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91.
Fox JT Renter DG Sanderson MW Thomson DU Lechtenberg KF Nagaraja TG 《Applied and environmental microbiology》2007,73(16):5253-5260
Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log10 CFU/g. The preenrichment streak detected >3.0 log10 CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log10 CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments. 相似文献
92.
The co-crystal structure of Thermus aquaticus elongation factor Tu.guanosine 5'- [beta,gamma-imido]triphosphate (EF-Tu.GDPNP) bound to yeast Phe-tRNA(Phe) reveals that EF-Tu interacts with the tRNA body primarily through contacts with the phosphodiester backbone. Twenty amino acids in the tRNA binding cleft of Thermus Thermophilus EF-Tu were each mutated to structurally conservative alternatives and the affinities of the mutant proteins to yeast Phe-tRNA(Phe) determined. Eleven of the 20 mutations reduced the binding affinity from fourfold to >100-fold, while the remaining ten had no effect. The thermodynamically important residues were spread over the entire tRNA binding interface, but were concentrated in the region which contacts the tRNA T-stem. Most of the data could be reconciled by considering the crystal structures of both free EF-Tu.GTP and the ternary complex and allowing for small (1.0 A) movements in the amino acid side-chains. Thus, despite the non-physiological crystallization conditions and crystal lattice interactions, the crystal structures reflect the biochemically relevant interaction in solution. 相似文献
93.
To assess the sensitivity of direct plating of bovine fecal samples for detection of Escherichia coli O157:H7, calves (n = 28) were orally inoculated with 109 colony-forming units (cfu) per calf of a mixture of three strains of nalidixic acid-resistant E. coli O157:H7, and fecal samples were collected for analysis. One-gram samples from inoculated calves were mixed with 9 mL of Gram-negative
broth with vancomycin, cefixime, and cefsoludin. From this suspension, serial dilutions were made (10−1 to 10−4) and spread plated in triplicate on Sorbitol MacConkey agar with nalidixic acid for enumeration of E. coli O157:H7 in fecal samples. Direct plating samples were streaked for isolation on Sorbitol MacConkey agar with cefixime, and
tellurite (SMACct). After incubation overnight at 37°C, morphologically typical colonies from direct streak plates were plated
onto blood agar and incubated overnight at 37°C; then an indole test was performed on each colony. Indole-positive colonies
were confirmed by O157 agglutination and were then plated on SMAC agar with 20 μg/mL nalidixic acid (SMACnal) to confirm nalidixic
acid resistance. Overall sensitivity of detection was 32.5% (110/338 samples). Sensitivity to detect fecal samples shedding
at above 5 × 104 cfu/g was 83% (71/86 samples). Based on these data, direct plating of fecal samples might be an effective way to identify
cattle that are likely to be shedding E. coli O157 at high levels. 相似文献
94.
Simone de Souza Martins James G. Sanderson José de Sousa e Silva-Júnior 《Biodiversity and Conservation》2007,16(4):857-870
The need for long-term biodiversity monitoring using standardized protocols led to the creation of the Tropical Ecology, Assessment and Monitoring (TEAM) initiative. At some 50 field stations in tropical forests around the world, TEAM will monitor various taxa such as ants, birds, butterflies, medium and large terrestrial mammals, primates, litter fall, and trees, as well as landscape change in nine tropical biodiversity hotspots and three tropical wilderness areas. The TEAM terrestrial mammal program calls for using a grid of camera phototraps to monitor long-term trends in densities and occupancy rates of species that can or cannot be uniquely identified, respectively. We describe the TEAM camera phototrapping program and provide results for the first TEAM site–Caxiuanã National Forest in northern Brazil. An intensive one year camera trapping effort was carried out to determine which months were most suitable for long-term monitoring. Fifteen species of medium and large terrestrial mammals and two large birds were recorded, including three xenarthrans, five carnivores, one perissodactyle, three artiodactyles, two rodents, and one marsupial. The medium and large terrestrial mammal diversity was well represented during two consecutive wet and dry months, respectively. We also recorded activity patterns for all species photographed by our camera traps more than 10 times. 相似文献
95.
96.
Background
Most studies inferring species phylogenies use sequences from single copy genes or sets of orthologs culled from gene families. For taxa such as plants, with very high levels of gene duplication in their nuclear genomes, this has limited the exploitation of nuclear sequences for phylogenetic studies, such as those available in large EST libraries. One rarely used method of inference, gene tree parsimony, can infer species trees from gene families undergoing duplication and loss, but its performance has not been evaluated at a phylogenomic scale for EST data in plants.Results
A gene tree parsimony analysis based on EST data was undertaken for six angiosperm model species and Pinus, an outgroup. Although a large fraction of the tentative consensus sequences obtained from the TIGR database of ESTs was assembled into homologous clusters too small to be phylogenetically informative, some 557 clusters contained promising levels of information. Based on maximum likelihood estimates of the gene trees obtained from these clusters, gene tree parsimony correctly inferred the accepted species tree with strong statistical support. A slight variant of this species tree was obtained when maximum parsimony was used to infer the individual gene trees instead.Conclusion
Despite the complexity of the EST data and the relatively small fraction eventually used in inferring a species tree, the gene tree parsimony method performed well in the face of very high apparent rates of duplication.97.
