Genome Sequence of the Lanthanide-Responsive Methylotrophic Bacterium Methylorubrum extorquens Strain GM97.

We present the complete genome sequence of Methylorubrum extorquens strain GM97, which formed large colonies on a 1/100 nutrient plate with samarium (Sm3+). The genome for strain GM97 was estimated to be 7,608,996 bp, which suggests that the strain is closely related to Methylorubrum extorquens strains.

Treatment with Interleukin-25 Suppresses Short-Term High-Fructose Diet-Induced Hepatic Gene Expression and Activities of Fatty Acid Synthesis Enzymes in Rats.

This study aimed to investigate the effects of interleukin-25, which belongs to the interleukin-17 family, on short-term high-fructose diet-induced hepatic triacylglycerol accumulation. Rats were fed a high-starch (control) or high-fructose diet for 7 d, with or without intraperitoneal administration of recombinant interleukin-25 from days 3-7. Treatment with interleukin-25 significantly reduced the mRNA levels and activity of fatty acid synthesis enzymes and caused a nominal reduction in hepatic triacylglycerol levels in rats fed a high-fructose diet but not in those fed a control diet. Interleukin-25 treatment did not affect the mRNA levels of ß-oxidation enzymes in either the control or fructose-fed rats. These results suggest that treatment with interleukin-25 suppresses short-term high-fructose diet-induced fatty acid synthesis and leads to the alleviation of triacylglycerol accumulation in the liver.

Tetraploidy-linked sensitization to CENP-E inhibition in human cells.

Tetraploidy is a hallmark of cancer cells, and tetraploidy-selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti-proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP-E) than are diploids. Treatment with a CENP-E inhibitor preferentially diminished the tetraploid cell population in a diploid-tetraploid co-culture at optimum conditions. Live imaging revealed that a tetraploidy-linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP-E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy-selective cell growth suppression. In contrast, the microtubule-stabilizing compound paclitaxel caused tetraploidy-selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP-E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP-E inhibitors in tetraploidy-selective suppression and their potential use in the development of tetraploidy-targeting interventions in cancer.

Characterization of the bacterial community structure in traditional Gifu ayu-narezushi (fermented sweetfish).

In this study, we aimed to elucidate the bacterial biota of ayu-nazushi, which is a fermented salted fish dish made in Gifu City, Japan. In traditional Gifu ayu-nazushi, Lactobacillaceae (mainly Latilactobacillus sakei) was the most dominant family, followed by Enterobacteriaceae. Moreover, fermentation bacteria in ayu-nazushi came from the salted fish, and the bacterial biota in the ayu-nazushi transferred as the fermentation process progressed. In the early stage of fermentation, Leuconostoc mesenteroides was main species, and then in the late stage, L. sakei became predominant. We also observed that when non-salted fish was used for the manufacture of ayu-nazushi, Aeromonas veronii, which is a pathogen for humans, was observed in significant quantities. These results indicate that L. sakei and L. mesenteroides were influential lactic acid bacteria for the fermentation of Gifu ayu-narezushi, and that salting treatment of the fish is an indispensable step in the manufacturing process in order to suppress the growth of Aeromonas species.

Dietary Supplementation with Sodium Butyrate Reduces High-sucrose Diet-induced Hepatic Accumulation of Triacylglycerols and Expression of Fatty Acid Synthesis Enzymes in Rats.

We investigated the effects of dietary supplementation with sodium butyrate (NaB) on the lipid levels, gene expression, and proteins related to lipid metabolism in nonalcoholic fatty liver disease (NAFLD) rat models fed a high-sucrose diet for 3 weeks. Supplementation with 1% and 3% NaB reduced high-sucrose-induced hepatic triacylglycerol levels and expression of genes and proteins related to fatty acid synthesis, such as fatty acid synthase and malic enzyme, in a dose-dependent manner. NaB supplementation did not affect hepatic cholesterol levels or expression of genes related to ß-oxidation. NaB may prevent high-sucrose-induced NAFLD by repressing the fatty acid synthesis pathway.

The potential and capability of the methylotrophic yeast Ogataea methanolica in a "methanol bioeconomy".

