Background and ObjectiveAllergic fungal airway diseases caused by environmental fungal exposure are often difficult to manage with medical therapy alone. We performed a pilot study to investigate fungal load and diversity recontaminating ductless minisplit air conditioners (ACs) within 1 year of high-pressure washing.MethodsFollow-up surveys of fungi on the internal parts of AC, AC cleaning effluent, indoor air, and house dust were conducted 1 year after high-pressure AC cleaning in 17 Japanese residences using quantitative PCR, mycobiome analysis, and culturing. The AC operating rates during summer were estimated based on the temperature and humidity in the AC air vents and the room.ResultsOne year after high-pressure AC cleaning, the estimated fungal genome copy numbers in the internal AC parts returned to prewashing levels. AC operating rates during the summer correlated significantly with fungal burden in AC effluents (rho = 0.60, p = 0.01), modestly with those in indoor air (rho = 0.48, p = 0.05), but not with house dust. beta-diversities of mycobiome in AC cleaning effluent, indoor air, and house dust were distinct (p = 0.001). The frequency of AC use was associated with the proportions of Ascomycota in AC effluent (p = 0.009) and indoor air (p = 0.02), whereas house dust communities remained consistent regardless of AC use. Fungal cultures at 37 degrees C identified viable fungi, including Aspergillus spp., in AC.ConclusionACs can be substantially recontaminated 1 year after cleaning, underscoring the importance of regular AC maintenance to prevent indoor fungal exposure.

It is known that several endogenous retroviruses, remnants of ancient retroviral integrations, retain envelope (env) genes encoding fusogenic proteins in primates. Whilst most env genes are degraded, a few have been co-opted by hosts, yet the entire evolutionary dynamics of env sequences remain poorly understood. To explore this, we screened and compared env open reading frames (ORFs) from 247 primate genomes. In total, 8683 nearly intact env-ORFs encoding 400 or more amino acids were identified, and their copy numbers ranged from 3 to 429 across primate species. Sequence similarity clustering revealed a clear evolutionary signature that distinguishes the small set of long-term co-opted env-derived genes, which are retained at low copy numbers across lineages, from the broader pool of rapidly turning-over env-ORFs. Applying this framework, we identified a previously unrecognized, single-copy env ortholog conserved across Tarsiiformes, named env-Tar1, and experimentally demonstrated its cell fusion activity in vitro, providing the first functional evidence of an env-derived gene co-opted in a tarsier lineage. Further, we found that certain co-opted env-derived genes may have lost their functions due to nonsense or indel mutations within specific primate lineages. Considering that many env genes tend to be maintained at low copy numbers and are reported to be under purifying selection, such dynamic evolutionary turnover of env-derived genes may be driven by host-virus arms races, as viruses and endogenous retroviruses often share cell-surface receptors.

Understanding the genetic diversity and evolutionary history of domestic horses (Equus caballus) is essential for reconstructing their population dynamics and origins. In this study, we analyzed mitochondrial DNA variation (COI and cytb) in six Kazakhstani horse populations representing four native breeds (Kazakh, Kostanay, Adai, and Mugalzhar) to assess genetic diversity, population structure, and evolutionary relationships within a broader phylogenetic framework. High haplotype diversity combined with low nucleotide diversity revealed population expansion and admixture. Population structure analyses revealed weak genetic differentiation and a lack of breed-specific structuring, with most variation occurring within populations. Phylogenetic reconstruction and haplotype network analyses showed that Kazakhstani horses are interspersed among global domestic lineages, reflecting extensive historical connectivity and admixture. Demographic analyses based on neutrality tests and mismatch distributions support signals of ancient population expansion, while the multimodal distribution patterns suggest a complex demographic history involving population substructure and multiple expansion events rather than a single, sudden expansion. Times since expansion were calculated to be around 205 Kya. Divergence time estimates place the diversification of all caballine horses, including Kazakhstani horses, within the Pleistocene (0.89 Mya) with the majority of them grouping with different horse breeds across the world. These results indicate that the maternal genetic structure of Kazakhstani horses has been shaped by a combination of ancient evolutionary processes and more recent demographic dynamics, including recurrent gene flow across Eurasian steppe environments. Overall, this study highlights a reticulate evolutionary history of domestic horses, emphasizing the role of long-term connectivity and population expansion in shaping mitochondrial diversity.

