Chromosome-level genome assemblies of Cutaneotrichosporon spp. (Trichosporonales, Basidiomycota) reveal imbalanced evolution between nucleotide sequences and chromosome synteny.

BACKGROUND: Since DNA information was first used in taxonomy, barcode sequences such as the internal transcribed spacer (ITS) region have greatly aided fungal identification; however, a barcode sequence alone is often insufficient. Thus, multi-gene- or whole-genome-based methods were developed. We previously isolated Basidiomycota yeasts classified in the Trichosporonales. Some strains were described as Cutaneotrichosporon cavernicola and C. spelunceum, whereas strain HIS471 remained unidentified. We analysed the genomes of these strains to elucidate their taxonomic relationship and genetic diversity. RESULTS: The long-read-based assembly resulted in chromosome-level draft genomes consisting of seven chromosomes and one mitochondrial genome. The genome of strain HIS471 has more than ten chromosome inversions or translocations compared to the type strain of C. cavernicola despite sharing identical ITS barcode sequences and displaying an average nucleotide identity (ANI) above 93%. Also, the chromosome synteny between C. cavernicola and the related species, C. spelunceum, showed significant rearrangements, whereas the ITS sequence identity exceeds 98.6% and the ANI is approximately 82%. Our results indicate that the relative evolutionary rates of barcode sequences, whole-genome nucleotide sequences, and chromosome synteny in Cutaneotrichosporon significantly differ from those in the model yeast Saccharomyces. CONCLUSIONS: Our results revealed that the relative evolutionary rates of nucleotide sequences and chromosome synteny are different among fungal clades, likely because different clades have diverse mutation/repair rates and distinct selection pressures on their genomic sequences and syntenic structures. Because diverse syntenic structures can be a barrier to meiotic recombination and may lead to speciation, the non-linear relationships between nucleotide and synteny diversification indicate that sequence-level distances at the barcode or whole-genome level are not sufficient for delineating species boundaries.

Hyphal Growth in Trichosporon asahii Is Accelerated by the Addition of Magnesium.

Fungal dimorphism involves two morphologies: a unicellular yeast cell and a multicellular hyphal form. Invasion of hyphae into human cells causes severe opportunistic infections. The transition between yeast and hyphal forms is associated with the virulence of fungi; however, the mechanism is poorly understood. Therefore, we aimed to identify factors that induce hyphal growth of Trichosporon asahii, a dimorphic basidiomycete that causes trichosporonosis. T. asahii showed poor growth and formed small cells containing large lipid droplets and fragmented mitochondria when cultivated for 16 h in a nutrient-deficient liquid medium. However, these phenotypes were suppressed via the addition of yeast nitrogen base. When T. asahii cells were cultivated in the presence of different compounds present in the yeast nitrogen base, we found that magnesium sulfate was a key factor for inducing cell elongation, and its addition dramatically restored hyphal growth in T. asahii. In T. asahii hyphae, vacuoles were enlarged, the size of lipid droplets was decreased, and mitochondria were distributed throughout the cell cytoplasm and adjacent to the cell walls. Additionally, hyphal growth was disrupted due to treatment with an actin inhibitor. The actin inhibitor latrunculin A disrupted the mitochondrial distribution even in hyphal cells. Furthermore, magnesium sulfate treatment accelerated hyphal growth in T. asahii for 72 h when the cells were cultivated in a nutrient-deficient liquid medium. Collectively, our results suggest that an increase in magnesium levels triggers the transition from the yeast to hyphal form in T. asahii. These findings will support studies on the pathogenesis of fungi and aid in developing treatments. IMPORTANCE Understanding the mechanism underlying fungal dimorphism is crucial to discern its invasion into human cells. Invasion is caused by the hyphal form rather than the yeast form; therefore, it is important to understand the mechanism of transition from the yeast to hyphal form. To study the transition mechanism, we utilized Trichosporon asahii, a dimorphic basidiomycete that causes severe trichosporonosis since there are fewer studies on T. asahii than on ascomycetes. This study suggests that an increase in Mg2+, the most abundant mineral in living cells, triggers growth of filamentous hyphae and increases the distribution of mitochondria throughout the cell cytoplasm and adjacent to the cell walls in T. asahii. Understanding the mechanism of hyphal growth triggered by Mg2+ increase will provide a model system to explore fungal pathogenicity in the future.

[Malassezia Display a Hyphae-like "Spaghetti-and-Meatballs" Configuration in Keratotic Plugs].

