Pathway analysis in metabolomics: Recommendations for the use of over-representation analysis

Over-representation analysis (ORA) is one of the commonest pathway analysis approaches used for the functional interpretation of metabolomics datasets. Despite the widespread use of ORA in metabolomics, the community lacks guidelines detailing its best-practice use. Many factors have a pronounced impact on the results, but to date their effects have received little systematic attention. Using five publicly available datasets, we demonstrated that changes in parameters such as the background set, differential metabolite selection methods, and pathway database used can result in profoundly different ORA results. The use of a non-assay-specific background set, for example, resulted in large numbers of false-positive pathways. Pathway database choice, evaluated using three of the most popular metabolic pathway databases (KEGG, Reactome, and BioCyc), led to vastly different results in both the number and function of significantly enriched pathways. Factors that are specific to metabolomics data, such as the reliability of compound identification and the chemical bias of different analytical platforms also impacted ORA results. Simulated metabolite misidentification rates as low as 4% resulted in both gain of false-positive pathways and loss of truly significant pathways across all datasets. Our results have several practical implications for ORA users, as well as those using alternative pathway analysis methods. We offer a set of recommendations for the use of ORA in metabolomics, alongside a set of minimal reporting guidelines, as a first step towards the standardisation of pathway analysis in metabolomics.     

Seedling Survival from Locally and Commercially Obtained Seeds on Two Semiarid Sites

Local populations of plants are likely to be better adapted to a site than populations from elsewhere. Thus, local seeds should yield higher survival in restoration attempts than commercial seed stocks. We compared seedling survival from locally and commercially obtained seeds of seven species, Pseudoroegneria spicata (bluebunch wheatgrass), Elymus elymoides (squirreltail), Pascopyrum smithii (western wheatgrass), Stipa hymenoides (Indian ricegrass), Stipa comata (needle‐and‐thread), Chrysothamnus nauseosus (rubber rabbitbrush) and Ephedra nevadensis (Mormon tea) over three years on two sites in Utah (Dugway and Tintic) that were dominated by the introduced annual Bromus tectorum (cheatgrass). At the Dugway site we included burned and unburned seedbed treatments. For all species at Dugway, seedling survival to the first summer was higher on burned plots where B. tectorum densities were greatly reduced. First‐year seedling survival was 20–30% for most species on the Dugway burned plots and at Tintic. At the drier Dugway site, only S. hymenoides and Ephedra had substantial third‐year survival. Elymus and Pascopyrum survived to the third year only at the moister Tintic site. Survival to the third year was less than 3% for all species except S. comata (6% survival), and densities were low (0.2–1.0 plants/m²). However, third‐year plants were well established and the grasses flowered. Pseudoroegneria and Ephedra at Dugway (on burned plots) and S. comata and Elymus at Tintic had higher first‐year survival or higher survival based on survival curves from local than from commercial seeds. However, final survival was never significantly higher, although such a trend was suggested. Seed dormancy traits could also provide advantages to local populations, and we observed differences in dormancy between local and commercial S. comata and S. hymenoides seeds that may be an example.     

Comparison of antifouling properties of native and invasive Sargassum (Fucales,Phaeophyceae) species

The invasiveness of algal species can be facilitated by chemo-ecological traits that allow the establishment of invasive species in a highly competitive environment. Anti-bacterial, anti-quorum sensing, anti-diatom and anti-larval properties of the invasive brown macroalga Sargassum muticum and three native Sargassum species from Oman waters were compared in laboratory and field experiments to assess whether these traits have the potential to facilitate the invasion process. Only the extract of S. muticum inhibited bacterial growth of four marine bacterial strains and quorum sensing in the reporter strain Chromobacterium violaceum CV017. Settlement, growth and survival of the diatom Cylindrotheca closterium and larvae of the bryozoan Bugula neritina were significantly inhibited by all Sargassum extracts in laboratory experiments. However, crude extracts of S. muticum had the strongest antifouling effect. Natural tissue-level concentrations of S. muticum extract reduced diatom density to about 20% compared with the controls. Larval mortality increased by 80–90% compared with controls with S. muticum extract diluted to one-third natural levels. Significant anti-diatom activity of S. muticum was confirmed in the field experiments with Sargassum extracts embedded in a phytagel matrix. Comparison of non-polar compounds by gas chromatography–mass spectrometry demonstrated that S. muticum extracts had overall fewer secondary metabolites but more species-unique compounds than extracts of native Sargassum spp. The greater antifouling defence of invasive vs. native Sargassum species indicates a selective trait that may contribute to the invasion success of S. muticum.     

