Deficiency of ß-xylosidase activity in Aspergillus luchuensis mut. kawachii IFO 4308.

In this study, we investigated a deleterious mutation in the ß-xylosidase gene, xylA (AkxylA), in Aspergillus luchuensis mut. kawachii IFO 4308 by constructing an AkxylA disruptant and complementation strains of AkxylA and xylA derived from A. luchuensis RIB2604 (AlxylA), which does not harbor the mutation in xylA. Only the AlxylA complementation strain exhibited significantly higher growth and substantial ß-xylosidase activity in medium containing xylan, accompanied by an increase in XylA expression. This resulted in lower xylobiose and higher xylose concentrations in the mash of barley shochu. These findings suggest that the mutation in xylA affects xylose level during the fermentation process. Because the mutation in xylA was identified not only in the genome of strain IFO 4308 but also the genomes of other industrial strains of A. luchuensis and A. luchuensis mut. kawachii, these findings enhance our understanding of the genetic factors that affect the fermentation characteristics.

Expression of heterochromatin protein 1 affects citric acid production in Aspergillus luchuensis mut. kawachii.

A putative methyltransferase, LaeA, controls citric acid production through epigenetic regulation of the citrate exporter gene, cexA, in the white koji fungus Aspergillus luchuensis mut. kawachii. In this study, we investigated the role of another epigenetic regulator, heterochromatin protein 1, HepA, in citric acid production. The ΔhepA strain exhibited reduced citric acid production in liquid culture, although to a lesser extent compared to the ΔlaeA strain. In addition, the ΔlaeA ΔhepA strain showed citric acid production similar to the ΔlaeA strain, indicating that HepA plays a role in citric acid production, albeit with a less-significant regulatory effect than LaeA. RNA-seq analysis revealed that the transcriptomic profiles of the ΔhepA and ΔlaeA strains were similar, and the expression level of cexA was reduced in both strains. These findings suggest that the genes regulated by HepA are similar to those regulated by LaeA in A. luchuensis mut. kawachii. However, the reductions in citric acid production and cexA expression observed in the disruptants were mitigated in rice koji, a solid-state culture. Thus, the mechanism by which citric acid production is regulated differs between liquid and solid cultivation. Further investigation is thus needed to understand the regulatory mechanism in koji.

Bioinformatics Analysis of the Molecular Networks Associated with the Amelioration of Aberrant Gene Expression by a Tyr-Trp Dipeptide in Brains Treated with the Amyloid-ß Peptide.

Short-chain peptides derived from various protein sources have been shown to exhibit diverse bio-modulatory and health-promoting effects in animal experiments and human trials. We recently reported that the oral administration of the Tyr-Trp (YW) dipeptide to mice markedly enhances noradrenaline metabolism in the brain and ameliorates the working-memory deficits induced by the ß-amyloid 25-35 peptide (Aß25-35). In the current study, we performed multiple bioinformatics analyses of microarray data from Aß25-35/YW-treated brains to determine the mechanism underlying the action of YW in the brain and to infer the molecular mechanisms and networks involved in the protective effect of YW in the brain. We found that YW not only reversed inflammation-related responses but also activated various molecular networks involving a transcriptional regulatory system, which is mediated by the CREB binding protein (CBP), EGR-family proteins, ELK1, and PPAR, and the calcium-signaling pathway, oxidative stress tolerance, and an enzyme involved in de novo l-serine synthesis in brains treated with Aß25-35. This study revealed that YW has a neuroprotective effect against Aß25-35 neuropathy, suggesting that YW is a new functional-food-material peptide.

Development of a chub mackerel with less-aggressive fry stage by genome editing of arginine vasotocin receptor V1a2.

Genome editing is a technology that can remarkably accelerate crop and animal breeding via artificial induction of desired traits with high accuracy. This study aimed to develop a chub mackerel variety with reduced aggression using an experimental system that enables efficient egg collection and genome editing. Sexual maturation and control of spawning season and time were technologically facilitated by controlling the photoperiod and water temperature of the rearing tank. In addition, appropriate low-temperature treatment conditions for delaying cleavage, shape of the glass capillary, and injection site were examined in detail in order to develop an efficient and robust microinjection system for the study. An arginine vasotocin receptor V1a2 (V1a2) knockout (KO) strain of chub mackerel was developed in order to reduce the frequency of cannibalistic behavior at the fry stage. Video data analysis using bioimage informatics quantified the frequency of aggressive behavior, indicating a significant 46% reduction (P = 0.0229) in the frequency of cannibalistic behavior than in wild type. Furthermore, in the V1a2 KO strain, the frequency of collisions with the wall and oxygen consumption also decreased. Overall, the manageable and calm phenotype reported here can potentially contribute to the development of a stable and sustainable marine product.