Malassezia are lipophilic yeasts in the skin microbiome that abundantly colonize all parts of human skin except for the soles of the feet. Fungal microbiome analysis of keratotic plugs from the noses of 10 healthy individuals identified Malassezia restricta as the predominant species, followed by Malassezia globosa. Malassezia hyphae were observed in 5 of the 10 individuals. The hyphae were curved and thick-walled with spherical thick-walled and grouped blastoconidia, described as a "spaghetti-and-meatballs" configuration. In this study, we observed Malassezia hyphae in keratotic plugs of healthy subjects, although abundant Malassezia hyphae have previously only been observed in lesional sites of patients with pityriasis versicolor.

Draft Genome Sequence of Pandoravirus japonicus Isolated from the Sabaishi River, Niigata, Japan.

"Pandoraviridae" is a proposed family of the phylum Nucleocytoviricota Its features include an amphora-shaped capsid and the largest genome among all viruses. We report the isolation and genome sequencing of a new member of this family, named Pandoravirus japonicus, the third strain discovered in Japan.

Co-continuous structural effect of size-controlled macro-porous glass membrane on extracellular vesicle collection for the analysis of miRNA.

Recent studies have shown that extracellular vesicles (EVs) can be utilized as appropriate and highly specific biomarkers in liquid biopsy for the diagnosis and prognosis of serious illness. However, there are few methods that can collect and isolate miRNA in EVs simply, quickly and efficiently using general equipment such as a normal centrifuge. In this paper, we developed an advanced glass membrane column (AGC) device incorporating a size-controlled macro-porous glass (MPG) membrane with a co-continuous structure to overcome the limitations of conventional EV collection and miRNA extraction from the EVs. The size of macro-pores in the MPG membrane could be accurately controlled by changing the heating temperature and time on the basis of spinodal decomposition of B2O3, Na2O, and SiO2 in phase separation. The AGC device with an MPG membrane could collect the EVs simply and quickly (< 10 min) from cell culture supernatant, serum and urine. This AGC device could extract miRNA from the EVs captured in the MPG membrane with high efficiency when combined with a miRNA extraction solution. We suggest that the AGC device with an MPG membrane can be useful for the diagnosis and prognosis of serious illness using of EVs in various kinds of body fluids.

Marseilleviridae Lineage B Diversity and Bunch Formation Inhibited by Galactose.

Marseilleviridae is a family of large double-stranded DNA viruses that is currently divided into five subgroups, lineages A-E. Hokutovirus and kashiwazakivirus, both of which belong to lineage B, have been reported to induce host acanthamoeba cells to form aggregations called "bunches". This putatively results in increased opportunities to infect acanthamoeba cells, in contrast to lineage A, which has been reported to not form "bunches". In the present study, we isolated 14 virus strains of the family Marseilleviridae from several Japanese water samples, 11 of which were identified as lineage B viruses. All 11 lineage B strains caused infected amoeba cells to form bunches. We then investigated the involvement of monosaccharides in bunch formation by amoeba cells infected with hokutovirus. Galactose inhibited bunch formation, thereby allowing amoeba cells to delay the process, whereas mannose and glucose did not. A kinetic image analysis of hokutovirus-infected amoeba cells confirmed the inhibition of bunch formation by galactose. The number of hokutovirus-infected amoeba cells increased more rapidly than that of tokyovirus-infected cells, which belongs to lineage A. This result suggests that bunch formation by infected amoeba cells is advantageous for lineage B viruses.

Genetic Diversity and Phylogeography of Thottapalayam thottimvirus (Hantaviridae) in Asian House Shrew (Suncus murinus) in Eurasia.

Murid and cricetid rodents were previously believed to be the principal reservoir hosts of hantaviruses. Recently, however, multiple newfound hantaviruses have been discovered in shrews, moles, and bats, suggesting a complex evolutionary history. Little is known about the genetic diversity and geographic distribution of the prototype shrew-borne hantavirus, Thottapalayam thottimvirus (TPMV), carried by the Asian house shrew (Suncus murinus), which is widespread in Asia, Africa, and the Middle East. Comparison of TPMV genomic sequences from two Asian house shrews captured in Myanmar and Pakistan with TPMV strains in GenBank revealed that the Myanmar TPMV strain (H2763) was closely related to the prototype TPMV strain (VRC66412) from India. In the L-segment tree, on the other hand, the Pakistan TPMV strain (PK3629) appeared to be the most divergent, followed by TPMV strains from Nepal, then the Indian-Myanmar strains, and finally TPMV strains from China. The Myanmar strain of TPMV showed sequence similarity of 79.3-96.1% at the nucleotide level, but the deduced amino acid sequences showed a high degree of conservation of more than 94% with TPMV strains from Nepal, India, Pakistan, and China. Cophylogenetic analysis of host cytochrome b and TPMV strains suggested that the Pakistan TPMV strain was mismatched. Phylogenetic trees, based on host cytochrome b and cytochrome c oxidase subunit I genes of mitochondrial DNA, and on host recombination activating gene 1 of nuclear DNA, suggested that the Asian house shrew and Asian highland shrew (Suncus montanus) comprised a species complex. Overall, the geographic-specific clustering of TPMV strains in Asian countries suggested local host-specific adaptation. Additional in-depth studies are warranted to ascertain if TPMV originated in Asian house shrews on the Indian subcontinent.

