Testing the reliability of genetic methods of species identification via simulation

Although genetic methods of species identification, especially DNA barcoding, are strongly debated, tests of these methods have been restricted to a few empirical cases for pragmatic reasons. Here we use simulation to test the performance of methods based on sequence comparison (BLAST and genetic distance) and tree topology over a wide range of evolutionary scenarios. Sequences were simulated on a range of gene trees spanning almost three orders of magnitude in tree depth and in coalescent depth; that is, deep or shallow trees with deep or shallow coalescences. When the query's conspecific sequences were included in the reference alignment, the rate of positive identification was related to the degree to which different species were genetically differentiated. The BLAST, distance, and liberal tree-based methods returned higher rates of correct identification than did the strict tree-based requirement that the query was within, but not sister to, a single-species clade. Under this more conservative approach, ambiguous outcomes occurred in inverse proportion to the number of reference sequences per species. When the query's conspecific sequences were not in the reference alignment, only the strict tree-based approach was relatively immune to making false-positive identifications. Thresholds affected the rates at which false-positive identifications were made when the query's species was unrepresented in the reference alignment but did not otherwise influence outcomes. A conservative approach using the strict tree-based method should be used initially in large-scale identification systems, with effort made to maximize sequence sampling within species. Once the genetic variation within a taxonomic group is well characterized and the taxonomy resolved, then the choice of method used should be dictated by considerations of computational efficiency. The requirement for extensive genetic sampling may render these techniques inappropriate in some circumstances.     

In Vivo CD8+ T-Cell Suppression of SIV Viremia Is Not Mediated by CTL Clearance of Productively Infected Cells

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.     

Conformational preferences of 1,4,7-trithiacyclononane: a molecular mechanics and density functional theory study

Conformational preferences of 1,4,7-trithiacyclononane were studied using a highly efficient sampling technique based on local nonstochastic deformations and the MM2(91) force field. The results show that conformers that the molecule adopts in the crystal state were found to be low-energy conformers (LECs) within 5 kcal mol(-1) of the global minimum. A conformation with C1 symmetry was the global minimum and the C3 and C2 conformations were calculated to be 0.03 and 1.78 kcal mol(-1) higher in energy, respectively. The structures were further minimized using Density Functional Theory (DFT) calculations with two different functionals. The C2 and the C1 conformations were found to be LECs with the C3 conformation more than 4.0 kcal mol(-1) above the global minimum. The relative energies and structural ordering obtained using the BP86 functional are in agreement with the previously reported relative energies calculated using second-order Moller-Plesset (MP2) ab initio calculations. With the energy ordering being dependent on the molecular mechanics force field used, the approach of MM-->DFT (searching exhaustively the available conformational space at the MM level followed by generating the energy ordering through DFT calculations) appears to be appropriate for thiacrown ethers.     

Cloning of the Atlantic salmon (Salmo salar) IL-1 receptor associated protein

In mammals, the pro-inflammatory cytokine interleukin-1 signals through a receptor complex containing a type I interleukin-1 receptor (IL-1RI) and a receptor associated protein (IL-1RAcP). Previously, we have described a cDNA from Atlantic salmon encoding a molecule with homology to the mammalian IL-RI. This molecule was named IL-1 receptor like protein (IL-1RLP) in the absence of functional data to support its proposed role as the salmon IL-1RI. Here, we describe the cloning and characterisation of a cDNA encoding salmon IL-1RAcP. Like other members of the IL-1R family, the salmon IL-1RAcP encodes three extracellular immunoglobulin-like domains and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain involved in signalling. Specific binding of salmon IL-1RAcP to IL-1RLP was shown by co-immunoprecipitation studies.     

X-ray microanalysis of elements of VA mycorrhizal and non-mycorrhizal Pennisetum pedicellatum roots

Chemical composition of inter and intracellular hyphae was elucidated in Pennisetum pedicellatum roots colonized by Gigaspora margarita by employing energy dispersive X-ray microanalysis. Maximum amount of phosphate was estimated in polyphosphate granules present in the roots. Amount of phosphate was low in fungal hyphae, arbuscular trunks and degenerated arbuscules. Elements S, K and Ca were present in the hyphae, arbuscular trunk and degenerated arbuscules.     

Natural plant enzyme inhibitors. VI. Studies on trypsin inhibitors of Colocasia antiquorum tubers.

A trypsin inhibitor was purified from the tubers of Colocasia antiquorum. The inhibitor acted on bovine trypsin, human trypsin and weakly on bovine chymotrypsin. The inhibitor, which had a molecular weight of 40 000, contained trace amounts of carbohydrates. The purified inhibitor was stable over a pH range of 2.0--12.0 and was more thermostable than the crude preparations. Trinitrobenzene sulphonate treatment resulted in the inactivation of the inhibitor. Chymotrypsin, pepsin and pronase digested the inhibitor. Pretreatment with trypsin at neutral pH resulted in the partial loss of antitryptic activity, whereas treatment at pH 3.7 led to complete inactivation. Evidence for the formation of a trypsin-inhibitor complex at pH 7.6 is provided. During the plant growth, in the early phase (0--40 days) there was a gradual increase in protein content and in antitryptic activity. The middle phase (40--55 days) was characterized by a rapid fall and abolition of the antitryptic activity and a diminution in protein content in the tubers. The immature tubers had low antitryptic activity compared to the mature ones. Mild heat treatment caused a sharp rise in antitryptic activity in the extracts of immature tubers but not with the mature tuber preparations.     

Genetic control of α‐farnesene production in apple fruit and its role in fungal pathogenesis

Terpenes are important compounds in plant trophic interactions. A meta‐analysis of GC‐MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)‐α‐farnesene. Four quantitative trait loci (QTLs) for α‐farnesene production in ripe fruit were identified in a segregating ‘Royal Gala’ (RG) × ‘Granny Smith’ (GS) population with one major QTL on linkage group 10 co‐locating with the MdAFS1 (α‐farnesene synthase‐1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC‐MS analysis of headspace and solvent‐extracted terpenes showing that cold‐treated GS apples produced higher levels of (E,E)‐α‐farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)‐α‐farnesene. To evaluate the role of (E,E)‐α‐farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post‐harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)‐α‐farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post‐inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)‐α‐farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.     

Expression levels of 10 candidate genes in lung tissue of vaccinated and TB‐infected cynomolgus macaques

The expression of ten tuberculosis candidate genes in lung and lymph nodes of cynomologus macaques vaccinated and experimentally infected with Mycobacterium tuberculosis (Mtb) was quantified. The expression of TNFα, IL10, IL1β, TLR4, IL17, IL6, IL12, and iNOS in the lungs of vaccinated animals was higher than that of non‐vaccinated animals.