Curtis FA Reed P Wilson LA Bowers LY Yeo RP Sanderson JM Walmsley AR Sharples GJ 《Journal of molecular recognition : JMR》2011,24(2):333-340
Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA. To help identify interacting domains we analysed Orf and SSB proteins carrying mutations or truncations in the C-terminal region. A cluster of acidic residues at the carboxy-terminus of SSB is known to attract multiple protein partners to assist in DNA replication and repair. In this case an alternative domain must be utilized since Orf association with SSB was unaffected by an SSB113 point mutant (P176S) or removal of the last ten residues (ΔC10). Structurally the Orf C-terminus consists of a helix with a flexible tail that protrudes from each side of the dimer and could serve as a binding site for either SSB or DNA. Eliminating the six residue flexible tail (ΔC6) or the entire helix (ΔC19) had no significant impact on the Orf-SSB interaction. However, the OrfΔC6 protein exhibited reduced DNA binding, a feature shared by single amino acid substitutions within (W141F) or adjacent (R140A) to this region. The OrfΔC19 mutant bound poorly to DNA and secondary structure analysis in solution revealed that this truncation induces protein misfolding and aggregation. The results show that the carboxy-terminus of Orf is involved in nucleic acid recognition and also plays an unexpected role in maintaining structural integrity. 相似文献
98.
The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNA(Thr)(UGU) were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound to mutations in three T-stem base pairs in a quantitatively identical manner. However, tRNA(Thr) differs from other tRNAs by also using its rare A52-C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNA(Thr)(UGU) T-stem changes are either unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNA(Thr) can substantially increase or decrease its affinity to EF-Tu, the observed affinities do not correlate with the growth phenotype of the mutations in any simple way. This may either reflect the different conditions used in the two assays or indicate that the T-stem mutants affect another step in the translation mechanism. 相似文献
99.
Brown NF Coombes BK Bishop JL Wickham ME Lowden MJ Gal-Mor O Goode DL Boyle EC Sanderson KL Finlay BB 《PloS one》2011,6(3):e17824
Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions. 相似文献
100.
Omeje Edwin Ogechukwu Osadebe Patience Ogoamaka Nworu Chukwuemeka Sylvester Amal Hassan Abdessamad Debbab Esimone Charles Okechukwu Akira Kawamura Proksch Peter 《Phytochemistry letters》2011,4(3):357-362
In search of immunomodulatory constituents from the Eastern Nigeria mistletoe, Loranthus micranthus Linn, two new stigmastane steroids: stigmast-7,20 (21)-diene-3β-hydroxy-6-one (1) and 3β-hydroxy-stigmast-23-ene (2); three (two new and one known) lupeol-based triterpenoid esters: 7β,15α-dihydroxyl-lup-20(29)-ene-3β-palmitate (3), 7β,15α-dihydroxyl-lup-20(29)-ene-3β-stearate (4) and 7β,15α-dihydroxyl-lup-20(29)-ene-3β-eicosanoate (5) were isolated and characterized following bioactivity-guided fractionation. The new compounds, 1, 2, 4 and 5 at concentrations of 10, 25 and 100 μg/ml were subjected to cell proliferation and early activation marker (CD69) expression studies in C57Bl/6 mice splenocytes using flow cytometry techniques against Lipopolysaccharide (LPS; 10 μg/ml) and Concanavalin A (ConA; 2 μg/ml) standards. The stigmastane steroids (1 and 2) at the highest concentration of 100 μg/ml showed statistically significantly (p < 0.05) stimulatory activity on the C57B1/6 splenocytes compared to the controls with values of 46 ± 0.76% and 43 ± 0.46% compared to 7.69 ± 0.41% recorded for the negative control. The novel lupeol esters, 4 and 5 at same concentration of 100 μg/ml exhibited lower stimulations of 30 ± 0.41% and 29 ± 0.17% respectively compared to the controls above. The CD69 expression assay at the above doses showed that all the compounds have minimal stimulation. The present study supports the observed immunomodulatory property of the Eastern Nigeria mistletoe and thus confirms the efficacy of this plant in mitigating against wide array of disease conditions orchestrated by immunodeficiency. 相似文献