LsSpt23p is a regulator of triacylglycerol synthesis in the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi has considerable potential in industrial application, since it can accumulate a large amount of triacylglycerol (TAG), which is produced from sugars under nitrogen limitation condition. However, the regulation of lipogenesis in L. starkeyi has not been investigated in depth. In this study, we compared the genome sequences of wild-type and mutants with increased TAG productivity, and identified a regulatory protein, LsSpt23p, which contributes to the regulation of TAG synthesis in L. starkeyi. L. starkeyi mutants overexpressing LsSPT23 had increased TAG productivity compared with the wild-type strain. Quantitative real-time PCR analysis showed that LsSpt23p upregulated the expression of GPD1, which encodes glycerol 3-phosphate dehydrogenase; the Kennedy pathway genes SCT1, SLC1, PAH1, DGA1, and DGA2; the citrate-mediated acyl-CoA synthesis pathway-related genes ACL1, ACL2, ACC1, FAS1, and FAS2; and OLE1, which encodes ∆9 fatty acid desaturase. Chromatin immunoprecipitation-quantitative PCR assays indicated that LsSpt23p acts as a direct regulator of SLC1 and PAH1, all the citrate-mediated acyl-CoA synthesis pathway-related genes, and OLE1. These results indicate that LsSpt23p regulates TAG synthesis. Phosphatidic acid is a common substrate of phosphatidic acid phosphohydrolase, which is used for TAG synthesis, and phosphatidate cytidylyltransferase 1 for phospholipid synthesis in the Kennedy pathway. LsSpt23p directly regulated PAH1 but did not affect the expression of CDS1, suggesting that the preferred route of carbon is the Pah1p-mediated TAG synthesis pathway under nitrogen limitation condition. The present study contributes to understanding the regulation of TAG synthesis, and will be valuable in future improvement of TAG productivity in oleaginous yeasts. KEY POINTS: LsSpt23p was identified as a positive regulator of TAG biosynthesis LsSPT23 overexpression enhanced TAG biosynthesis gene expression and TAG production LsSPT23M1108T overexpression mutant showed fivefold higher TAG production than control.

Prdm12 regulates inhibitory neuron differentiation in mouse embryonal carcinoma cells.

The epigenetic regulatory system significant influences the fate determination of cells during developmental processes. Prdm12 is a transcriptional regulator that modulates gene expression epigenetically. The Prdm12 gene has been shown to be expressed in neural tissues, specifically during development, but its detailed function is not fully understood. This study investigated the function of the Prdm12 gene in P19 mouse embryonic tumor cells as a model for neural differentiation. A decrease in the expression of neuron-specific genes and the alterations of dendrites and axons morphology was confirmed in Prdm12-knockout P19 cells. In addition, almost no astrocytes were generated in Prdm12-knockout P19 cells. Comprehensive gene expression analysis revealed that there was a reduction in the expression of the inhibitory neuron-specific genes Gad1/2 and Glyt2, but not the excitatory neuron-specific gene VGLUT2, in Prdm12-knockout P19 cells. Furthermore, the expression of inhibitory neuron-related factors, Ptf1a, Dbx1, and Gsx1/2, decreased in Prdm12-knockout P19 cells. Gene expression analysis also revealed that the Ptf1a, Hic1, and Foxa1 genes were candidate targets of Prdm12 during neurogenesis. These results suggest that Prdm12 regulates the differentiation of inhibitory neurons and astrocytes by controlling the expression of these genes during the neural differentiation of P19 cells.

Chromosome-level genome sequence data and analysis of the white koji fungus, Aspergillus luchuensis mut. kawachii IFO 4308.