Efficient bioconversion of methanol, which can be generated from greenhouse gases, into valuable resources contributes to achieving climate goals and developing a sustainable economy. The methylotrophic yeast Ogataea methanolica is considered to be a suitable host for efficient methanol bioconversion because it has outstanding characteristics for the better adaptive potential to a high methanol environment (i.e., greater than 5%). This capacity represents a huge potential to construct an innovative carbon-neutral production system that converts methanol into value-added chemicals under the control of strong methanol-induced promoters. In this review, we discuss what is known about the regulation of methanol metabolism and adaptation mechanisms for 5% methanol conditions in O. methanolica in detail. We also discuss about the potential to breed "super methylotrophic yeast," which has potent growth characteristics under high methanol conditions.

Draft Genome Sequence of a Lanthanide-Responsive Bacterium, Bradyrhizobium sp. Strain Ce-3.

We present the draft genome sequence of Bradyrhizobium sp. strain Ce-3, which produced exopolysaccharide (EPS) and oxidized methanol in the presence of Ce3+. The genome for strain Ce-3 was estimated at 7,608,996 bp and showed that the strain is closely related to Bradyrhizobium erythrophlei MT12 and Bradyrhizobium sp. strain C9.

Transition and regulation mechanism of bacterial biota in Kishu saba-narezushi (mackerel narezushi) during its fermentation step.

The transition of the bacterial biota of Kishu saba-narezushi (mackerel-narezushi) in the Hidaka region of Wakayama prefecture, Japan, was analyzed using amplicon sequencing based on the V3-V4 variable region of the 16S rRNA gene. In the non-fermented sample (0 day), the major genus with the highest abundance ratio was Staphylococcus. In the early stage (fermentation for 2 days), however, the genus Lactococcus became a dominant species, and in the later stage (fermentation for 5 days), the abundance ratio of the genus Lactobacillus increased significantly. Lactococcus lactis strains isolated from the narezushi samples had the ability to suppress the growth of not only Staphylococcus genera but also Lactobacillus. Moreover, the isolates produced a bacteriocin, which was identified as nisin Z. On the basis of these results, it is concluded that L. lactis plays an important role in preparing the fermentation conditions of Kishu saba-narezushi in the early stage by suppressing unwanted microorganisms using lactic acid and nisin Z.

Fatty acid composition of the methylotrophic yeast Komagataella phaffii grown under low- and high-methanol conditions.

In this study, we analysed the intracellular fatty acid profiles of Komagataella phaffii during methylotrophic growth. K. phaffii grown on methanol had significantly lower total fatty acid contents in the cells compared with glucose-grown cells. C18 and C16 fatty acids were the predominant fatty acids in K. phaffii, although the contents of odd-chain fatty acids such as C17 fatty acids were also relatively high. Moreover, the intracellular fatty acid composition of K. phaffii changed in response to not only carbon sources but also methanol concentrations: C17 fatty acids and C18:2 content increased significantly as methanol concentration increased, whereas C18:1 and C18:3 contents were significantly lower in methanol-grown cells. The intracellular content of unidentified compounds (Cn H2n O4 ), on the other hand, was significantly greater in cells grown on methanol. As the intracellular contents of these Cn H2n O4 compounds were significantly higher in a gene-disrupted strain for glutathione peroxidase (gpx1Δ) than in the wild-type strain, we presume that the Cn H2n O4 compounds are fatty acid peroxides. These results indicate that K. phaffii can coordinate intracellular fatty acid composition during methylotrophic growth in order to adapt to high-methanol conditions and that certain fatty acid species such as C17:0, C17:1, C17:2 and C18:2 may be related to the physiological functions by which K. phaffii adapts to high-methanol conditions.

Metabolic regulation adapting to high methanol environment in the methylotrophic yeast Ogataea methanolica.

Since methylotrophic yeasts such as Ogataea methanolica can use methanol as a sole carbon feedstock, they could be applied to produce valuable products from methanol, a next-generation energy source synthesized from natural gases, using genetic engineering tools. In this study, metabolite profiling of O. methanolica was conducted under glucose (Glc) and low and high methanol (L- and H-MeOH) conditions to show the adaptation mechanism to a H-MeOH environment. The yeast strain responded not only to the presence of methanol but also to its concentration based on the growth condition. Under H-MeOH conditions, O. methanolica downregulated the methanol utilization, glycolytic pathway and alcohol oxidase (AOD) isozymes and dihydroxyacetone synthase (DAS) expression compared with L-MeOH-grown cells. However, levels of energy carriers, such as ATP, were maintained to support cell survival. In H-MeOH-grown cells, reactive oxygen species (ROS) levels were significantly elevated. Along with increasing ROS levels, ROS scavenging system expression was significantly increased in H-MeOH-grown cells. Thus, we concluded that formaldehyde and H2 O2 , which are products of methanol oxidation by AOD isozymes in the peroxisome, are overproduced in H-MeOH-grown cells, and excessive ROS derived from these cells is generated in the cytosol, resulting in upregulation of the antioxidant system and downregulation of the methanol-utilizing pathway to suppress overproduction of toxic intermediates.

