Metagenomic analysis of the gut microbiota of hooded cranes (Grus monacha) on the Izumi plain in Japan.

Recent advances in DNA sequencing technology have dramatically improved our understanding of the gut microbiota of various animal species. However, research on the gut microbiota of birds lags behind that of many other vertebrates, and information about the gut microbiota of wild birds such as migratory waterfowl is particularly lacking. Because the ecology of migratory waterfowl (e.g., lifestyle, diet, physiological characteristics) differs from that of other birds, the gut microbiota of migratory waterfowl likely also differs, but much is still unknown. The hooded crane (Grus monacha) is an important representative migratory waterbird species and is listed as endangered on the International Union for Conservation of Nature and Natural Resources Red List of Threatened Species. In this study, we analyzed the bacterial and viral microbiota in the gut of hooded cranes by using deep sequencing data from fecal samples of hooded cranes that winter on the Izumi plain in Japan, and found that Cetobacterium, Clupeiformes, and Pbunavirus were clearly present in the fecal samples of hooded cranes. These findings advance our understanding of the ecology of hooded cranes.

Effect of a FOXO1 inhibitor on trophoblast differentiation from human pluripotent stem cells and ERV-associated gene expression.

Introduction: In human placental development, the trophectoderm (TE) appears in blastocysts on day 5 post-fertilization and develops after implantation into three types of trophoblast lineages: cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT). CDX2/Cdx2 is expressed in the TE, and Cdx2 expression is upregulated by knockdown of Foxo1 in mouse ESCs. However, the significance of FOXO1 in trophoblast lineage differentiation during the early developmental period remains unclear. In this study, we examined the effect of FOXO1 inhibition on the differentiation of naive human induced pluripotent stem cells (iPSCs) into TE and trophoblast lineages. Methods: We induced TE differentiation from naive iPSCs in the presence or absence of a FOXO1 inhibitor, and the resulting cells were subjected to trophoblast differentiation procedures without the FOXO1 inhibitor. The cells obtained in these processes were assessed for morphology, gene expression, and hCG secretion using phase-contrast microscopy, reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (RT-qPCR), RNA-seq, immunochromatography, and a chemiluminescent enzyme immunoassay. Results: In the induction of trophoblast differentiation from naive iPSCs, treatment with a FOXO1 inhibitor resulted in the enhanced expression of TE markers, CDX2 and HAND1, but conversely decreased the expression of ST markers, such as ERVW1 (Syncytin-1) and GCM1, and an EVT marker, HLA-G. The proportion of cells positive for an early TE marker TACSTD2 and negative for a late TE marker ENPEP was higher in FOXO1 inhibitor-treated cells than in non-treated cells. The expressions of ERVW1 (Syncytin-1), ERVFRD-1 (Syncytin-2), and other endogenous retrovirus (ERV)-associated genes that have been reported to be expressed in trophoblasts were suppressed in the cells obtained by differentiating the TE cells treated with FOXO1 inhibitor. Conclusions: Treatment with a FOXO1 inhibitor during TE induction from naive iPSCs promotes early TE differentiation but hinders the progression of differentiation into ST and EVT. The suppression of ERV-associated genes may be involved in this process.

Buccal Swab Samples from Japanese Brown Cattle Fed with Limonite Reveal Altered Rumen Microbiome.

The areas of the Mount Aso grasslands in Kumamoto, Japan, are the primary location for the breeding of the Kumamoto strain of Japanese Brown cattle (JBRK). Although Aso limonite, deposited by volcanic ash and magma, has been commonly fed to pregnant JBRK in this area, the mechanisms of its salutary effects on pregnant JBRK have not yet been elucidated. Approximately 100 days before the expected day of calf delivery, seven JBRK (four supplemented with limonite and three controls without limonite) were assigned to this study, from which a buccal swab was collected at the highest rumination every 30 days for 90 days. DNA extracted from these swabs was then analyzed using a 16S rRNA gene amplicon sequence analysis. Statistically significant differences between the two groups were discovered through beta-diversity analysis, though results from alpha-diversity analysis were inconclusive. The microbiota identified were classified into six clusters, and three of the main clusters were core-rumen bacteria, primarily cellulose digestion in cluster 1, oral bacteria in cluster 2, and non-core-rumen bacteria in cluster 3. In the limonite group, core-rumen bacteria decreased while non-core-rumen bacteria increased, suggesting that limonite feeding alters rumen microbiota, particularly activation of non-core-rumen microbiota.

Amino acid influx via LAT1 regulates iron demand and sensitivity to PPMX-T003 of aggressive natural killer cell leukemia.