DNA replication machinery prevents Rad52-dependent single-strand annealing that leads to gross chromosomal rearrangements at centromeres.

Homologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). At fission yeast centromeres, Rad51-dependent conservative recombination predominantly occurs between inverted repeats, thereby suppressing formation of isochromosomes whose arms are mirror images. However, it is unclear how GCRs occur in the absence of Rad51 and how GCRs are prevented at centromeres. Here, we show that homology-mediated GCRs occur through Rad52-dependent single-strand annealing (SSA). The rad52-R45K mutation, which impairs SSA activity of Rad52 protein, dramatically reduces isochromosome formation in rad51 deletion cells. A ring-like complex Msh2-Msh3 and a structure-specific endonuclease Mus81 function in the Rad52-dependent GCR pathway. Remarkably, mutations in replication fork components, including DNA polymerase α and Swi1/Tof1/Timeless, change the balance between Rad51-dependent recombination and Rad52-dependent SSA at centromeres, increasing Rad52-dependent SSA that forms isochromosomes. Our results uncover a role of DNA replication machinery in the recombination pathway choice that prevents Rad52-dependent GCRs at centromeres.

Author Correction: Dakrông virus, a novel mobatvirus (Hantaviridae) harbored by the Stoliczka's Asian trident bat (Aselliscus stoliczkanus) in Vietnam.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanism of pancreatic juice reflux in pancreaticobiliary maljunction: A fluid dynamics model experiment.

BACKGROUND: Pancreatic juice reflux to the common bile duct and gallbladder is observed in the pancreaticobiliary maljunction (PBM), and various pathological conditions occur in the biliary tract. However, the mechanism of pancreatic juice reflux has not been discussed yet. This study aimed to investigate the mechanism of this phenomenon from the perspective of the fluid dynamics theory. METHODS: A fluid dynamics model of PBM without biliary dilatation having gallbladder function and of the pressure of sphincter of Oddi was developed. Water (as bile juice and pancreatic juice) was flowed to these models with a flow rate similar to that in humans. Pancreatic and bile juice flow and bile duct pressure were observed in three phases of gallbladder function. Moreover, the same experiment was performed in the PBM without biliary dilatation model without gallbladder. RESULTS: Pancreatic juice reflux could be observed when the gallbladder was passively expanded with the pressure in the bile duct lower than that in the sphincter of Oddi. However, pancreatic juice reflux was not observed in the model without gallbladder. CONCLUSIONS: Gallbladder function may be strongly involved in pancreatic juice reflux in PBM without biliary dilatation. Cholecystectomy may be able to stop the reflux of pancreatic juice.

Dakrông virus, a novel mobatvirus (Hantaviridae) harbored by the Stoliczka's Asian trident bat (Aselliscus stoliczkanus) in Vietnam.

The recent discovery of genetically distinct shrew- and mole-borne viruses belonging to the newly defined family Hantaviridae (order Bunyavirales) has spurred an extended search for hantaviruses in RNAlater®-preserved lung tissues from 215 bats (order Chiroptera) representing five families (Hipposideridae, Megadermatidae, Pteropodidae, Rhinolophidae and Vespertilionidae), collected in Vietnam during 2012 to 2014. A newly identified hantavirus, designated Dakrông virus (DKGV), was detected in one of two Stoliczka's Asian trident bats (Aselliscus stoliczkanus), from Dakrông Nature Reserve in Quang Tri Province. Using maximum-likelihood and Bayesian methods, phylogenetic trees based on the full-length S, M and L segments showed that DKGV occupied a basal position with other mobatviruses, suggesting that primordial hantaviruses may have been hosted by ancestral bats.

Fifteen Marseilleviruses Newly Isolated From Three Water Samples in Japan Reveal Local Diversity of Marseilleviridae.