Effects of Dietary Supplementation with Myo-inositol on Hepatic Expression of Glycolytic and Fructolytic Enzyme Genes in Rats Fed a High-sucrose Diet.

We examined effects of a major lipotrope, myo-inositol, on the expression of primary glycolytic (glucokinase and phosphofructokinase) and fructolytic enzyme (ketohexokinase [KHK] and aldolase B) genes in the livers of rats fed a control diet, high-sucrose diet, or high-sucrose diet supplemented with 0.5% myo-inositol for 14 d. Supplementation with myo-inositol decreased the hepatic expression of fructolytic enzyme genes, but not that of glycolytic enzyme genes, and the levels of triglycerides, fatty acid synthase, and KHK proteins in high-sucrose diet-induced fatty liver. The study results suggest that myo-inositol represses primary fructlysis, but not glycolysis, in high-sucrose diet-induced fatty liver.

High-fructose diet-induced hepatic expression of the Scd1 gene is associated with increased acetylation of histones H3 and H4 and the binding of ChREBP at the Scd1 promoter in rats.

Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in the biosynthesis of monounsaturated fatty acids, and the expression of the Scd1 gene is induced by the intake of the lipogenic sugar fructose. We examined the effects of a high-fructose diet on hepatic acetylation of histones H3 and H4 and the binding of carbohydrate response element-binding protein (ChREBP) on the Scd1 gene promoter in rats. Rats were fed a control diet or a high-fructose diet for 10 days. The intake of a high-fructose diet significantly increased histone H3 and H4 acetylation and ChREBP binding to the Scd1 gene promoter as well as the amount of triglyceride and the expression of the Scd1 gene. These results suggest that short-term intake of high fructose upregulates expression of Scd1 by enhancing acetylation of histones H3 and H4 and binding of ChREBP at the Scd1 promoter.

Dietary supplementation with myo-inositol reduces high-fructose diet-induced hepatic ChREBP binding and acetylation of histones H3 and H4 on the Elovl6 gene in rats.

ELOVL fatty acid elongase 6 (ELOVL6) is a long-chain fatty acid elongase, and the hepatic expression of the Elovl6 gene and accumulation of triglycerides (TG) are enhanced by long-term high-fructose intake. Fatty acid synthesis genes, including Elovl6, are regulated by lipogenic transcription factors, sterol regulatory element-binding protein 1c (SREBP-1c) and carbohydrate-responsive element-binding protein (ChREBP). In addition, carbohydrate signals induce the expression of fatty acid synthase not only via these transcription factors but also via histone acetylation. Since a major lipotrope, myo-inositol (MI), can repress short-term high-fructose-induced fatty liver and the expression of fatty acid synthesis genes, we hypothesized that MI might influence SREBP-1c, ChREBP, and histone acetylation of Elovl6 in fatty liver induced by even short-term high-fructose intake. This study aimed to investigate whether dietary supplementation with MI affects Elovl6 expression, SREBP-1 and ChREBP binding, and acetylation of histones H3 and H4 at the Elovl6 promoter in short-term high-fructose diet-induced fatty liver in rats. Rats were fed a control diet, high-fructose diet, or high-fructose diet supplemented with 0.5% MI for 10 days. This study showed that MI supplementation reduced short-term high-fructose diet-induced hepatic expression of the Elovl6 gene, ChREBP binding, but not SREBP-1 binding, and acetylation of histones H3 and H4 at the Elovl6 promoter.

Regulation of lanthanide-dependent methanol oxidation pathway in the legume symbiotic nitrogen-fixing bacterium Bradyrhizobium sp. strain Ce-3.

