth (BD, Sparks, MD) or (ii) C. jejuni (strain NZRM 1958) grown for 48 h at 42°C in Exeter broth (11) to obtain the following concentrations: set 1, 0 CFU g−1 (unseeded) and 3.5 log10, 4.5 log10, and 5.5 log10 CFU of E. coli O157:H7 g−1, and set 2, 0 CFU g−1 (unseeded) to 5.2 log10 CFU of E. coli O157:H7 g−1. Set 3 and 4 concentrations varied from 0 CFU g−1 (unseeded) to 6.4 log10 C. jejuni CFU g−1. DNA was either extracted directly from fresh samples or extracted from samples after lyophilization. Lyophilization involved mixing of prepared fecal samples in phosphate-buffered saline (145 mM NaCl, 59 mM Na2HPO4, 8 mM KH2PO4, pH 7.5) at a ratio of 1:10 (wt/vol), homogenization with a lab blender model 400 (Seward Medical, London, United Kingdom), cooling to −35°C, and concentration using a 1015GP lyophilizer (Cuddon Ltd., Blenheim, New Zealand). Total DNA was extracted from 0.2 g of a fresh or lyophilized fecal sample by using a QIAamp DNA stool minikit (Qiagen Inc., Mississauga, Canada). DNA was amplified using either a TaqMan E. coli O157:H7 detection kit (Applied Biosystems, Foster City, CA) or mapA primers and a corresponding probe (1). Amplification and fluorescence data were collected with optical-grade 96-well plates by using a TaqMan 7300 PCR system (Applied Biosystems). For each DNA sample, a mean threshold cycle (CT) value for triplicate qPCR runs was calculated. When no CT value was obtained, an arbitrary CT value of 40 was assigned. All data were reported as equivalent concentrations in fresh feces. Significance levels were determined by one-way analysis of variance. The relationship between the log10 numbers of CFU g−1 fresh feces (viable-cell counts) and CT values was analyzed using GenStat software (version 10.2.0.175; VSN International, Oxford, United Kingdom). Confidence intervals were obtained using the software program Flexi (21).Lyophilized samples were associated with significantly improved sensitivity (P < 0.001) at seeding levels of 4.5 and 5.5 log10 E. coli O157:H7 CFU g−1 (Table (Table1).1). At 3.5 log10 CFU g−1, the rate of E. coli O157:H7 detection was also higher, with all lyophilized samples producing a CT value of <40 (Table (Table1).1). Individual CT values for the three qPCR amplification runs were sufficiently similar to allow averaging (P > 0.05). Regression analysis of the averaged set 2 and 3 data (Fig. (Fig.1)1) demonstrated that the detection of both E. coli O157:H7 and C. jejuni was linear for seeding levels ranging from ca. 2 log10 to 6 log10 CFU g−1 fresh feces. The range of concentrations used reflects the reported range of concentrations of these bacteria in feces (i.e., 0 to 6 log10 CFU g−1) as determined by conventional culture (3, 4, 18, 19). The high coefficients of correlation for the relationships between the log10 numbers of CFU g−1 feces and the CT values indicated the specific amplification of the target DNA. The reproducibility of detection of E. coli O157:H7 was reduced at the lowest seeding concentration (i.e., 2.2 log10 CFU g−1 feces), with 75% of the samples giving a CT value of <40. The limit for 100% successful detection after lyophilization was 2.9 log10 E. coli O157:H7 CFU g−1. The detection of C. jejuni by qPCR varied between sets. For set 3, 100% reproducibility occurred at 2.2 log10 C. jejuni CFU g−1. For set 4, satisfactory detection was obtained only after dilution of the DNA extract prior to qPCR. Despite this requirement for dilution, C. jejuni was still detected in 80% of the samples of set 4 seeded at a density of 2.2 log10 C. jejuni CFU g−1.Open in a separate windowFIG. 1.Ranges of quantification of E. coli O157:H7 (A) and C. jejuni (B) bacteria obtained from lyophilized fecal samples by real-time PCR. Each point represents the average CT value for triplicate runs of one fecal sample at one seeding concentration. The hatched areas represent the 95% confidence intervals.
