Is qualitative and quantitative metabarcoding of dung fauna biodiversity feasible?

In biodiversity assessments, especially of small-bodied organisms for which taxonomic expertise is lacking, identification by genetic barcoding may be a cost-effective and efficient alternative to traditional identification of species by morphology, ecology, and behavior. The authors tested the feasibility and accuracy of such an approach using dung insects of practical relevance in ecotoxicological assessments of veterinary pharmaceutical residues in the environment. They produced 8 known mixtures that varied in absolute and relative composition of small-bodied and large-bodied species to see whether mitochondrial cytochrome c oxidase subunit 1 barcoding picks up all species qualitatively and quantitatively. As demonstrated before in other contexts, such metabarcoding of large numbers of dung insect specimens is principally possible using next-generation sequencing. The authors recovered most species in a sample (low type I error), at minimum permitting analysis of species richness. They obtained even quantitative responses reflecting the body size of the species, although the number of specimens was not well detected. The latter is problematic when calculating diversity indices. Nevertheless, the method yielded too many closely related false positives (type II error), thus generally overestimating species diversity and richness. These errors can be reduced by refining methods and data filtering, although this requires bioinformatics expertise often unavailable where such research is carried out. Identification by barcoding foremost hinges on a good reference database, which does not yet exist for dung organisms but would be worth developing for practical applications. Environ Toxicol Chem 2016;35:1970-1977. © 2015 SETAC.

New microsatellite loci for the green sea urchin Strongylocentrotus droebachiensis using universal M13 labelled markers.

BACKGROUND: The green sea urchin Strongylocentrotus droebachiensis has a wide circumpolar distribution and plays a key role in coastal ecosystems worldwide by destructively grazing macroalgae beds and turn them into marine deserts, so-called barren grounds. In the past decades, large established kelp forests have been overgrazed and transformed to such barren grounds on the Norwegian coast. This has important repercussions for the coastal diversity and production, including reproduction of several fish species relying on the kelp forests as nurseries. Genetic diversity is an important parameter for the study and further anticipation of this large scale phenomenon. FINDINGS: Microsatellites were developed using a Norwegian S. droebachiensis individual primarily for the study of Northeast Atlantic populations. The 10 new microsatellite loci were amplified using M13 forward tails, enabling the use of M13 fluorescent tagged primers for multiplex reading. Among these loci, 2 acted polysomic and should therefore not be considered useful for population genetic analysis. We screened 96 individuals sampled from 4 different sites along the Norwegian coast which have shown unexpected diversity. CONCLUSIONS: The new microsatellite loci should be a useful resource for further research into connectivity among S. droebachiensis populations, and assessing the risks for spreading and new overgrazing events.

Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells.

Cell entry of Simian Virus 40 (SV40) involves caveolar/lipid raft-mediated endocytosis, vesicular transport to the endoplasmic reticulum (ER), translocation into the cytosol, and import into the nucleus. We analyzed the effects of ER-associated processes and factors on infection and on isolated viruses and found that SV40 makes use of the thiol-disulfide oxidoreductases, ERp57 and PDI, as well as the retrotranslocation proteins Derlin-1 and Sel1L. ERp57 isomerizes specific interchain disulfides connecting the major capsid protein, VP1, to a crosslinked network of neighbors, thus uncoupling about 12 of 72 VP1 pentamers. Cryo-electron tomography indicated that loss of interchain disulfides coupled with calcium depletion induces selective dissociation of the 12 vertex pentamers, a step likely to mimic uncoating of the virus in the cytosol. Thus, the virus utilizes the protein folding machinery for initial uncoating before exploiting the ER-associated degradation machinery presumably to escape from the ER lumen into the cytosol.

Structure-function analysis of the endoplasmic reticulum oxidoreductase TMX3 reveals interdomain stabilization of the N-terminal redox-active domain.

Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b'). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb', we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb', the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.

Metabolic engineering of a genetic selection system with tunable stringency.

The biosynthesis of small molecules can be fine-tuned by (re)engineering metabolic flux within cells. We have adapted this approach to optimize an in vivo selection system for the conversion of prephenate to phenylpyruvate, a key step in the production of the essential aromatic amino acid phenylalanine. Careful control of prephenate concentration in a bacterial host lacking prephenate dehydratase, achieved through provision of a regulable enzyme that diverts it down a parallel biosynthetic pathway, provides the means to systematically increase selection pressure on replacements of the missing catalyst. Successful differentiation of dehydratases whose activities vary over a >50,000-fold range and the isolation of mechanistically informative prephenate dehydratase variants from large protein libraries illustrate the potential of the engineered selection strain for characterizing and evolving enzymes. Our approach complements other common methods for adjusting selection pressure and should be generally applicable to any selection system that is based on the conversion of an endogenous metabolite.

Identification and characterization of a novel thioredoxin-related transmembrane protein of the endoplasmic reticulum.

The endoplasmic reticulum (ER) contains a number of thiol-disulfide oxidoreductases of the protein-disulfide isomerase (PDI) family that catalyze the formation of disulfide bonds in newly synthesized proteins. Here we describe the identification and characterization of a novel member of the human PDI family, TMX3 (thioredoxin-related transmembrane protein 3). The TMX3 gene encodes a protein of 454 amino acid residues that contains a predicted N-terminal signal sequence, a single domain with sequence similarity to thioredoxin and a CGHC active site sequence, a potential transmembrane domain, and a C-terminal KKKD tetrapeptide sequence that matches the classical KKXX-type consensus sequence for ER retrieval of type I transmembrane proteins. Endogenous TMX3 contains endoglycosidase H-sensitive glycans, localizes to the ER by immunofluorescence microscopy, and is present in the membrane fraction after alkaline extraction of the ER luminal content. The TMX3 transcript is found in a variety of tissues and is not up-regulated by the unfolded protein response. Circular dichroism spectroscopy of the recombinantly expressed luminal domain of TMX3 showed features typical of a properly folded protein of the alpha/beta type. The redox potential of recombinant luminal TMX3 was determined to -0.157 V, similar to the values previously found for PDI and ERp57. Interestingly, TMX3 showed oxidase activity, and in human tissue-culture cells the protein was found partially in the oxidized form, potentially suggesting a function of the protein as a dithiol oxidase.