A crucial step in infection isTuberculosis (TB) remains a significant therapeutic challenge for the early 21st century. The global incidence and prevalence of infection and multidrug resistance and the impact of AIDS and poverty diminish the likelihood that TB will be controlled without a more effective vaccine (9, 15). Therefore, innovative approaches to discovering improved diagnostic methods, therapies, and vaccine candidates are very much needed.

Detection of chimeric artifacts formed when PCR is used to retrieve naturally occurring small-subunit (SSU) rRNA sequences may rely on demonstrating that different sequence domains have different phylogenetic affiliations. We evaluated the CHECK CHIMERA method of the Ribosomal Database Project and another method which we developed, both based on determining nearest neighbors of different sequence domains, for their ability to discern artificially generated SSU rRNA chimeras from authentic Ribosomal Database Project sequences. The reliability of both methods decreases when the parental sequences which contribute to chimera formation are more than 82 to 84% similar. Detection is also complicated by the occurrence of authentic SSU rRNA sequences that behave like chimeras. We developed a naive statistical test based on CHECK CHIMERA output and used it to evaluate previously reported SSU rRNA chimeras. Application of this test also suggests that chimeras might be formed by retrieving SSU rRNAs as cDNA. The amount of uncertainty associated with nearest-neighbor analyses indicates that such tests alone are insufficient and that better methods are needed.

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The use of cup-loading for sample application has become widely used in two-dimensional electrophoresis (2-DE) for resolution of basic proteins, but no side-by-side quantitative study has been published which compares cup-loading with the alternative passive and active rehydration methods to fully promote one type of loading method over another. Replicate 2-D gels from each loading method were quantitatively evaluated for gel-to-gel reproducibility using IPG 6-11 strips and semipreparative protein loads (300 microg). Gels were stained with SYPRO Ruby and analyzed with PDQuest. An inexpensive home-made assembly for cup-loading was used with the Protean IEF Cell for separation of whole cell extracts from the archaeon, Sulfolobus solfataricus. Cup-loading was determined to be far superior for IPG 6-11 separations than active or passive rehydration methods. Cup-loading consistently produced the greatest number of detectable spots, the best spot matching efficiency (56%), lowest spot quantity variations (28% coefficient of variation, CV), and the best-looking gels qualitatively. The least satisfactory results were obtained with active rehydration, followed closely by passive rehydration in off-line tubes. Passive rehydration experiments, performed using an on-line isoelectric focusing (IEF) tray, produced comparable spot numbers to cup-loading (84%), with 55% of the spots having higher intensity but 10% more spot quantity variance than cup-loading.

Worldwide, the landscape homogeneity of extensive monocultures that characterizes conventional agriculture has resulted in the development of specialized and interacting multitrophic pest complexes. While integrated pest management emphasizes the need to consider the ecological context where multiple species coexist, management recommendations are often based on single-species tactics. This approach may not provide satisfactory solutions when confronted with the complex interactions occurring between organisms at the same or different trophic levels. Replacement of the single-species management model with more sophisticated, multi-species programs requires an understanding of the direct and indirect interactions occurring between the crop and all categories of pests. We evaluated a modeling framework to make multi-pest management decisions taking into account direct and indirect interactions among species belonging to different trophic levels. We adopted a Bayesian decision theory approach in combination with path analysis to evaluate interactions between Bromus tectorum (downy brome, cheatgrass) and Cephus cinctus (wheat stem sawfly) in wheat (Triticum aestivum) systems. We assessed their joint responses to weed management tactics, seeding rates, and cultivar tolerance to insect stem boring or competition. Our results indicated that C. cinctus oviposition behavior varied as a function of B. tectorum pressure. Crop responses were more readily explained by the joint effects of management tactics on both categories of pests and their interactions than just by the direct impact of any particular management scheme on yield. In accordance, a C. cinctus tolerant variety should be planted at a low seeding rate under high insect pressure. However as B. tectorum levels increase, the C. cinctus tolerant variety should be replaced by a competitive and drought tolerant cultivar at high seeding rates despite C. cinctus infestation. This study exemplifies the necessity of accounting for direct and indirect biological interactions occurring within agroecosystems and propagating this information from the statistical analysis stage to the management stage.