Aspergillus luchuensis mut. kawachii is used primarily in the production of shochu, a traditional Japanese distilled alcoholic beverage. Here, we report the chromosome-level genome sequence of A. luchuensis mut. kawachii IFO 4308 (NBRC 4308) and a comparison of the sequence with that of A. luchuensis RIB2601. The genome of strain IFO 4308 was assembled into nine contigs consisting of eight chromosomes and one mitochondrial DNA segment. The nearly complete genome of strain IFO 4308 comprises 37,287,730 bp with a GC content of 48.85% and 12,664 predicted coding sequences and 267 tRNAs. Comparison of the IFO 4308 and RIB2601 genomes revealed a highly conserved structure; however, the IFO 4308 genome is larger than that of RIB2601, which is primarily attributed to chromosome 5. The genome sequence of IFO 4308 was deposited in DDBJ/ENA/GenBank under accession numbers AP024425-AP024433.

PUFA synthase-independent DHA synthesis pathway in Parietichytrium sp. and its modification to produce EPA and n-3DPA.

The demand for n-3 long-chain polyunsaturated fatty acids (n-3LC-PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), will exceed their supply in the near future, and a sustainable source of n-3LC-PUFAs is needed. Thraustochytrids are marine protists characterized by anaerobic biosynthesis of DHA via polyunsaturated fatty acid synthase (PUFA-S). Analysis of a homemade draft genome database suggested that Parietichytrium sp. lacks PUFA-S but possesses all fatty acid elongase (ELO) and desaturase (DES) genes required for DHA synthesis. The reverse genetic approach and a tracing experiment using stable isotope-labeled fatty acids revealed that the ELO/DES pathway is the only DHA synthesis pathway in Parietichytrium sp. Disruption of the C20 fatty acid ELO (C20ELO) and ∆4 fatty acid DES (∆4DES) genes with expression of ω3 fatty acid DES in this thraustochytrid allowed the production of EPA and n-3docosapentaenoic acid (n-3DPA), respectively, at the highest level among known microbial sources using fed-batch culture.

Hepatocyte-Specific Phgdh-Deficient Mice Culminate in Mild Obesity, Insulin Resistance, and Enhanced Vulnerability to Protein Starvation.

l-Serine (Ser) is synthesized de novo from 3-phosphoglycerate via the phosphorylated pathway committed by phosphoglycerate dehydrogenase (Phgdh). A previous study reported that feeding a protein-free diet increased the enzymatic activity of Phgdh in the liver and enhanced Ser synthesis in the rat liver. However, the nutritional and physiological functions of Ser synthesis in the liver remain unclear. To clarify the physiological significance of de novo Ser synthesis in the liver, we generated liver hepatocyte-specific Phgdh KO (LKO) mice using an albumin-Cre driver. The LKO mice exhibited a significant gain in body weight compared to Floxed controls at 23 weeks of age and impaired systemic glucose metabolism, which was accompanied by diminished insulin/IGF signaling. Although LKO mice had no apparent defects in steatosis, the molecular signatures of inflammation and stress responses were evident in the liver of LKO mice. Moreover, LKO mice were more vulnerable to protein starvation than the Floxed mice. These observations demonstrate that Phgdh-dependent de novo Ser synthesis in liver hepatocytes contributes to the maintenance of systemic glucose tolerance, suppression of inflammatory response, and resistance to protein starvation.

Chromosome-Level Genome Sequence of Aspergillus chevalieri M1, Isolated from Katsuobushi.

In this study, we report the chromosome-level genome sequence of the osmophilic filamentous fungus Aspergillus chevalieri M1, which was isolated from a dried bonito, katsuobushi. This fungus plays a significant role in the fermentation and ripening process. Thus, elucidating the sequence data for this fungus will aid in subsequent genomic research on the fungi involved in katsuobushi production.

Chromosome-Level Genome Sequence of Aspergillus puulaauensis MK2, a Fungus Isolated from a Dead Hard Tick.

Aspergillus puulaauensis strain MK2 was isolated from a dead hard tick (Haemaphysalis longicornis). Here, we determined the chromosome-level genome sequence of A. puulaauensis MK2.

Analysis of the Segregation Distortion of FcRAN1 Genotypes Based on Whole-Genome Resequencing of Fig (Ficus carica L.) Breeding Parents.

