Crucial role of iron in epigenetic rewriting during adipocyte differentiation mediated by JMJD1A and TET2 activity.

Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, 　including Pparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.

α,ß-trehalose, an intracellular substance in resting cyst of colpodid ciliates as a key to environmental tolerances.

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,ß-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,ß-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance (1H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,ß-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment.

Deep-learning algorithm to detect fibrosing interstitial lung disease on chest radiographs.

BACKGROUND: Antifibrotic therapies are available to treat chronic fibrosing interstitial lung diseases (CF-ILDs), including idiopathic pulmonary fibrosis. Early use of these treatments is recommended to slow deterioration of respiratory function and to prevent acute exacerbation. However, identifying patients in the early stages of CF-ILD using chest radiographs is challenging. In this study, we developed and tested a deep-learning algorithm to detect CF-ILD using chest radiograph images. METHOD: From the image archive of Sapporo Medical University Hospital, 653 chest radiographs from 263 patients with CF-ILDs and 506 from 506 patients without CF-ILD were identified; 921 were used for deep learning and 238 were used for algorithm testing. The algorithm was designed to output a numerical score ranging from 0 to 1, representing the probability of CF-ILD. Using the testing dataset, the algorithm's capability to identify CF-ILD was compared with that of doctors. A second dataset, in which CF-ILD was confirmed using computed tomography images, was used to further evaluate the algorithm's performance. RESULTS: The area under the receiver operating characteristic curve, which indicates the algorithm's detection capability, was 0.979. Using a score cut-off of 0.267, the sensitivity and specificity of detection were 0.896 and 1.000, respectively. These data showed that the algorithm's performance was noninferior to that of doctors, including pulmonologists and radiologists; performance was verified using the second dataset. CONCLUSIONS: We developed a deep-learning algorithm to detect CF-ILDs using chest radiograph images. The algorithm's detection capability was noninferior to that of doctors.

Role of hypoxanthine-guanine phosphoribosyltransferase in the metabolism of fairy chemicals in rice.

Fairy chemicals (FCs), 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH), are molecules with many diverse functions in plants. The defined biosynthetic pathway for FCs is a novel purine metabolism in which they are biosynthesized from 5-aminoimidazole-4-carboxamide. Here, we show that one of the purine salvage enzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), recognizes AHX and AOH as substrates. Two novel compounds, AOH ribonucleotide and its ribonucleoside which are the derivatives of AOH, were enzymatically synthesized. The structures were determined by mass spectrometry, 1D and 2D NMR spectroscopy, and X-ray single-crystal diffraction analysis. This report demonstrates the function of HGPRT and the existence of novel purine metabolism associated with the biosynthesis of FCs in rice.

Expression and localization of Bombyx mori nucleopolyhedrovirus GP37.

Mitochondria play an essential role in intracellular energy metabolism. This study described the involvement of Bombyx mori nucleopolyhedrovirus (BmNPV) GP37 (BmGP37) in host mitochondria. Herein, the proteins associated with host mitochondria isolated from BmNPV-infected or mock-infected cells by two-dimensional gel electrophoresis were compared. One mitochondria-associated protein in virus-infected cells was identified as BmGP37 by liquid chromatography-mass spectrometry analysis. Furthermore, the BmGP37 antibodies were generated, which could react specifically with BmGP37 in the BmNPV-infected BmN cells. Western blot experiments showed that BmGP37 was expressed at 18 h post-infection and was verified as a mitochondria-associated protein. Immunofluorescence analysis demonstrated that BmGP37 localized to the host mitochondria during BmNPV infection. Furthermore, western blot analysis revealed that BmGP37 is a novel component protein of the occlusion-derived virus (ODV) of BmNPV. The present results indicated that BmGP37 is one of the ODV-associated proteins and may have important roles in host mitochondria during BmNPV infection.

Identification of Biosynthetic and Metabolic Genes of 2-Azahypoxanthine in Lepista sordida Based on Transcriptomic Analysis.