Lanthanide (Ln)-dependent XoxF-type methanol dehydrogenase (MDH) genes can be found in bacteria that are not believed to be methylotrophs, and studies on their methylotrophic pathways and their use of Ln are now emerging. Ln-dependent methanol utilization in Bradyrhizobium sp. strain Ce-3, which belongs to the Bradyrhizobium elkanii superclade (clade II), was investigated in this study. Strain Ce-3 was able to grow in a media containing methanol as a sole carbon source and light Ln (L-Ln, i.e., La3+, Ce3+, Pr3+, and Nd3+), whereas the strain did not show any growth with Ca2+ or the heavy Ln, Sm3+. We found that the uptake of L-Ln is enhanced mainly by methanol and L-Ln species, and the strain incorporates each L-Ln species evenly into the cell. The genome of strain Ce-3 encodes the xox cluster for Ln-dependent methanol dehydrogenase (xoxF) and the enzymes participating in the methanol oxidation pathway (xoxG, fldA, and gfaA) and regulation (xoxR), but the gene encoding formate dehydrogenase (FDH) was not found in the cluster. MDH, formaldehyde dehydrogenase, and FDH activities were induced by methanol/Ln. Moreover, expression of the genes on the xox cluster was upregulated by methanol/Ln. Based on these results, we concluded that strain Ce-3 possesses a complete L-Ln-dependent methanol oxidation pathway, which is dissimilar to plant phyllospheric bacteria, Methylobacterium species, with a transport system for L-Ln species.

Preference for particular lanthanide species and thermal stability of XoxFs in Methylorubrum extorquens strain AM1.

XoxF-type methanol dehydrogenase was recently found to be lanthanide-dependent, while its counterpart MxaF is Ca2+-dependent. The lanthanide (Ln) series consists of 15 different elements, all of which exist in nature, although at different relative abundances. XoxF from Methylorubrum extorquens strain AM1 has been shown to be induced by four light Ln species (La3+ to Nd3+). The preference of XoxFs for certain co-existing Ln species and the catalytic activity and stability of XoxF metallated with different Ln species have not been well investigated. In this study, we found that (i) strain AM1 cells preferentially utilize La3+ rather than Nd3+ for growth, (ii) XoxF purified from cells grown with a La3+ and Nd3+ mixture contained a larger proportion of La3+, and (iii) La3+-metallated XoxF has higher activity and thermal stability than Nd3+-metallated XoxF, although (iv) both enzymes showed unchanged surface charges. Thermal shift assay in particular revealed that metallation affects the temperature for subunit denaturation but not for subunit dissociation. We concluded that, although La3+ and Nd3+ have similar distributions in nature, XoxF could chose La3+ preferentially.

Maltobionic acid accelerates recovery from iron deficiency-induced anemia in rats.

In experiments 1 and 2, effect of ingestion of maltobionic acid calcium salt (MBCa) on recovery of rats from a latent iron deficiency and from iron deficiency anemia was examined, respectively. After grouping rats into control and iron-deficiency groups, a latent iron deficiency or iron-deficiency anemia was induced in the latter group. And recovery from these states by MBCa containing diets (0%, 3%, and 6% MBCa in diet, classified into MBCa-0, MBCa-3, and MBCa-6 groups) was compared for convalescence period in light of iron sufficient control group. In experiment 1, MBCa ingestion significantly increased the iron concentration in the serum and liver, and promoted recovery from a latent iron deficiency. In experiment 2, hemoglobin and hematocrit levels increased significantly with MBCa intake, and recovery from iron-deficiency anemia was promoted. MBCa effectively promoted the recovery of rats from a subclinical iron deficiency and iron-deficiency anemia.Abbreviations: ANOVA: analysis of variance; DMT1: divalent metal transporter 1; EDTA-2Na: disodium salt of ethylenediaminetetraacetic acid; Fpn: feroportin; Hb: hemoglobin; Ht: hematocrit; ICP-OES: inductivity coupled plasma optical emission spectrometer; MBCa: maltobionic acid calcium salt; nitroso-PSAP: 2-nitroso-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol; SE: standard error; SI: serum-iron concentration; TSAT: transferrin saturation; TIBC: total iron-binding capacity; UIBC: unsaturated iron-binding capacity.

Lanthanide-dependent methanol dehydrogenase from the legume symbiotic nitrogen-fixing bacterium Bradyrhizobium diazoefficiens strain USDA110.

