New mitochondrial primers for metabarcoding of insects,designed and evaluated using in silico methods

Insect metabarcoding has been mainly based on PCR amplification of short fragments within the “barcoding region” of the gene cytochrome oxidase I (COI). However, because of the variability of this gene, it has been difficult to design good universal PCR primers. Most primers used today are associated with gaps in the taxonomic coverage or amplification biases that make the results less reliable and impede the detection of species that are present in the sample. We identify new primers for insect metabarcoding using computational approaches (ecoprimers and degeprime ) applied to the most comprehensive reference databases of mitochondrial genomes of Hexapoda assembled to date. New primers are evaluated in silico against previously published primers in terms of taxonomic coverage and resolution of the corresponding amplicons. For the latter criterion, we propose a new index, exclusive taxonomic resolution, which is a more biologically meaningful measure than the standard index used today. Our results show that the best markers are found in the ribosomal RNA genes (12S and 16S); they resolve about 90% of the genetically distinct species in the reference database. Some markers in protein‐coding genes provide similar performance but only at much higher levels of primer degeneracy. Combining two of the best individual markers improves the effective taxonomic resolution with up to 10%. The resolution is strongly dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S primers detect 12% of Collembola. Our results indicate that amplicon‐based metabarcoding of insect samples can be improved by choosing other primers than those commonly used today.     

Simplified Intestinal Microbiota to Study Microbe-Diet-Host Interactions in a Mouse Model

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Synthesis of the 'primer-adaptor' trisaccharide moiety of Escherichia coli O8, O9, and O9a lipopolysaccharide

Described is the synthesis of the trisaccharide alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-beta-D-GlcpNAcO(CH2)8N3, the glycan portion of which corresponds to the 'adaptor-primer' moiety linking the O-chain and core oligosaccharide in the lipopolysaccharide of several Escherichia coli and Klebsiella pneumoniae serotypes. This report represents the first synthesis of this trisaccharide motif, and in the route involved, a key step is a [2+1] coupling of a protected Manp-(1-->3)-alpha-D-Manp glycosyl donor with a GlcpNAc acceptor. The azido group was included in the target to facilitate future preparation of neoglycoconjugates.     

Growth and survival trade-offs and outbreeding depression in rainbow trout (Oncorhynchus mykiss)

The purpose of this study was to examine, using a rainbow trout (Oncorhynchus mykiss) model system, the fitness consequences of three generations of introgression of genotypes adapted to two different environments (culture and nature). The experiments also isolated the influence of competitive interactions and risk of predation on the relative growth and survival of the wild and backcrossed lines. Line crosses representing fast-growing pure domestic (D), slow-growing pure wild (W), domestic x wild hybrids (F1), F1 x wild backcrosses (B1), and B1 x wild backcrosses (B2) were generated and reared under (1) culture conditions, (2) seminatural conditions with competition among genotypes, and (3) seminatural conditions under risk of predation. Survival of the fry in a seminatural environment with competition fit an additive model of gene action with the domestic fish having the highest survival and the wild fish the lowest, but under risk of predation outbreeding depression was suggested by low survival of the B2 lines. Evidence of a trade-off in growth and survival under risk of predation along with observations of genetically determined behavioral differences among the strains may provide some explanation for the observed differences in survival among the strains. This information is relevant to improving our evolutionary understanding of the interaction among genomes, and the influence of environment, during hybridization events. Results from this experiment indicate that alteration of phenotype likely played a prominent role in the reduced fitness experienced by progeny produced after three generations of introgression, supporting the theory that disruption of genotypes selected for adaptation to local conditions may be a primary cause of outbreeding depression in species such as salmon.     

Deriving respiratory cell types from stem cells

The reported pluripotential capabilities of many human stem cell types has made them an attractive area of research, given the belief they may hold considerable therapeutic potential for treating a wide range of human diseases and injuries. Although the bulk of stem cell based research has focused on developing procedures for the treatment of pancreatic, neural, cardiovascular and haematopoietic diseases, the potential for deriving respiratory cell types from stem cells for treatment of respiratory specific diseases has also been explored. It is suggested that stem cell derivatives may be used for lung replacement/regeneration therapeutics and high though-put pharmacological screening strategies for a variety of respiratory injuries and diseases including: cystic fibrosis, chronic obstructive pulmonary disease, respiratory distress syndrome, pulmonary fibrosis and pulmonary edema. This review will explore recent progress in characterizing adult respiratory and bone marrow derived stem cells with respiratory potential as well as the endogenous mechanisms directing the homing of these cells to the diseased and injured lung. In addition, the potential for embryonic stem cell based therapies in this domain as well as the histological, anatomical and molecular aspects of respiratory development will be summarized.     

Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.     

Taxonomic distinctness and species richness as measures of functional structure in bird assemblages

Most traditional "biodiversity" indices have an uncertain ecological interpretation, unfavourable sampling properties, and excessive data requirements. A new index of taxonomic distinctness (the average evolutionary distance between species in an assemblage) has many advantages over traditional measures, but its ecological interpretation remains unclear. We used published behavioural species data in conjunction with bird atlas data to quantify simple functional metrics (the fraction of species engaged in non-competitive interactions, and the average between-species disparity in habitat preferences) for breeding-bird assemblages in Europe and North America. We then analysed correlations of functional metrics with taxonomic distinctness and species richness, respectively. All functional metrics had weak, positive correlations with species richness. In contrast, correlations between functional metrics and taxonomic distinctness ranged from slightly negative to strongly positive, depending on the relative habitat heterogeneity, and on the resource involved in the between-species interaction. Strong positive correlations between taxonomic distinctness and the fraction of interactive species occurred for resources with few producer species per consumer species, and we suggest that taxonomic distinctness is consistently correlated with conservation worth. With its favourable sampling properties and data requirements, this taxonomic distinctness measure is a promising tool for biodiversity research and for environmental monitoring and management.     

Sequence context effect on the structure of nitrous acid induced DNA interstrand cross-links

In the preceding paper in this journal, we described the solution structure of the nitrous acid cross-linked dodecamer duplex [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined). The structure revealed that the cross-linked guanines form a nearly planar covalently linked 'G:G base pair', with the complementary partner cytidines flipped out of the helix. Here we explore the flanking sequence context effect on the structure of nitrous acid cross-links in [d(CG)]2 and the factors allowing the extrahelical cytidines to adopt such fixed positions in the minor groove. We have used NMR spectroscopy to determine the solution structure of a second cross-linked dodecamer duplex, [d(CGCTACGTAGCG)]2, which shows that the identity of the flanking base pairs significantly alters the stacking patterns and phosphate backbone conformations. The cross-linked guanines are now stacked well on adenines preceding the extrahelical cytidines, illustrating the importance of purine- purine base stacking. Observation of an imino proton resonance at 15.6 p.p.m. provides evidence for hydrogen bonding between the two cross-linked guanines. Preliminary structural studies on the cross-linked duplex [d(CGCGACGTCGCG)]2 show that the extrahelical cytidines are very mobile in this sequence context. We suggest that favorable van der Waals interactions between the cytidine and the adenine 2 bp away from the cross-link localize the cytidines in the previous cross-linked structures.     

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.