Biological Control of Sheep Parasites using Duddingtonia flagrans: Trials on Commercial Farms in Sweden

   

Trials were conducted on 3 commercial sheep farms in Sweden to assess the effect of administering spores of the nematode trapping fungus, Duddingtonia flagrans, together with supplementary feed to lactating ewes for the first 6 weeks from turn-out on pastures in spring. Also control groups of ewes, receiving only feed supplement, were established on all 3 farms. Groups were monitored by intensive parasitological investigation. The ewes and their lambs were moved in late June to saved pastures for summer grazing, the lambs receiving an anthelmintic treatment at this time. After approximately 6 weeks on summer pasture the lambs were weaned, treated a second time with anthelmintic, and returned to their original lambing pastures for finishing. Decisions as to when lambs were to be marketed were entirely at the discretion of the farmer co-operators. No difference in lamb performance was found between the two treatments on all three farms. This was attributed to the high levels of nutrition initially of the ewes limiting their post-partum rise in nematode faecal egg counts in spring, which in turn resulted in low levels of nematode infection on pastures throughout the autumn period. Additionally, pastures were of good quality for the lambs during the finishing period, so they grew at optimal rates as far as the farmers were concerned.     

Improved analyses of human mtDNA sequences support a recent African origin for Homo sapiens

New quantitative methods are applied to the 135 human mitochondrial sequences from the Vigilant et al. data set. General problems in analyzing large numbers of short sequences are discussed, and an improved strategy is suggested. A key feature is to focus not on individual trees but on the general "landscape" of trees. Over 1,000 searches were made from random starting trees with only one tree (a local optimum) being retained each time, thereby ensuring optima were found independently. A new tree comparison metric was developed that is unaffected by rearrangements of trees around many very short internal edges. Use of this metric showed that downweighting hypervariable sites revealed more evolutionary structure than studies that weighted all sites equally. Our results are consistent with convergence toward a global optimum. Crucial features are that the best optima show very strong regional differentiation, a common group of 49 African sequences is found in all the best optima, and the best optima contain the 16 !Kung sequences in a separate group of San people. The other 86 sequences form a heterogeneous mixture of Africans, Europeans, Australopapuans, and Asians. Thus all major human lineages occur in Africa, but only a subset occurs in the rest of the world. The existence of these African-only groups strongly contradicts multiregional theories for the origin of Homo sapiens that require widespread migration and interbreeding over the entire range of H. erectus. Only when the multiregional model is rejected is it appropriate to consider the root, based on a single locus, to be the center of origin of a population (otherwise different loci could give alternative geographic positions for the root). For this data, several methods locate the root within the group of 49 African sequences and are thus consistent with the recent African origin of H. sapiens. We demonstrate that the time of the last common ancestor cannot be the time of major expansion in human numbers, and our results are thus also consistent with recent models that differentiate between the last common ancestor, expansion out of Africa, and the major expansion in human populations. Such a two-phase model is consistent with a wide range of molecular and archeological evidence.      

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.      

Fibrillar and tubular fine structures in the cytoplasm of Entamoeba histolytica (author's transl)]

The electron-microscopical study of conventional fixed and contrasted Entamoeba histolytica-trophozoites from Diamonds monoxenic TTY-medium revealed "thick fibrils" in the vesicular cytoplasm of the parasites. They are 9-14 nm in diameter and are therefore considered to be myosin-like filaments, that are spread mainly in the tail or the uroid of the moving ameba. No association with microfilaments (MF) or other organelles could be observed. During the same investigation microtubules (MT) of variable length are described having a diameter of 35 nm, surrounded by a 19-25 nm wide capsule. They are distributed singly or in bundles with a maximal diameter of 350 nm. Some of the MT were coiled up to a helical shape.     

Structural changes on Entamoeba histolytica trophozoites after cryopreservation in liquid nitrogen.

Trophozoites of Entamoeba histolytica cultures which had been deep-frozen in the presence of 5% DMSO, along with untreated cells and cells treated with DMSO (5%), were examined for fine-structural changes. After deep-freezing in liquid nitrogen only a few amoebae exhibited normal nuclear and cytoplasmic structure. One frequently observed but unspecific finding pertaining to recovered cells is the separation of the cytoplasm into large vacuolated (coarse-granular) and electron-optically fine-granular (hyaline) zones. The glycogen which normally lies in the cytoplasm is always eluted. In many cases numerous short RNP helices are scattered unevenly in the vesicular plasma, but they are also found in larger masses adjacent to the membranes of still intact and already damaged nuclei. Moderately damaged nuclei have a poorly folded membrane and their chromatin is markedly denatured. More heavily damaged nuclei have a membrane which has partly fibrillated or ruptured and then formed conspicuous folds, where the nuclear membrane has ruptured nucleoplasmic remnants of chromatin and button-like bodies appear to pour into the surrounding cytoplasm. The final destruction of the cell is marked by coalescing autolytic zones, first in the vacuolated and later in the fine-granular cytoplasm. Finally only remnants of the nuclear membrane and of the membranes of numerous vacuoles remain. It is assumed that most of the changes in the cytoplasm are of a secondary nature and are caused by the early functional disturbance of the nucleus.