The legume symbiotic nitrogen-fixing bacterium, B. diazoefficiens strain USDA110, utilizes methanol for growth in the presence of light lanthanides, such as La3+, Ce3+, Pr3+ or Nd3+, and its cells possess significant methanol dehydrogenase (MDH) activity. We purified MDH to homogeneity from B. diazoefficiens strain USDA110 grown in a methanol/Ce3+ medium; the protein was identified as XoxF5-type MDH (blr6213). The purified XoxF contained 0.58 cerium atoms per enzyme subunit. Moreover, the in-solution structure of XoxF was analyzed by small angle X-ray scattering (SAXS) analysis; the radius of gyration (Rg) and maximum particle dimension (Dmax) of XoxF were calculated to be 32.3 and 96.8 Å, respectively, suggesting that XoxF adopts a dimer structure in solution. These results show that B. diazoefficiens strain USDA110 has XoxF, a lanthanides-dependent MDH, which has methanol oxidation activity and is induced by methanol/lanthanaides, and that lanthanide is one of the important factors in methanol utilization by the strain.

Treatment with myo-inositol attenuates binding of the carbohydrate-responsive element-binding protein to the ChREBP-ß and FASN genes in rat nonalcoholic fatty liver induced by high-fructose diet.

Dietary supplementation with the major lipotrope myo-inositol (MI) potently reduces triglyceride (TG) content and expression levels of the fatty acid synthesis genes, for example, fatty acid synthase (FASN), in rat nonalcoholic fatty liver induced by high-fructose diet. Fatty acid synthesis genes are regulated by the carbohydrate-responsive element-binding protein (ChREBP) that exists in 2 isoforms: ChREBP-α and ChREBP-ß. The gene encoding the latter isoform is more responsive to fructose. Because MI repressed the induction of fatty acid synthesis gene expression by high-fructose diet, we hypothesized that MI may reduce binding of ChREBP to the carbohydrate response elements (ChoREs) in the ChREBP-ß gene as well as in fatty acid synthesis genes in the liver. Rats were fed high-glucose, high-fructose, or high-fructose diets supplemented with MI (0.05% and 0.25%) for 2 weeks. Hepatic TG content and expression levels of the glucose-6-phosphate dehydrogenase, malic enzyme 1, FASN, acetyl-CoA carboxylase alpha, S14, and ChREBP-ß were remarkably elevated in rats fed with high fructose compared with the corresponding levels in high-glucose group. Notably, elevated values of these parameters in high-fructose group were reduced by MI. Similarly, high-fructose-induced ChREBP binding to the ChoREs of the ChREBP-ß and FASN genes was nominally decreased by MI. This study showed that treatment with MI reduced elevated TG content and expression of genes related to fatty acid synthesis, such as FASN and ChREBP-ß, in rat nonalcoholic fatty liver induced by high-fructose diet. Furthermore, MI treatment nominally decreased increased binding of ChREBP to the ChoREs of ChREBP-ß and FASN genes.

Tetraploidy-associated centrosome overduplication in mouse early embryos.

Recently, we observed that tetraploidization of certain types of human cancer cells resulted in upregulation of centrosome duplication cycles and chronic generation of the extra centrosome. Here, we investigated whether tetraploidy-linked upregulation of centrosome duplication also occurs in non-cancer cells using tetraploidized parthenogenetic mouse embryos. Cytokinesis blockage at early embryonic stage before de novo centriole biogenesis provided the unique opportunity in which tetraploidization can be induced without transient doubling of centrosome number. The extra numbers of the centrioles and the centrosomes were observed more frequently in tetraploidized embryos during the blastocyst stage than in their diploid counterparts, demonstrating the generality of the newly found tetraploidy-driven centrosome overduplication in mammalian non-cancer systems.

Ploidy-dependent change in cyclin D2 expression and sensitization to cdk4/6 inhibition in human somatic haploid cells.

Near-haploidy is observed in certain cancer types, but ploidy-dependent alterations in gene regulation in the haploid state remain elusive. Here, by comparative transcriptome analysis between human isogenic haploid and diploid cell lines, we found lowering of cyclin D2 level in haploids. Acute genome duplication in haploids restored cyclin D2 expression to diploid level, indicating that the regulation of cyclin D2 expression is directly linked to ploidy. Downstream pathways of cyclin D2, such as Rb phosphorylation and p27 sequestration remained intact in haploids, suggesting that they adapt to lowered cyclin D level. Interestingly, however, haploid cells were more susceptible to cdk4/6 inhibition compared to diploids. Our finding indicates feasibility of selective growth suppression of haploid cells based on ploidy-linked gene regulation.