Open in a separate windowaOnly one fecal sample gave a CT value of <40.Overall, the removal of water by lyophilization provided an approximately 10-fold increase in the amount of fecal material used. Consequently, the test sensitivity was 10-fold greater than that reported previously (17, 7). Lyophilization of feces has been reported to be useful for PCR-based studies of pigs (14), and our results indicate a useful role for the quantification of E. coli O157:H7 bacteria in cattle feces. Indeed, the slopes and the linear regression coefficients for the qPCR signal (CT values) and the known concentrations of microbial pathogen cells in the feces are in agreement with published values (2). Our methodology shows a lower limit of C. jejuni quantification by qPCR (ca. 2 log10 CFU g−1 in seeded fresh feces) than that reported previously (8), demonstrating the usefulness of lyophilization to improve detection and quantification of bacteria in feces.In our study, the accurate detection of C. jejuni after DNA extraction from lyophilized feces was adversely affected for some samples. Interference due to partial removal of PCR inhibitors after DNA extraction using the QIAamp DNA stool minikit has been reported by other workers (10, 15). For lyophilized samples, the inhibition was successfully overcome by dilution of DNA. Recent reports confirmed the importance of diluting DNA (up to 3 log) to increase the accuracy of detection by real-time PCR (6, 13). Lyophilization presents the advantage that lyophilized material can be stored for long periods at room temperature, is easy to transport, and can also be used for complementary chemical analysis. 相似文献
TABLE 1.
Difference in CT values obtained for real-time PCR detection of E. coli O157:H7 in seeded fecal samples (n = 5) with and without lyophilizationSeeding level (log10 CFU g−1 fresh feces) or status | Average CT value (range) | |
---|---|---|
Without lyophilization | With lyophilization | |
5.5 | 31.50 (31.02-32.18) | 28.34 (28.04-29.03) |
4.5 | 34.79 (33.43-35.75) | 31.33 (31.01-31.89) |
3.5 | 35.45a | 33.52 (33.21-33.87) |
Unseeded | >40 | >40 |
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Jim R. Muirhead Brian Leung Colin van Overdijk David W. Kelly Kanavillil Nandakumar Kenneth R. Marchant Hugh J. MacIsaac 《Diversity & distributions》2006,12(1):71-79
Limiting the damage by non-indigenous species requires rapid determination of current and potential distributions and vectors of dispersal, and development of appropriate management measures. The emerald ash borer ( Agrilus planipennis ), a wood-boring beetle native to South-East Asia, was first reported in the Great Lakes region during summer 2002. The beetle poses an enormous threat to native ash ( Fraxinus ) species of North America, as untreated trees in infested areas of Ontario, Michigan and Ohio suffer high mortality. We demonstrate that the borer has spread in North America through a combination of diffusive range extension, associated with local flights, and by long-distance 'jump' dispersal associated with human movement of infested sapling or contaminated firewood. Probability of infestation was inversely related to distance from borer epicentres but positively related to the size of human population centres. At least 9 of 39 populations that were first reported in Michigan during 2004 cannot be accounted for by local diffusion, raising the possibility that other unidentified mechanisms may be contributing to the dispersal of the beetle. In the absence of quarantine, by 2005 all of Michigan's lower peninsula was contained within the boundaries of potential diffusive range expansion. Infested ash saplings also were introduced from Michigan to Maryland during 2003, and subsequently transplanted to five sites in Maryland and Virginia. Quarantine and eradication measures have had mixed results: in the south-central USA, the species appears on the brink of eradication, whereas its distribution has continued to spread during 2005 in the Great Lakes region despite extensive containment and quarantine measures. Quarantine success in the Great Lakes region is encumbered by multiple dispersal vectors, larger borer population sizes and by the more extensive geographical distribution that was achieved prior to implementation of control measures. 相似文献
59.
Little MP Muirhead CR Haylock RG Thomas JM 《Radiation and environmental biophysics》1999,38(4):267-283
In this paper the radiation-associated relative risks of second primary cancer incidence in groups treated for first primary
cancer by radiotherapy are compared with radiation-associated relative risk estimates in the Japanese atomic bomb survivor
cancer incidence data. For four cancer sites, namely lung cancer, bone cancer, ovarian cancer and leukaemia, the relative
risks in the comparable (age at exposure, time since exposure, sex matched) subsets of the Japanese data are significantly
greater than those in the majority of second cancer studies. Even when the differences between the relative risks in the Japanese
atomic bomb survivors and the medical series do not approach conventional levels of statistical significance, relative risks
tend to be higher in the Japanese data than in the second cancer studies. At least for leukaemia, the discrepancy between
the Japanese and second cancer risks can be largely explained by cell- sterilisation effects. There are few indications of
modification of radiation-associated second cancer relative risk among those treated with adjuvant chemotherapy, nor are there
strong indications of modification of radiation- associated relative risk by heritable genetic factors. If anything, there
is evidence that second cancer relative excess risks are lower among those patients with cancer-prone disorders than among
non-susceptible patients. However, the higher underlying cancer risk in some of these medically exposed populations should
also be considered, in particular for those with cancer-prone conditions, so that the absolute excess risk is sometimes higher
than in the Japanese data.
Received: 14 May 1999 / Accepted in revised form: 17 September 1999 相似文献
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