The common fig (Ficus carica L.) has a gynodioecious breeding system, and its sex phenotype is an important trait for breeding because only female plant fruits are edible. During breeding to select for female plants, we analyzed the FcRAN1 genotype, which is strongly associated with the sex phenotype. In 12 F1 populations derived from 13 cross combinations, the FcRAN1 genotype segregation ratio was 1:1, whereas the M119-226 × H238-107 hybridization resulted in an extremely male-biased segregation ratio (178:7 = male:female). This finding suggests that the segregation distortion was caused by some genetic factor(s). A whole-genome resequencing of breeding parents (paternal and maternal lines) identified 9,061 high-impact SNPs in the parents. A genome-wide linkage analysis exploring the gene(s) responsible for the distortion revealed 194 high-impact SNPs specific to Caprifig6085 (i.e., seed parent ancestor) and 215 high-impact SNPs specific to H238-107 (i.e., pollen parent) in 201 annotated genes. A comparison between the annotated genes and the genes required for normal embryo or gametophyte development and function identified several candidate genes possibly responsible for the segregation distortion. This is the first report describing segregation distortion in F. carica.

Chromosome-Level Genome Sequence of the Black Koji Fungus Aspergillus luchuensis RIB2601.

Aspergillus luchuensis is used for the production of awamori and shochu, which are traditional Japanese distilled alcoholic beverages. Here, we determined the chromosome-level genome sequence of A. luchuensis RIB2601.

Obesogenic and developmental effects of TBT on the gene expression of juvenile Japanese medaka (Oryzias latipes).

The widely used antifoulant tributyltin chloride (TBT) is highly toxic to aquatic organisms. In the present study, four-week-old Japanese medaka (Oryzias latipes) juveniles were orally exposed to TBT at 1 and 10 ng/g bw/d for 1, 2, and 4 weeks, respectively. Half of the tested medaka juveniles showed bone morphology alterations in both 1 and 10 ng/g bw/d TBT 4-week exposure groups. Nile Red (NR) staining showed that the juveniles exposed to 1 ng/g bw/d TBT for 2 and 4 weeks had significantly enlarged adipocyte areas. The mRNA-Seq analysis indicated that 1 ng/g bw/d TBT exposure for 2 weeks affected bone morphology through developmental processes. The GO and KEGG analyses suggested that the adipogenic effect of TBT observed in this study may be induced by metabolic processes, oxidative phosphorylation, and fatty acid degradation and metabolism pathways. Therefore, both morphological observation and mRNA-Seq analysis showed obesogenic effects and developmental toxicity of TBT to juvenile Japanese medaka.

Analysis of the light regulatory mechanism in carotenoid production in Rhodosporidium toruloides NBRC 10032.

Light stimulates carotenoid production in an oleaginous yeast Rhodosporidium toruloides NBRC 10032 by promoting carotenoid biosynthesis genes. These genes undergo two-step transcriptional activation. The potential light regulator, Cryptochrome DASH (CRY1), has been suggested to contribute to this mechanism. In this study, based on KU70 (a component of nonhomologous end joining (NHEJ)) disrupting background, CRY1 disruptant was constructed to clarify CRY1 function. From analysis of CRY1 disruptant, it was suggested that CRY1 has the activation role of the carotenogenic gene expression. To obtain further insights into the light response, mutants varying carotenoid production were generated. Through analysis of mutants, the existence of the control two-step gene activation was proposed. In addition, our data analysis showed the strong possibility that R. toruloides NBRC 10032 is a homo-diploid strain.

Complex Modulating Effects of Dietary Calcium Intake on Obese Mice.

BACKGROUND/AIM: Οverweight and obesity are risk factors for chronic diseases. Dietary calcium has been reported to exert anti-obesity effects. However, the complex modulating effects of calcium intake on obese mice have not been clarified. MATERIALS AND METHODS: The effects of calcium intake on body weight/visceral fat mass were examined in the obese mouse model, KK-Ay Results: Body weight gain decreased in mice fed a diet containing 0.4 to 3.2% calcium at the age of 11 and 13 weeks, but not at 12 weeks after normalization for food intake. Calcium intake also decreased serum insulin levels and increased the amount of feces excreted. Fecal deoxycholate levels were lower in the high-calcium group than in the normal diet control group. Furthermore, the ratio of the deoxycholate-producing microbiome in feces decreased. CONCLUSION: Dietary calcium has anti-obesity effects in obese KK-Ay mice. Inhibition of insulin production and an increased amount of feces excreted with calcium intake may affect body weight.

Development of a time-series shotgun metagenomics database for monitoring microbial communities at the Pacific coast of Japan.