Given the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continuous analysis of its genomic variations at the nucleotide level is imperative to monitor the emergence of novel variants of concern. The Global Initiative on Sharing All Influenza Data (GISAID) serves as the de facto standard database for the genomic information of SARS-CoV-2. However, limitations of its data-sharing policy hinder the comprehensive analysis of genomic variations. To address this problem, we developed SGV-caller, a bioinformatics pipeline for analyzing the frequently updated GISAID database. SGV-caller compares input datasets with pre-existing databases and generates local databases encompassing nucleotide, amino acid, and codon-level genomic variations for each SARS-CoV-2 genome. Furthermore, SGV-caller accommodates SARS-CoV-2 genomes from non-GISAID sources as well as other viral genomes. SGV-caller source code and test data are available at https://github.com/wujiaqi06/SGV-caller.

Finding early trigger genes involved in cell-fate-determining processes is important for understanding molecular mechanisms of, e.g., differentiation and disease progression. One of the powerful tools for the finding is hypothesis-free omics measurements, e.g., gene expression analysis (transcriptome analysis) by RNA sequencing (RNA-seq). However, because whole single-cell RNA-seq requires cell disruption and the fate of the disrupted cell is generally unknown, it is difficult to find fate-related genes by single-cell RNA-seq profiles, especially in the early stages of cell-fate determination. Meanwhile, deep learning has successfully predicted cell fates using individual cell images. Here, we developed an approach by integrating image-based cell-fate prediction using deep learning and single-cell whole-transcriptome analysis to find differentially expressed genes (DEGs) between different predicted fates. As a proof of principle, we applied this approach to cells fated to die and survive. First, we applied temporary heat stress to a mammalian cell line to induce a certain fraction of cells to die, and performed time-lapse imaging to observe this process. Second, we made image-based deep learning models trained with our dataset for the cell fate prediction (survival and death). Third, we picked the cells after another time-lapse imaging and performed single-cell RNA-seq. Finally, we compared the transcriptomes between cells predicted to die and survive. We successfully detected the DEGs when the transcriptomic profiles did not show clear multiple clusters that may correspond to the heat-induced different fates in a dimension-reduced plane. Our approach may contribute to a deeper understanding of cell-fate regulation and new molecular marker detection.

Probiotics refer to living microorganisms that exert a variety of beneficial effects on human health. On the contrary, they also can cause infection, produce toxins within the body, and transfer antibiotic-resistant genes to the other microorganisms in the digestive tract necessitating a comprehensive safety assessment. This study aimed to conduct functional genomic analysis and some relevant biochemical tests to uncover the probiotic potentials of Lactobacillus delbrueckii subsp. indicus TY-11 isolated from native yogurt in Bangladesh. We also performed transmission electron microscopic (TEM) analysis, comparative genomic study as well as phylogenetic tree construction with 332 core genes from 262 genomes. The strain TY-11 was identified as Lactobacillus delbrueckii subsp. indicus, whose genome (1,916,674 bp) contained 1911 CDS, and no gene was identified for either antibiotic resistance or toxic metabolites. It carried genes for the degradation of toxic metabolites, treatment of lactose intolerance, toll-like receptor 2-dependent innate immune response, heat and cold shock, bile salts tolerance, and acidic pH tolerance. Genes were annotated for inhibiting pathogenic bacteria by inhibitory substances [bacteriocin: Helveticin-J (331 bp) and Enterolysin-A (275 bp), hydrogen peroxide, and acid]; blockage of adhesion sites; and competition for nutrients. The genes involved in its metabolic pathway were detected as suitable for digesting indigestible nutrients in the human gut. The TY-11 genome possessed an additional 37 core genes of subspecies indicus which were deficient in the core genome of the most popular subsp. bulgaricus. During the phenotypic testing, the isolate TY-11 demonstrated high antagonistic activity (inhibition zone of 21.33 ± 1.53 mm) against Escherichia coli ATCC 8739 and was not sensitive to any of the 10 tested antibiotics. This study was the first study to explore the molecular insights into probiotic roles, including antimicrobial activities and antibiotic sensitivity, of a representative strain (TY-11) of Lactobacillus delbrueckii subsp. indicus. IMPORTANCE:This study aimed to conduct functional genomic analysis to uncover the probiotic potential of Lactobacillus delbrueckii subsp. indicus TY-11 isolated from native yogurt in Bangladesh. We also performed transmission electron microscopic (TEM) analysis, comparative genomic study as well as phylogenetic tree construction with 332 core genes from 262 genomes. In our current investigation, we revealed a number of common and unique excellences of the probiotic Lactobacillus delbrueckii subsp. indicus TY-11 that are likely to be important to illustrate its intestinal residence and probiotic roles. This is the first study to explore the molecular insights into intestinal residence and probiotic roles, including antimicrobial activities and antibiotic sensitivity, of a representative strain (TY-11) of Lactobacillus delbrueckii subsp. indicus.