Aggressive natural killer cell leukemia (ANKL) is a rare hematological malignancy with a fulminant clinical course. Our previous study revealed that ANKL cells proliferate predominantly in the liver sinusoids and strongly depend on transferrin supplementation. In addition, we demonstrated that liver-resident ANKL cells are sensitive to PPMX-T003, an anti-human transferrin receptor 1 inhibitory antibody, whereas spleen-resident ANKL cells are resistant to transferrin receptor 1 inhibition. However, the microenvironmental factors that regulate the iron dependency of ANKL cells remain unclear. In this study, we first revealed that the anti-neoplastic effect of PPMX-T003 was characterized by DNA double-strand breaks in a DNA replication-dependent manner, similar to conventional cytotoxic agents. We also found that the influx of extracellular amino acids via LAT1 stimulated sensitivity to PPMX-T003. Taken together, we discovered that the amount of extracellular amino acid influx through LAT1 was the key environmental factor determining the iron dependency of ANKL cells via adjustment of their mTOR/Myc activity, which provides a good explanation for the different sensitivity to PPMX-T003 between liver- and spleen-resident ANKL cells, as the liver sinusoid contains abundant amino acids absorbed from the gut.

Genes involved in the limited spread of SARS-CoV-2 in the lower respiratory airways of hamsters may be associated with adaptive evolution.

Novel respiratory viruses can cause a pandemic and then evolve to coexist with humans. The Omicron strain of severe acute respiratory syndrome coronavirus 2 has spread worldwide since its emergence in late 2021, and its sub-lineages are now established in human society. Compared to previous strains, Omicron is markedly less invasive in the lungs and causes less severe disease. One reason for this is that humans are acquiring immunity through previous infection and vaccination, but the nature of the virus itself is also changing. Using our newly established low-volume inoculation system, which reflects natural human infection, we show that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain. Furthermore, by characterizing chimeric viruses with the Omicron gene in the Wuhan strain genetic background and vice versa, we found that viral genes downstream of ORF3a, but not the S gene, were responsible for the limited spread of the Omicron strain in the lower airways of the virus-infected hamsters. Moreover, molecular evolutionary analysis of SARS-CoV-2 revealed a positive selection of genes downstream of ORF3a (M and E genes). Our findings provide insight into the adaptive evolution of the virus in humans during the pandemic convergence phase.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread worldwide since its emergence in late 2021, and its sub-lineages are established in human society. Compared to previous strains, the Omicron strain is less invasive in the lower respiratory tract, including the lungs, and causes less severe disease; however, the mechanistic basis for its restricted replication in the lower airways is poorly understood. In this study, using a newly established low-volume inoculation system that reflects natural human infection, we demonstrated that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain and found that viral genes downstream of ORF3a are responsible for replication restriction in the lower respiratory tract of Omicron-infected hamsters. Furthermore, we detected a positive selection of genes downstream of ORF3a (especially the M and E genes) in SARS-CoV-2, suggesting that these genes may undergo adaptive changes in humans.

Rare CRHR2 and GRM8 variants identified as candidate factors associated with eating disorders in Japanese patients by whole exome sequencing.

Eating disorders (EDs) are a type of psychiatric disorder characterized by pathological eating and related behavior and considered to be highly heritable. The purpose of this study was to explore rare variants expected to display biological functions associated with the etiology of EDs. We performed whole exome sequencing (WES) of affected sib-pairs corresponding to disease subtype through their lifetime and their parents. From those results, rare single nucleotide variants (SNVs) concordant with sib-pairs were extracted and estimated to be most deleterious in the examined families. Two non-synonymous SNVs located on corticotropin-releasing hormone receptor 2 (CRHR2) and glutamate metabotropic receptor 8 (GRM8) were identified as candidate disease susceptibility factors. The SNV of CRHR2 was included within the cholesterol binding motif of the transmembrane helix region, while the SNV of GRM8 was found to contribute to hydrogen bonds for an α-helix structure. CRHR2 plays important roles in the serotoninergic system of dorsal raphe nuclei, which is involved with feeding and stress-coping behavior, whereas GRM8 modulates glutamatergic neurotransmission. Moreover, GRM8 modulates glutamatergic neurotransmission, and is also considered to have effects on dopaminergic and adrenergic neurotransmission. Thus, identification of rare and deleterious variants in this study is expected to increase understanding and treatment of affected individuals. Further investigation regarding the biological function of these variants may provide an opportunity to elucidate the pathogenesis of EDs.

Two-step evolution of HIV-1 budding system leading to pandemic in the human population.