Although numerous metagenome, amplicon sequencing-based studies have been conducted to date to characterize marine microbial communities, relatively few have employed full metagenome shotgun sequencing to obtain a broader picture of the functional features of these marine microbial communities. Moreover, most of these studies only performed sporadic sampling, which is insufficient to understand an ecosystem comprehensively. In this study, we regularly conducted seawater sampling along the northeastern Pacific coast of Japan between March 2012 and May 2016. We collected 213 seawater samples and prepared size-based fractions to generate 454 subsets of samples for shotgun metagenome sequencing and analysis. We also determined the sequences of 16S rRNA (n = 111) and 18S rRNA (n = 47) gene amplicons from smaller sample subsets. We thereafter developed the Ocean Monitoring Database for time-series metagenomic data ( http://marine-meta.healthscience.sci.waseda.ac.jp/omd/ ), which provides a three-dimensional bird's-eye view of the data. This database includes results of digital DNA chip analysis, a novel method for estimating ocean characteristics such as water temperature from metagenomic data. Furthermore, we developed a novel classification method that includes more information about viruses than that acquired using BLAST. We further report the discovery of a large number of previously overlooked (TAG)n repeat sequences in the genomes of marine microbes. We predict that the availability of this time-series database will lead to major discoveries in marine microbiome research.

Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification.

Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n = 36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.

Fingerprinting of hatchery haplotypes and acquisition of genetic information by whole-mitogenome sequencing of masu salmon, Oncorhynchus masou masou, in the Kase River system, Japan.

Stocking hatchery fish can lead to disturbance and extinction of the local indigenous population. Masu salmon Oncorhynchus masou masou, which is endemic across Japan, is a commonly stocked fish for recreational fishing in Japan. To conserve the indigenous resource, their genetic information is required, however, especially on Kyushu Island, the paucity of genetic information for this species has hindered proper resource management. Here, to identify hatchery mitogenome haplotypes of this species, stocked in the Kase River system, Kyushu Island, Japan, and to provide mitogenomic information for the resource management of this species, we analyzed the whole-mitogenome of masu salmon in this river system and several hatcheries potentially used for stocking. Whole-mitogenome sequencing clearly identified hatchery haplotypes, like fingerprints: among the 21 whole-mitogenome haplotypes obtained, six were determined to be hatchery haplotypes. These hatchery haplotypes were distributed in 13 out of 17 sites, suggesting that informal stocking of O. m. masou has been performed widely across this river system. The population of no hatchery haplotypes mainly belonged to clade I, a clade not found in Hokkaido Island in previous studies. Sites without hatchery haplotypes, and the non-hatchery haplotypes in clade I might be candidates for conservation as putative indigenous resources. The whole-mitogenome haplotype analysis also clarified that the same reared strain was used in multiple hatcheries. Analysis of molecular variance suggested that stocked hatchery haplotypes reduce the genetic variation among populations in this river system. It will be necessary to pay attention to genetic fluctuations so that the resources of this river system will not deteriorate further. The single nucleotide polymorphism data obtained here could be used for resource management in this and other rivers: e.g., for monitoring of informal stocking and stocked hatchery fishes, and/or putative indigenous resources.

Identification of gene expression markers and development of evaluation method using cell-based and RT-PCR-based assay for skin sensitising potential of chemicals.

Recently, alternatives to animal testing have been used to evaluate skin sensitisers in cosmetic products. However, testing is still complicated and expensive. To develop a simpler, cost-effective and more accurate evaluation method for the skin sensitising chemicals, we employed cell-based and RT-PCR-based assay. Representative sensitiser specific gene expression in THP-1 cells was analysed by microarray. Gene ontology (GO) analysis revealed that 26 genes induced by the sensitisers were associated with immune function. First, seven of the 26 genes were chosen arbitrarily as candidate markers for our sensitisation assay. Then, THP-1 cells were exposed to 13 reference chemicals with known sensitising potential, and real-time RT-PCR assays targeting the candidate marker genes were performed. Among them, six markers were able to properly evaluate the sensitisation potential by classifying the gene induction rates with appropriate criteria. Especially, the results of the assay using TREM1 and TNFRSF12A gene markers showed 100% sensitivity and specificity. An existing test method, h-CLAT, requires a flow cytometer and is complicated to operate. In contrast, our method is relatively simpler and more cost-effective. Therefore, our method is a promising one to evaluate sensitising chemicals.