Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis

Recent advances in environmental DNA (eDNA) analysis using high‐throughput sequencing (HTS) enable evaluation of intraspecific genetic diversity in a population. As the intraspecific genetic diversity provides invaluable information for wildlife conservation and management, there is an increasing demand to apply eDNA analysis to population genetics and the phylogeography by quantitative evaluation of intraspecific diversity. However, quantitative evaluations of intraspecific genetic diversity using eDNA is not straightforward because the number of eDNA sequence reads obtained by HTS may not be an index of the quantity of eDNA. In this study, to quantitatively evaluate genetic diversity using eDNA analysis, we applied a quantitative eDNA metabarcoding method using the internal standard DNAs. We targeted Ayu (Plecoglossus altivelis altivelis) and added internal standard DNAs with known copy numbers to each eDNA sample obtained from three rivers during the library preparation process. The sequence reads of each Ayu haplotype were successfully converted to DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In all rivers, the calculated copy number of each haplotype showed a significant positive correlation with the haplotype frequency estimated by a capture‐based survey. Furthermore, estimates of genetic indicators such as nucleotide diversity based on the eDNA copy numbers were comparable with those estimated based on a capture‐based study. Our results demonstrate that eDNA analysis with internal standard DNAs enables reasonable quantification of intraspecific genetic diversity, and this method could thus be a promising tool in the field of population genetics and phylogeography.     

Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

Environmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA‐based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high‐throughput sequencing, denoising was performed using two of the most widely used denoising packages, unoise3 and dada2 . Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (unoise3 ) and 41 (dada2 ) haplotypes were detected by eDNA analysis. When dada2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. Accordingly, although it is important to note that eDNA‐based method has some limitations and some risk of false positive and false negative, this study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large‐scale capture‐based conventional methods. Our results suggest that eDNA‐based methods could become a more efficient survey method for investigating intraspecific genetic diversity in the field.     

A synergy of activity,stability, and inhibitor‐interaction of HIV‐1 protease mutants evolved under drug‐pressure

A clinically‐relevant, drug‐resistant mutant of HIV‐1 protease (PR), termed Flap+_(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug‐PR interactions, compared to wild‐type PR. A similar mutant, Flap+_(I54A), which evolves from Flap+_(I54V) and contains the single change at residue 54 relative to Flap+_(I54V), does not. Yet, how Flap+_(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+_(I54V), Flap+_(I54A), and Flap+_(I54), a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.     

Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling

To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na⁺/H⁺ exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase–Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.     

Effect of Acridine with Various Linker Arms Attached to C5 Position of 2′-Deoxyuridine on the Stability of DNA/DNA and DNA/RNA Duplexes

Abstract

Acridine-modified oligodeoxyribonucleotides (ODNs) at the C5-position of a 2′-deoxyuridine via different lengths of linker arms were synthesized. Reaction of 5-(N-aminoalkyl)carbamoylmethyl-2′-deoxyuridines with 9-phenoxyacridine gave the acridine-modified 2′-deoxyuridines which were incorporated into ODNs. The duplexes containing the acridine-modified strands and their complementary DNA or RNA were thermally more stable than that containing the unmodified strand. Thermal stability of the duplexes of the modified ODNs varied depending on the length of the linker arms.

     

2-Bromoadenosine-Substituted 2′,5′-Oligoadenylates Modulate Binding and Activation Abilities of Human Recombinant RNase L

Abstract

2-Bromoadenosine-substituted analogues of 2–5A, p5′A2′p-5′A2′p5′(br²A), p5′(br²A)2′p5′A2′p5′A, and p5′(br²A)2′p5′(br²A)2′p-S′(br²A), were prepared via a modification of a lead ion-catalyzed ligation reaction and were subsequently converted into the corresponding 5′-triphosphates. Both binding and activation of human recombinant RNase L by various 2-bromoadenosine-substituted 2–5A analogues were examined. Among the 2-bromoadenosine-substituted 2–5A analogues, the analogue with 2-bromoadenosine residing in the 2′-terminal position, p5′A2′p5′A2′p-5′(br²A), showed the strongest binding affinity and was as effective as 2–5A itself as an activator of RNase L. The CD spectrum of p5′A2′p-5′A2′p5′(br²A) was superimposable on that of p5′A2′p5′A2′p5′A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2–5A.     

Synthesis of 2′,3′-Dideoxypurinenucleosides via the Palladium Catalyzed Reduction of 9-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-d-xylofuranosyl)purine Derivatives

Abstract

Practical method to produce 2′,3′-dideoxypurinenucleosides from 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)purines (1) was developed. High ratio of 2′,3′-dideoxynucleoside to 3′-deoxyribonucleoside was obtained by selecting the reaction conditions (solvent, pH and/or base), or changing 2′-acyloxy leaving group. The reaction mechanism was studied by deuteration experiments of 1a and 1-(3,5-di-O-acety1-2-bromo-2-deoxy-β-D-ribofuranosyl)thymine (12).

     

Effects of remnant primary forests on ant and dung beetle species diversity in a secondary forest in Sarawak, Malaysia

Tropical landscape structures have been transformed into mosaic structures consisting of small patches of primary and secondary forests, and areas of other land use. Diversity of insect assemblages is often higher in primary forests than in surrounding secondary forests. However, little is known about how the primary forests affect diversity in surrounding secondary forests in a landscape. In Sarawak, Malaysia, the typical landscape in areas from which lowland tropical rainforests had originally spread consists mainly of primary and secondary forests, with small areas of cultivation. In this study, we examined how the proportion of remnant primary forests in a landscape affects species diversity and species composition of ants and dung beetles in Macaranga-dominated secondary forests. The proportions were quantified based on remote-sensing data at various spatial scales, ranging from 100- to 5,000-m radius from each of the target forests. We found that the proportions of remnant primary forests within a 100-m radius had a significant positive effect on ant species diversity, and those within 100-, 300-, and 500-m radii significantly affected species compositions. However, the proportions of remnant primary forests had no significant relationship with dung beetle diversity, while those within 100- and 1,000-m radii had significant effects on species composition. The different responses to the remnant primary forests are likely to be related to differences in the movement and dispersal traits between the two taxa.