The pandemic HIV-1, HIV-1 group M, emerged from a single spillover event of its ancestral lentivirus from a chimpanzee. During human-to-human spread worldwide, HIV-1 diversified into multiple subtypes. Here, our interdisciplinary investigation mainly sheds light on the evolutionary scenario of the viral budding system of HIV-1 subtype C (HIV-1C), a most successfully spread subtype. Of the two amino acid motifs for HIV-1 budding, the P(T/S)AP and YPxL motifs, HIV-1C loses the YPxL motif. Our data imply that HIV-1C might lose this motif to evade immune pressure. Additionally, the P(T/S)AP motif is duplicated dependently of the level of HIV-1 spread in the human population, and >20% of HIV-1C harbored the duplicated P(T/S)AP motif. We further show that the duplication of the P(T/S)AP motif is caused by the expansion of the CTG triplet repeat. Altogether, our results suggest that HIV-1 has experienced a two-step evolution of the viral budding process during human-to-human spread worldwide.

Multiple mutations of SARS-CoV-2 Omicron BA.2 variant orchestrate its virological characteristics.

IMPORTANCE: Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.

SARS-CoV-2 HaploGraph: visualization of SARS-CoV-2 haplotype spread in Japan.

Since the early phase of the coronavirus disease 2019 (COVID-19) pandemic, a number of research institutes have been sequencing and sharing high-quality severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes to trace the route of infection in Japan. To provide insight into the spread of COVID-19, we developed a web platform named SARS-CoV-2 HaploGraph to visualize the emergence timing and geographical transmission of SARS-CoV-2 haplotypes. Using data from the GISAID EpiCoV database as of June 4, 2022, we created a haplotype naming system by determining the ancestral haplotype for each epidemic wave and showed prefecture- or region-specific haplotypes in each of four waves in Japan. The SARS-CoV-2 HaploGraph allows for interactive tracking of virus evolution and of geographical prevalence of haplotypes, and aids in developing effective public health control strategies during the global pandemic. The code and the data used for this study are publicly available at: https://github.com/ktym/covid19/.

Current trends in RNA virus detection through metatranscriptome sequencing data.

With advances in sequencing technology, metatranscriptome sequencing from a variety of environmental and biological sources has revealed the existence of various previously unknown RNA viruses. This review presents recent major RNA virome studies sampled from invertebrate and vertebrate species as well as aquatic environments. In particular, we focus on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and related RNA virus identification through metatranscriptome sequencing analyses. Recently developed bioinformatics software and databases for RNA virus identification are introduced. A relationship between newly identified RNA viruses and endogenous viral elements in host genomes is also discussed.

The hepatic niche leads to aggressive natural killer cell leukemia proliferation through the transferrin-transferrin receptor 1 axis.

Aggressive natural killer cell leukemia (ANKL) is a rare lymphoid neoplasm frequently associated with Epstein-Barr virus, with a disastrously poor prognosis. Owing to the lack of samples from patients with ANKL and relevant murine models, comprehensive investigation of its pathogenesis including the tumor microenvironment (TME) has been hindered. Here we established 3 xenograft mice derived from patients with ANKL (PDXs), which enabled extensive analysis of tumor cells and their TME. ANKL cells primarily engrafted and proliferated in the hepatic sinusoid. Hepatic ANKL cells were characterized by an enriched Myc-pathway and proliferated faster than those in other organs. Interactome analyses and in vivo CRISPR-Cas9 analyses revealed transferrin (Tf)-transferrin receptor 1 (TfR1) axis as a potential molecular interaction between the liver and ANKL. ANKL cells were rather vulnerable to iron deprivation. PPMX-T003, a humanized anti-TfR1 monoclonal antibody, showed remarkable therapeutic efficacy in a preclinical setting using ANKL-PDXs. These findings indicate that the liver, a noncanonical hematopoietic organ in adults, serves as a principal niche for ANKL and the inhibition of the Tf-TfR1 axis is a promising therapeutic strategy for ANKL.

Dynamic Evolution of Retroviral Envelope Genes in Egg-Laying Mammalian Genomes.

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

Functional molecular evolution of a GTP sensing kinase: PI5P4Kß.

Over 4 billion years of evolution, multiple mutations, including nucleotide substitutions, gene and genome duplications and recombination, have established de novo genes that translate into proteins with novel properties essential for high-order cellular functions. However, molecular processes through which a protein evolutionarily acquires a novel function are mostly speculative. Recently, we have provided evidence for a potential evolutionary mechanism underlying how, in mammalian cells, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) evolved into a GTP sensor from ATP-utilizing kinase. Mechanistically, PI5P4Kß has acquired the guanine efficient association (GEA) motif by mutating its nucleotide base recognition sequence, enabling the evolutionary transition from an ATP-dependent kinase to a distinct GTP/ATP dual kinase with its KM for GTP falling into physiological GTP concentrations-the genesis of GTP sensing activity. Importantly, the GTP sensing activity of PI5P4Kß is critical for the manifestation of cellular metabolism and tumourigenic activity in the multicellular organism. The combination of structural, biochemical and biophysical analyses used in our study provides a novel framework for analysing how a protein can evolutionarily acquire a novel activity, which potentially introduces a critical function to the cell.