Partial separation of a peripheral population may lead to its divergence and, potentially, speciation due to genetic drift followed by selection and geographic isolation. This process may cause taxonomic uncertainty because reproductive isolation in allopatry cannot be verified directly. The two Nearctic allopatric species of magpies (Aves, Corvidae: Pica) serve as a good example of these problems. The Black-billed magpie Pica hudsonia is widely distributed in North America, whereas the Yellow-billed Magpie Pica nuttalli is endemic to a restricted range in California. Their relationships with Palearctic species have been little studied. We obtained complete mitochondrial genomes of both Nearctic magpie species, along with the Eurasian Magpie (Pica pica) and the Oriental Magpie (Pica serica), 20 mitogenomes in total. Phylogenetic analysis reveals a basal position of P. serica, and P. pica as a sister clade to the two Nearctic species. P. hudsonia and P. nuttalli form reciprocal monophyletic subclades, showing recent divergence between and within them. Our data show that the Nearctic magpie lineage diverged from the common ancestor with P. pica, with a single migration wave via the Beringia. Within the Nearctic, we hypothesize a peripatric mode of speciation among Pica taxa due to the divergence and separation of the small marginal population in California below the Sierra-Nevada mountains. Diversifying amino acid substitutions in ND4-ND5-ND6 genes along the branch leading to the New World clade may indicate selection for heat-tolerance. Considering the clear phenotypic differences between P. hudsonia and P. nuttalli, our data, showing their reciprocal monophylies and genetic distinctness, is consistent with the two-species taxonomy.

Improvements in DNA sequencing technology are allowing the dramatic increase of whole genome data for a wide variety of species. Such genome sequence data can assist the monitoring of intraspecific genetic diversity, but is often lacking for threatened species. In this project, we focused on the national Red List, a catalog of extinct and threatened species, issued by the Japanese government. We combined the data included in it with the record of genome assembly in NCBI and tabulated the assembly availability of the species in the list. The combined data shows a low percentage (2.1%) of the availability of whole genome sequence data for the taxa ranked on the Japanese Red List as well as a strong bias towards mammals and birds in Animalia and vascular plants in Plantae. Our data presentation highlights potential systematic limitations in genome sequencing (e.g., budget for sequencing large genomes of amphibians) and instructs future policies including which taxon needs more effort for genome sequencing. The resultant tables are available in the original website https://treethinkers.nig.ac.jp/redlist/ and are regularly updated.

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.

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.

We conducted a global-scale study to identify H. pylori antimicrobial-resistant genes (ARG), address their global distribution, and understand their effect on the antimicrobial resistance (AMR) phenotypes of the clinical isolates. We identified ARG using several well-known tools against extensive bacterial ARG databases, then analyzed their correlation with clinical antibiogram data from dozens of patients across countries. This revealed that combining multiple tools and databases, followed by manual selection of ARG from the annotation results, produces more conclusive results than using a single tool or database alone. After curation, the results showed that H. pylori has 42 ARG against 11 different antibiotic classes (16 genes related to single antibiotic class resistance and 26 genes related to multidrug resistance). Further analysis revealed that H. pylori naturally harbors ARG in the core genome, called the 'Set of ARG commonly found in the Core Genome of H. pylori (ARG-CORE)', while ARG-ACC-the ARG in the accessory genome-are exclusive to particular strains. In addition, we detected 29 genes of potential efflux pump-related AMR that were mostly categorized as ARG-CORE. The ARG distribution appears to be almost similar either by geographical or H. pylori populations perspective; however, some ARG had a unique distribution since they tend to be found only in a particular region or population. Finally, we demonstrated that the presence of ARG may not directly correlate with the sensitive/resistance phenotype of clinical patient isolates but may influence the minimum inhibitory concentration phenotype.

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.

Background Probiotics refer to living microorganisms that exerts a variety of beneficial effects on human health. On the contrary, they also can cause infection, produce toxins within the body, and transfer antibiotic resistant genes to the other microorganisms in the digestive tract necessitating a comprehensive safety assessment. This study aimed to conduct functional genomic analysis to uncover the probiotic potential of Lactobacillus delbrueckii subsp. indicus TY-11 isolated from native yogurt in Bangladesh. We also performed Transmission Electron Microscopic (TEM) analysis, comparative genomic study as well as phylogenetic tree construction with 332 core genes from 262 genomes. These experiments on subspecies indicus were not studied previously.Results The strain TY-11 was identified as Lactobacillus delbrueckii subsp. indicus, whose genome (1916674bp) contained genes to adapt to diverse and stressful environments of the human gut, and no gene was identified for either antibiotic resistance or toxic metabolites. It embraced genes for the degradation of toxic metabolites, treatment of lactose intolerance, toll-like receptor 2-dependent innate immune response, heat and cold shock, bile salts tolerance in the gut, and acidic pH tolerance in the stomach. Genes were annotated for inhibiting pathogenic bacteria in the gut by inhibitory substances, (bacteriocin: Helveticin-J (331bp) and Enterolysin-A (275bp), hydrogen peroxide, and acid); blockage of adhesion sites; and competition for nutrients. Its metabolic pathway helped the human gut to recover the digestive disorders and digest indigestible nutrients. The TY-11 genome possessed additional 37 core genes of subspecies indicus which were deficient in the core genome of the most popular subspecies bularicus .Conclusions This is the first study to explore the molecular insights into intestinal residence and probiotic roles including antimicrobial activities of a representative strain (TY-11) of Lactobacillus delbrueckii subsp. indicus .