The family Marseilleviridae, defined as a group of icosahedral double-stranded DNA viruses with particle size of approximately 250 nm and genome size of 350-380 kbp, belongs to the nucleo-cytoplasmic family of large DNA viruses. The family Marseilleviridae is currently classified into lineages A-E. In this study, we isolated 12 or 15 new members of the family Marseilleviridae from three sampling locations in Japan. Molecular phylogenetic analysis of the MCP genes showed that the new viruses could be further classified into three groups, hokutoviruses, kashiwazakiviruses, and kyotoviruses. Hokutoviruses were closely related to lineage B, kyotoviruses were related to lineage A, and kashiwazakiviruses were also classified into lineage B but a new putative subgroup of lineage B, revealing the diversity of this lineage. Interestingly, more than two viruses with slightly different MCP genes were isolated from a single water sample from a single location, i.e., two hokutoviruses and one kashiwazakivirus were isolated from a small reservoir, five kashiwazakiviruses from the mouth of a river, and five kyotoviruses from fresh water of a river, suggesting that several milliliters of water samples contain several types of giant viruses. Amoeba cells infected with hokutoviruses or kashiwazakiviruses exhibited a "bunch" formation consisting of normal and infected cells similarly to a tupanvirus, whereas cells infected with kyotoviruses or tokyovirus did not. These results suggest the previously unrecognized local diversity of the family Marseilleviridae in aquatic environments.

Visualization of giant virus particles and development of "VIRAMOS" for high school and university biology course.

Several educational trials on handling viruses and or virology have been reported. However, given their small size, direct visualization of these viruses under a microscope has been rarely performed. The so-called "giant viruses" are larger than other viruses with a particle size greater than 200-300 nm. This enables their direct visualization under a light microscope more easily than other viruses. In this study, we developed two new types of teaching material for learning about viruses and cellular organisms using mimivirus, one of the well-known giant viruses. One teaching material involves using glass slides with enclosed mimivirus particles, and another is a paper-based teaching material, named VIRAMOS (http://tlab-edusys.azurewebsites.net/content/viramos_en.pdf). Using these, students can investigate and learn about viruses and cellular organisms. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):426-431, 2019.

Molecular Phylogeny of Mobatviruses (Hantaviridae) in Myanmar and Vietnam.

The discovery of highly divergent lineages of hantaviruses (family Hantaviridae) in shrews, moles, and bats of multiple species raises the possibility that non-rodent hosts may have played a significant role in their evolutionary history. To further investigate this prospect, total RNA was extracted from RNAlater®-preserved lung tissues of 277 bats (representing five families, 14 genera and 40 species), captured in Myanmar and Vietnam during 2013⁻2016. Hantavirus RNA was detected in two of 15 black-bearded tomb bats (Taphozous melanopogon) and two of 26 Pomona roundleaf bats (Hipposideros pomona) in Myanmar, and in three of six ashy leaf-nosed bats (Hipposideros cineraceus) in Vietnam. Pair-wise alignment and comparison of coding regions of the S, M, and L segments of hantaviruses from Taphozous and Hipposideros bats revealed high nucleotide and amino acid sequence similarities to prototype Láibin virus (LAIV) and Xuân SÆ¡n virus (XSV), respectively. Phylogenetic analyses, generated by maximum-likelihood and Bayesian methods, showed a geographic clustering of LAIV strains from China and Myanmar, but not of XSV strains from China and Vietnam. These findings confirm that the black-bearded tomb bat is the natural reservoir of LAIV, and that more than one species of Hipposideros bats can host XSV.

Kinetic Analysis of the Motility of Giant Virus-Infected Amoebae Using Phase-Contrast Microscopic Images.

Tracking cell motility is a useful tool for the study of cell physiology and microbiology. Although phase-contrast microscopy is commonly used, the existence of optical artifacts called "halo" and "shade-off" have inhibited image analysis of moving cells. Here we show kinetic image analysis of Acanthamoeba motility using a newly developed computer program named "Phase-contrast-based Kinetic Analysis Algorithm for Amoebae (PKA3)," which revealed giant-virus-infected amoebae-specific motilities and aggregation profiles using time-lapse phase-contrast microscopic images. This program quantitatively detected the time-dependent, sequential changes in cellular number, size, shape, and direction and distance of cell motility. This method expands the potential of kinetic analysis of cultured cells using versatile phase-contrast images. Furthermore, this program could be a useful tool for investigating detailed kinetic mechanisms of cell motility, not only in virus-infected amoebae but also in other cells, including cancer cells, immune response cells, and neurons.