Genomic diversity of SARS-CoV-2 can be accelerated by mutations in the nsp14 gene.

Coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a proofreading exonuclease, nonstructural protein 14 (nsp14), that helps ensure replication competence at a low evolutionary rate compared with other RNA viruses. In the current pandemic, SARS-CoV-2 has accumulated diverse genomic mutations including in nsp14. Here, to clarify whether amino acid substitutions in nsp14 affect the genomic diversity and evolution of SARS-CoV-2, we searched for amino acid substitutions in nature that may interfere with nsp14 function. We found that viruses carrying a proline-to-leucine change at position 203 (P203L) have a high evolutionary rate and that a recombinant SARS-CoV-2 virus with the P203L mutation acquired more diverse genomic mutations than wild-type virus during its replication in hamsters. Our findings suggest that substitutions, such as P203L, in nsp14 may accelerate the genomic diversity of SARS-CoV-2, contributing to virus evolution during the pandemic.

Delivering mRNAs to mouse tissues using the SEND system.

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo . Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

Characterization of Megabat-Favored, CA-Dependent Susceptibility to Retrovirus Infection.

The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.

Nanopore Sequencing Data Analysis of 16S rRNA Genes Using the GenomeSync-GSTK System.

With the development of nanopore sequencing technology, long reads of DNA sequences can now be determined rapidly from various samples. This protocol introduces the GenomeSync-GSTK system for bacterial species identification in a given sample using nanopore sequencing data of 16S rRNA genes as an example. GenomeSync is a collection of genome sequences designed to provide easy access to genomic data of the species as demanded. GSTK (genome search toolkit) is a set of scripts for managing local homology searches using genomes obtained from the GenomeSync database. Based on this protocol, nanopore sequencing data analyses of metagenomes and amplicons could be efficiently performed. We also noted reanalysis in conjunction with future developments in nanopore sequencing technology and the accumulation of genome sequencing data.

Potentially reduced fusogenicity of syncytin-2 in New World monkeys.

Syncytin-2 is a membrane fusion protein involved in placenta development that is derived from the endogenous retrovirus envelope gene acquired in the common ancestral lineage of New World and Old World monkeys (OWMs). It is known that syncytin-2 is conserved between apes and OWMs, suggesting its functional importance; however, syncytin-2 of common marmosets (Callithrix jacchus) exhibits lower fusogenic activity than those of humans and OWMs in human cell lines. To obtain insight into the functional diversity of syncytin-2 genes in primates, we examined the syncytin-2 gene in New World monkeys (NWMs). We experimentally evaluated the cell fusion ability of syncytin-2 in humans, C. jacchus, and tufted capuchins (Sapajus apella). We found that the cell fusion ability of S. apella was lower than that of human syncytin-2. Chimeric syncytin-2 constructs revealed that the amino acid differences in the surface unit of S. apella syncytin-2 were responsible for the weak cell fusion activity. In addition, genomic sequence analyses of syncytin-2 revealed that the open reading frames (ORFs) of syncytin-2 were highly conserved in seven apes and 22 OWMs; however, the syncytin-2 ORFs of three of 12 NWM species were truncated. Our results suggest that syncytin-2 in several NWMs may be of less importance than in OWMs and apes, and other syncytin-like genes may be required for placental development in various NWM species.

The SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant.

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.

Monotreme-specific conserved putative proteins derived from retroviral reverse transcriptase.

Endogenous retroviruses (ERVs) have played an essential role in the evolution of mammals. ERV-derived genes are reported in the therians, many of which are involved in placental development; however, the contribution of the ERV-derived genes in monotremes, which are oviparous mammals, remains to be uncovered. Here, we conducted a comprehensive search for possible ERV-derived genes in platypus and echidna genomes and identified three reverse transcriptase-like genes named RTOM1, RTOM2, and RTOM3 clustered in the GRIP2 intron. Comparative genomic analyses revealed that RTOM1, RTOM2, and RTOM3 are strongly conserved and are under purifying selection between these species. These could be generated by tandem duplications before the divergence of platypus and echidna. All RTOM transcripts were specifically expressed in the testis, possibly suggesting their physiological importance. This is the first study reporting monotreme-specific de novo gene candidates derived from ERVs, which provides new insights into the unique evolution of monotremes.