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.     

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose

The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.

Methods

Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.

Results

Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.

Conclusion

The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.     

Environmental DNA as a ‘Snapshot’ of Fish Distribution: A Case Study of Japanese Jack Mackerel in Maizuru Bay,Sea of Japan

Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors (e.g., water flow) are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel (Trachurus japonicus) using echo sounder technology. A linear model showed a high R² value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10–150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a ‘snapshot’ of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions (e.g., patterns of release, degradation, and diffusion of eDNA) are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.     

Rabies and other lyssaviruses

Since rabies has not been reported in Japan for nearly the past 50 years, it has been relegated to the status of a forgotten infectious disease in this country. However,in the neighboring Asian countries, Africa, America, the number fo rabies cases had not decrease but on the contrary, seen an increasing trend. In Russia and the former Soviet Union countries (CIS countries), the number of information. Between 30,000 approximately 20,000 fatal cases of rabies in both humans and animals had been reported yearly butit was thought that the number might run up to hundred of thousands.japan, Taiwan, UK, Australia and New Zealand are rabies-free countries and should be considered the exception rather than the norm. Due to the long Lull in which rabies has not occurred in Japan,people tend to forget that the disease can infect all mammals including humans, with a mortality rate of 100% after manifestation of debilitating nervous symptoms and that is one of the most dangerous zoonotic viral diseases on earth.     

Diarrhea-inducing activity of avian rotavirus NSP4 glycoproteins, which differ greatly from mammalian rotavirus NSP4 glycoproteins in deduced amino acid sequence in suckling mice

Avian rotavirus NSP4 glycoproteins expressed in Escherichia coli acted as enterotoxins in suckling mice, as did mammalian rotavirus NSP4 glycoproteins, despite great differences in the amino acid sequences. The enterotoxin domain of PO-13 NSP4 exists in amino acid residues 109 to 135, a region similar to that reported in SA11 NSP4.     

Antigenic analysis of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13

In order to analyze the antigenic structure of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13, 25 monoclonal antibodies (MAbs) against NSP4 expressed in Escherichia coli were produced. All MAbs reacted with NSP4 on Western blotting, indicating that they recognized sequential epitopes. To determine the antigenic sites (ASs) recognized by the produced MAbs, seven truncated NSP4s were expressed in E. coli. Western blotting analysis showed that there are at least four major ASs on PO-13 NSP4, designated as AS I located in amino acids (aa) 151 to 169, AS II (aa 136 to 150), AS III (aa 112 to 133) and AS IV (aa 1 to 24). Two MAbs reacted exclusively with AS III encompassing the region that has been reported to be an enterotoxin domain. MAbs against ASs II, III and IV reacted with all avian rotaviruses tested by indirect immunofluorescent antibody assays. MAbs against AS I reacted with turkey strains, Ty-1 and Ty-3, but not with a chicken strain, Ch-1. Nine of 11 MAbs against AS II cross-reacted with NSP4 of mammalian rotavirus strains with different NSP4 genotypes. These results suggest that AS II on NSP4 is widely conserved among a variety of rotaviruses.     

Genetic characterization of rabies field isolates from Thailand

We sequenced 512 nucleotides in two variable regions of the N gene of 23 rabies isolates from the northeastern part of Thailand by direct sequencing of PCR-amplified products. The sequencing data revealed two new lineages in these rabies isolates. Based on the results of this study together with the findings of our earlier study, the rabies isolates in Thailand were divided into two genogroups, designated as T1 and T2, which were predominantly localized in the northern and northeastern areas, respectively. Each of these two genogroups consisted of four lineages. There was a correlation between these eight lineages and the geographical origins of the isolates. Two lineages belonging to the T2 genogroup from the northeastern area of Thailand were newly identified in this study. The isolates in both genogroups were also prevalent in the central area of Thailand. Each lineage in the T1 and T2 genogroups was found independently in dogs in the upper and lower southern areas of Thailand, respectively. These genetic data and the historical background suggest that rabies viruses belonging to the T2 genogroups were prevalent many years ago in the central and northeastern areas of Thailand and were later transferred to the lower southern parts of Thailand.