Imaging of angiogenesis of human umbilical vein endothelial cells by uptake of exosomes secreted from hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a typical hyper-vascular tumor, so the understanding the mechanisms of angiogenesis in HCC is very important for its treatment. However, the influence of the exosomes secreted from HCC cells (HCC-exosomes) on angiogenesis remains poorly understood. We herein examined the effects of the exosomes secreted from HepG2 cells (HepG2-exosomes) on the lumen formation of human umbilical vein endothelial cells (HUVECs) by the imaging of angiogenesis. The degree of lumen formation of HUVECs was dependent on the number of HepG2-exosomes. The HepG2-exosomes expressed NKG2D, an activating receptor for immune cells, and HSP70, a stress-induced heat shock protein associated with angiogenesis through the vascular endothelial growth factor (VEGF) receptor. In addition, the HepG2-exosomes contained several microRNAs (miRNAs) reported to exist in the serum of HCC patients. These results suggest that the HCC-exosomes play an important role in angiogenesis. Further studies on the function of HCC-exosomes may provide a new target for HCC treatment.

Genetic defects in SAPK signalling, chromatin regulation, vesicle transport and CoA-related lipid metabolism are rescued by rapamycin in fission yeast.

Rapamycin inhibits TOR (target of rapamycin) kinase, and is being used clinically to treat various diseases ranging from cancers to fibrodysplasia ossificans progressiva. To understand rapamycin mechanisms of action more comprehensively, 1014 temperature-sensitive (ts) fission yeast (Schizosaccharomyces pombe) mutants were screened in order to isolate strains in which the ts phenotype was rescued by rapamycin. Rapamycin-rescued 45 strains, among which 12 genes responsible for temperature sensitivity were identified. These genes are involved in stress-activated protein kinase (SAPK) signalling, chromatin regulation, vesicle transport, and CoA- and mevalonate-related lipid metabolism. Subsequent metabolome analyses revealed that rapamycin upregulated stress-responsive metabolites, while it downregulated purine biosynthesis intermediates and nucleotide derivatives. Rapamycin alleviated abnormalities in cell growth and cell division caused by sty1 mutants (Δsty1) of SAPK. Notably, in Δsty1, rapamycin reduced greater than 75% of overproduced metabolites (greater than 2× WT), like purine biosynthesis intermediates and nucleotide derivatives, to WT levels. This suggests that these compounds may be the points at which the SAPK/TOR balance regulates continuous cell proliferation. Rapamycin might be therapeutically useful for specific defects of these gene functions.

Release of condensin from mitotic chromosomes requires the Ran-GTP gradient in the reorganized nucleus.

After mitosis, nuclear reorganization occurs together with decondensation of mitotic chromosomes and reformation of the nuclear envelope, thereby restoring the Ran-GTP gradient between the nucleus and cytoplasm. The Ran-GTP gradient is dependent on Pim1/RCC1. Interestingly, a defect in Pim1/RCC1 in Schizosaccharomyces pombe causes postmitotic condensation of chromatin, namely hypercondensation, suggesting a relationship between the Ran-GTP gradient and chromosome decondensation. However, how Ran-GTP interacts with chromosome decondensation is unresolved. To examine this interaction, we used Schizosaccharomyces japonicus, which is known to undergo partial breakdown of the nuclear membrane during mitosis. We found that Pim1/RCC1 was localized on nuclear pores, but this localization failed in a temperature-sensitive mutant of Pim1/RCC1. The mutant cells exhibited hypercondensed chromatin after mitosis due to prolonged association of condensin on the chromosomes. Conceivably, a condensin-dephosphorylation defect might cause hypercondensed chromatin, since chromosomal localization of condensin is dependent on phosphorylation by cyclin-dependent kinase (CDK). Indeed, CDK-phospho-mimic mutation of condensin alone caused untimely condensin localization, resulting in hypercondensed chromatin. Together, these results suggest that dephosphorylation of CDK sites of condensin might require the Ran-GTP gradient produced by nuclear pore-localized Pim1/RCC1.

Transformation of Schizosaccharomyces japonicus.

This protocol describes the use of electroporation to transform Schizosaccharomyces japonicus with plasmids or linear DNA. Plasmids are helpful for the complementation testing of mutations and for the expression of specific genes. Linear DNA fragments integrated into chromosomal DNA by homologous recombination are useful for gene deletion or to fuse a gene with a tag sequence (e.g., encoding a fluorescent protein). To introduce DNA into S. japonicus, electroporation methods are recommended because S. japonicus is sensitive to lithium acetate (LiOAc).