A comprehensive comparison of fecal microbiota in three ecological bird groups of raptors, waders, and waterfowl.

Gut microbiota plays a vital role in maintaining the health and immunity of wild birds. However, less is known about the comparison of fecal microbiota between different ecological groups of wild birds, particularly in the Yellow River National Wetland in Baotou, China, an important transit point for birds migrating all over the East Asia-Australian and Central Asian flyways. In this study, we characterized the fecal microbiota and potential microbial function in nine bird species of raptors, waders, and waterfowl using 16S rRNA gene amplicon sequencing to reveal the microbiota differences and interaction patterns. The results indicated that there was no significant difference in α-diversity, but a significant difference in ß-diversity between the three groups of birds. The fecal bacterial microbiota was dominated by Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes in all groups of birds. Furthermore, we identified five bacterial genera that were significantly higher in raptors, five genera that were significantly higher in waders, and two genera that were more abundant in waterfowl. The bacterial co-occurrence network results revealed 15 and 26 key genera in raptors and waterfowls, respectively. The microbial network in waterfowl exhibited a stronger correlation pattern than that in raptors. PICRUSt2 predictions indicated that fecal bacterial function was significantly enriched in the antibiotic biosynthesis pathway in all three groups. Metabolic pathways related to cell motility (bacterial chemotaxis and flagellar assembly) were significantly more abundant in raptors than in waders, whereas waders were enriched in lipid metabolism (synthesis and degradation of ketone bodies and fatty acid biosynthesis). The fecal microbiota in waterfowl harbored more abundant vitamin B6 metabolism, RNA polymerase, and tyrosine and tryptophan biosynthesis. This comparative study revealed the microbial community structure, microbial co-occurrence patterns, and potential functions, providing a better understanding of the ecology and conservation of wild birds. Future studies may focus on unraveling metagenomic functions and dynamics along with the migration routine or different seasons by metagenomics or metatranscriptomics.

Degradation of MicroRNA miR-466d-3p by Japanese Encephalitis Virus NS3 Facilitates Viral Replication and Interleukin-1ß Expression.

Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma (NA) cells after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly downregulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin-1ß (IL-1ß) expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotype 1 (DENV-1) and DENV-2 also decreased miR-466d-3p expression. Results from helicase-blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation.IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing the expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1ß expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA-binding sites, because we demonstrated that miRNA degradation was abolished if arginines at R226 and R202 were mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino acid sites that could be mutated for a JEV vaccine.

Histone Demethylase UTX is an Essential Factor for Zygotic Genome Activation and Regulates Zscan4 Expression in Mouse Embryos.

Following fertilization, the zygotic genome is activated through a process termed zygotic genome activation (ZGA), which enables zygotic gene products to replace the maternal products and initiates early embryonic development. During the ZGA period, the embryonic epigenome experiences extensive recodifications. The H3K27me3 demethylase UTX is essential for post-implantation embryonic development. However, it remains unclear whether UTX participates in preimplantation development, especially during the ZGA process. In the present study, we showed that either knockdown or overexpression of UTX led to embryonic development retardation, whereas simultaneous depletion of UTX and overexpression of ZSCAN4D rescued the embryonic development, indicating that UTX positively regulated Zscan4d expression. Using a transgenic mice model, we also found that UTX was required for preimplantation embryonic development. In conclusion, these results indicate that UTX functions as a novel regulator and plays critical roles during ZGA in addition to early embryonic development.

Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids.

Linoleic acid (18 : 2, n-6) and α-linolenic acid (18 : 3, n-3) are polyunsaturated fatty acids (PUFAs), which are essential for mammalian health, development and growth. However, the majority of mammals, including humans, are incapable of synthesizing n-6 and n-3 PUFAs. Mammals must obtain n-6 and n-3 PUFAs from their diet. Fatty acid desaturase (Fad) plays a critical role in plant PUFA biosynthesis. Therefore, we generated plant-derived Fad3 single and Fad2-Fad3 double transgenic mice. Compared with wild-type mice, we found that PUFA levels were greatly increased in the single and double transgenic mice by measuring PUFA levels. Moreover, the concentration of n-6 and n-3 PUFAs in the Fad2-Fad3 double transgenic mice were greater than in the Fad3 single transgenic mice. These results demonstrate that the plant-derived Fad2 and Fad3 genes can be expressed in mammals. To clarify the mechanism for Fad2 and Fad3 genes in transgenic mice, we measured the PUFAs synthesis-related genes. Compared with wild-type mice, these Fad transgenic mice have their own n-3 and n-6 PUFAs biosynthetic pathways. Thus, we have established a simple and efficient method for in vivo synthesis of PUFAs.

KDM6A and KDM6B play contrasting roles in nuclear transfer embryos revealed by MERVL reporter system.

Despite the success of animal cloning by somatic cell nuclear transfer (SCNT) in many species, the method is limited by its low efficiency. After zygotic genome activation (ZGA) during mouse development, a large number of endogenous retroviruses (ERVs) are expressed, including the murine endogenous retrovirus-L (MuERVL/MERVL). In this study, we generate a series of MERVL reporter mouse strains to detect the ZGA event in embryos. We show that the majority of SCNT embryos do not undergo ZGA, and H3K27me3 prevents SCNT reprogramming. Overexpression of the H3K27me3-specific demethylase KDM6A, but not of KDM6B, improves the efficiency of SCNT Conversely, knockdown of KDM6B not only facilitates ZGA, but also impedes ectopic Xist expression in SCNT reprogramming. Furthermore, knockdown of KDM6B increases the rate of SCNT-derived embryonic stem cells from Duchenne muscular dystrophy embryos. These results not only provide insight into the mechanisms underlying failures of SCNT, but also may extend the applications of SCNT.

The Maternal Effect Genes UTX and JMJD3 Play Contrasting Roles in Mus musculus Preimplantation Embryo Development.

During the process of embryonic development in mammals, epigenetic modifications must be erased and reconstructed. In particular, the trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcriptional repression and contributes to the maintenance of the pluripotent embryos. In this study, we determined that the global levels of the H3K27me3 marker were elevated in MII oocyte chromatin and decrease to minimal levels at the 8-cell and morula stages. When the blastocyst hatched, H3K27me3 was re-established in the inner cell mass. We also determined that H3K27me3-specific demethylases, UTX and JMJD3, were observed at high transcript and protein levels in mouse preimplantation embryos. In the activated oocytes, when the H3K27me3 disappeared at the 8-cell stage, the UTX (but not JMJD3) protein levels were undetectable. Using RNA interference, we suppressed UTX and JMJD3 gene expression in the embryos and determined that the functions of UTX and JMJD3 were complementary. When JMJD3 levels were decreased by RNA interference, the embryo development rate and quality were improved, but the knockdown of UTX produced the opposite results. Understanding the epigenetic mechanisms controlling preimplantation development is critical to comprehending the basis of embryonic development and to devise methods and approaches to treat infertility.