2-Azahypoxanthine was isolated from the fairy ring-forming fungus Lepista sordida as a fairy ring-inducing compound. 2-Azahypoxanthine has an unprecedented 1,2,3-triazine moiety, and its biosynthetic pathway is unknown. The biosynthetic genes for 2-azahypoxanthine formation in L. sordida were predicted by a differential gene expression analysis using MiSeq. The results revealed that several genes in the purine and histidine metabolic pathways and the arginine biosynthetic pathway are involved in the biosynthesis of 2-azahypoxanthine. Furthermore, nitric oxide (NO) was produced by recombinant NO synthase 5 (rNOS5), suggesting that NOS5 can be the enzyme involved in the formation of 1,2,3-triazine. The gene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT), one of the major phosphoribosyltransferases of purine metabolism, increased when 2-azahypoxanthine content was the highest. Therefore, we hypothesized that HGPRT might catalyze a reversible reaction between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. We proved the endogenous existence of 2-azahypoxanthine-ribonucleotide in L. sordida mycelia by LC-MS/MS for the first time. Furthermore, it was shown that recombinant HGPRT catalyzed reversible interconversion between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. These findings demonstrate that HGPRT can be involved in the biosynthesis of 2-azahypoxanthine via 2-azahypoxanthine-ribonucleotide generated by NOS5.

Pectin decomposition at the early stage of brown-rot decay by Fomitopsis palustris.

The sapwood of Japanese cedar (Cryptomeria japonica D. Don) was decayed by the brown-rot fungus Fomitopsis palustris under bright and dark conditions. Scanning electron microscopy revealed the presence of mycelia inside the wood even after 1 week from the start of fungal exposure. Moreover, holes were observed in the torus after fungal exposure. Ruthenium red staining revealed that the pectin in pits was largely absent for 3 weeks. These events occurred before the mass loss of wood samples was confirmed at the early stage. Moreover, FpPG28A was more highly expressed at the hyphal front on a pectin-containing medium under dark conditions compared with bright conditions. This up-regulation under dark conditions indicated that the pectin decomposition ability was promoted inside the wood where light could not reach. In conclusion, we suggest that the brown-rot fungus completed its hyphal expansion within the wood via pectin decomposition in pits before holocellulose decomposition.

Heterologous expression of the lectin CmRlec from Cordyceps militaris (Cordycipitaceae, Ascomycota) in Escherichia coli.

Ascomycete lectins may play an important role in their life cycle. In this report, we mined a ricin B-type lectin, named CmRlec, from the Cordyceps militaris genome by homology search. Furthermore, we succeeded in the soluble expression of CmRlec using ß-glucuronidase as a solubilization tag and demonstrated that this lectin is a novel chitin-recognizing lectin.

The role of xanthine dioxygenase in the biosynthetic pathway of 2-aza-8-oxohypoxanthine of Lepista sordida.

2-Azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), discovered as causal substances of fairy rings are known to be endogenous in the fairy ring-forming Lepista sordida. In this study, we showed that xanthine dioxygenase, an a-ketoglutarate-dependent dioxygenase, might catalyze the conversion of AHX to AOH in the fungus. Furthermore, this enzyme is the first reported molybdopterin-independent protein of hypoxanthine metabolism.

1,2,3-Triazine formation mechanism of the fairy chemical 2-azahypoxanthine in the fairy ring-forming fungus Lepista sordida.

2-Azahypoxanthine (AHX) was first isolated from the culture broth of the fungus Lepista sordida as a fairy ring-inducing compound. It has since been found that a large number of plants and mushrooms produce AHX endogenously and that AHX has beneficial effects on plant growth. The AHX molecule has an unusual, nitrogen-rich 1,2,3-triazine moiety of unknown biosynthetic origin. Here, we establish the biosynthetic pathway for AHX formation in L. sordida. Our results reveal that the key nitrogen sources that are responsible for the 1,2,3-triazine formation are reactive nitrogen species (RNS), which are derived from nitric oxide (NO) produced by NO synthase (NOS). Furthermore, RNS are also involved in the biochemical conversion of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranosyl 5'-monophosphate (AICAR) to AHX-ribotide (AHXR), suggesting that a novel biosynthetic route that produces AHX exists in the fungus. These findings demonstrate a physiological role for NOS in AHX biosynthesis as well as in biosynthesis of other natural products containing a nitrogen-nitrogen bond.

Forward-genetics analysis of sleep in randomly mutagenized mice.

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

T1R3 homomeric sweet taste receptor negatively regulates insulin-induced glucose transport through Gαs-mediated microtubules disassembly in 3T3-L1 adipocytes.