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/.

A virulence bacterium, Helicobacter pylori, evolved parallel to its host human, therefore, can work as a marker for tracing the human migration. We found H. pylori strains indigenous in the southernmost islands of Japanese Archipelago, Okinawa, and defined them as hspOkinawa and hpRyukyu. Genome data of the strains revealed that hspOkinawa diverged from other East Asian strains about 20,000 years ago, and that hpRyukyu diverged about 45,000 years ago. The closest strains of hpRyukyu were found from Afghanistan, Punjab, and Nepal, which suggest this strain originated in the central Asia and traveled across the Eurasian continent during Paleolithic era. The divergence date of hpRyukyu corresponds with human fossil records in Okinawa. Although it is controversial from human DNA analyses whether descendants of the Paleolithic migrants remain in the modern Japanese population, this study reveals that the bacterium of Paleolithic origin remains in the stomachs of current Japanese.

Single-cell whole-transcriptome analysis is the gold standard approach to identifying molecularly defined cell phenotypes. However, this approach cannot be used for dynamics measurements such as live-cell imaging. Here, we developed a multifunctional robot, the automated live imaging and cell picking system (ALPS) and used it to perform single-cell RNA sequencing for microscopically observed cells with multiple imaging modes. Using robotically obtained data that linked cell images and the whole transcriptome, we successfully predicted transcriptome-defined cell phenotypes in a noninvasive manner using cell image-based deep learning. This noninvasive approach opens a window to determine the live-cell whole transcriptome in real time. Moreover, this work, which is based on a data-driven approach, is a proof of concept for determining the transcriptome-defined phenotypes (i.e., not relying on specific genes) of any cell from cell images using a model trained on linked datasets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome data are essential for epidemiology, vaccine development, and tracking emerging variants. Millions of SARS-CoV-2 genomes have been sequenced during the pandemic. However, downloading SARS-CoV-2 genomes from databases is slow and unreliable, largely due to suboptimal choice of compression method. We evaluated the available compressors and found that Nucleotide Archival Format (NAF) would provide a drastic improvement compared with current methods. For Global Initiative on Sharing Avian Flu Data’s (GISAID) pre-compressed datasets, NAF would increase efficiency 52.2 times for gzip-compressed data and 3.7 times for xz-compressed data. For DNA DataBank of Japan (DDBJ), NAF would improve throughput 40 times for gzip-compressed data. For GenBank and European Nucleotide Archive (ENA), NAF would accelerate data distribution by a factor of 29.3 times compared with uncompressed FASTA. This article provides a tutorial for installing and using NAF. Offering a NAF download option in sequence databases would provide a significant saving of time, bandwidth, and disk space and accelerate biological and medical research worldwide.

Background It has been suggested that the local microbiota in the reproductive organs is relevant to women's health and may also affect pregnancy outcomes. Analysis of partial 16S ribosomal RNA (rRNA) gene sequences generated by short-read sequencers has been used to identify vaginal and endometrial microbiota, but it requires a long time to obtain the results, making it unsuitable for rapid bacterial identification from a small specimen amount in a clinical context. Methods We developed a simple workflow using the nanopore sequencer MinION that allows high-resolution and rapid differentiation of vaginal microbiota. Vaginal samples collected from 18 participants were subjected to DNA extraction and full-length 16S rRNA gene sequencing with MinION. Results The principal coordinate analysis showed no differences in the bacterial compositions regardless of the sample collection method. The analysis of vaginal microbiota could be completed with a total analysis time of approximately four hours, allowing same-day results. Taxonomic profiling by MinION sequencing revealed relatively low diversity of the vaginal bacterial community, identifying the prevailing Lactobacillus species and several causative agents of bacterial vaginosis. Conclusions Full-length 16S rRNA gene sequencing analysis with MinION provides a rapid means for identifying vaginal bacteria with higher resolution. Species-level profiling of human vaginal microbiota by MinION sequencing can allow the analysis of associations with conditions such as genital infections, endometritis, and threatened miscarriage.