T1R3 is a class C G protein-coupled receptor family member that forms heterodimeric umami and sweet taste receptors with T1R1 and T1R2, respectively, in the taste cells of taste buds. T1R3 is expressed in 3T3-L1 cells in homomeric form and negatively regulates adipogenesis in a Gαs-dependent but cAMP-independent manner. Although T1R3 expression is markedly upregulated during adipogenesis, its physiological role in mature adipocytes remains obscure. Here, we show that stimulation of T1R3 with sucralose or saccharin induces microtubule disassembly in differentiated 3T3-L1 adipocytes. The effect was reproduced by treatment with cholera toxin or isoproterenol but not with forskolin. Treatment with sucralose or saccharin for 3 h inhibited insulin-stimulated glucose uptake by 32% and 45% in differentiated adipocytes, respectively, similar to the inhibitory effect of nocodazole (by 33%). Isoproterenol treatment inhibited insulin-stimulated glucose transport by 26%, whereas sucralose did not affect the intrinsic activity of the glucose transporter, indicating that it inhibited insulin-induced GLUT4 translocation to the plasma membrane. Immunostaining analysis showed that insulin-stimulated GLUT4 accumulation on the plasma membrane was abrogated in sucralose-treated cells, in association with depolymerization of microtubules. Sucralose-mediated inhibition of GLUT4 translocation was reversed by the overexpression of dominant-negative Gαs (Gαs-G226A) or knockdown of Gαs. Additionally, membrane fractionation analysis showed that sucralose treatment reduced GLUT4 levels in the plasma membrane fraction from insulin-stimulated adipocytes. We have identified a novel non-gustatory role for homomeric T1R3 in adipocytes, and activation of the T1R3 receptor negatively regulates insulin action of glucose transport via Gαs-dependent microtubule disassembly.

Characterization of tulip streak virus, a novel virus associated with the family Phenuiviridae.

In Japan, tulip-growing areas have been plagued by viral diseases for decades, but the viruses causing the damage remain undescribed. In this study, Nicotiana benthamiana and Chenopodium quinoa plants mechanically inoculated with crude sap from a symptomatic tulip flower exhibited necrosis symptoms. Additionally, flexuous and filamentous virus particles were detected by electron microscopy analysis. Moreover, we determined the complete sequences of two genomic segments of the tulip streak virus (TuSV), which is a new virus associated with streaking symptoms, on the basis of a next-generation sequencing analysis. Homology analyses of the amino acid sequence of RNA-dependent RNA polymerase and the terminal sequence of the genomic RNA indicated that TuSV is associated with viruses in the family Phenuiviridae, but differs substantially from other reported viruses.

Chloroplast relocation movement in the liverwort Apopellia endiviifolia.

Changes in the subcellular localisation of chloroplasts help optimise photosynthetic activity under different environmental conditions. In many plants, this movement is mediated by the blue-light photoreceptor phototropin. A model organism with simple phototropin signalling that allows clear observation of chloroplasts would facilitate the study of chloroplast relocation movement. Here, we examined this process in the simple thalloid liverwort Apopellia endiviifolia. Transverse sections of the thallus tissue showed uniformly developed chloroplasts and no air chambers; these characteristics enable clear observation of chloroplasts and analysis of their movements under a fluorescence stereomicroscope. At 22°C, the chloroplasts moved to the anticlinal walls of cells next to the neighbouring cells in the dark (dark-positioning response), whereas they moved towards weak light (accumulation response) and away from strong light (avoidance response). When the temperature was reduced to 5°C, the chloroplasts moved away from weak light (cold-avoidance response). Hence, both light- and temperature-dependent chloroplast relocation movements occur in A. endiviifolia. Notably, the accumulation, avoidance and cold-avoidance responses were induced under blue-light but not under red-light. These results suggest that phototropin is responsible for chloroplast relocation movement in A. endiviifolia and that the characteristics are similar to those in the model liverwort Marchantia polymorpha. RNA sequencing and Southern blot analysis identified a single copy of the PHOTOTROPIN gene in A. endiviifolia, indicating that a simple phototropin signalling pathway functions in A. endiviifolia. We conclude that A. endiviifolia has great potential as a model system for elucidating the mechanisms of chloroplast relocation movement.

Construction of an infectious dahlia common mosaic virus clone.

Dahlia is a major ornamental plant that is cultivated worldwide. However, dahlia plants, which are mainly propagated through vegetative reproduction, are susceptible to widespread damage by viruses, and viral control requires that the nature of the infecting virus(es) be known. In this study, dahlia common mosaic virus (DCMV) was detected for the first time in Japan and sequenced. This is the first report of an infectious DCMV clone being constructed, and it will aid in the characterization of DCMV.

Intestinal Behçet's disease complicated by myelodysplastic syndrome and secondary pulmonary alveolar proteinosis: a case report.

BACKGROUND: Gastrointestinal lesions, which sometimes develop in Behçet's disease (BD), are referred to as intestinal BD. Although rare, intestinal BD can be accompanied by myelodysplastic syndrome (MDS) with abnormal karyotype trisomy 8, which is refractory to immunosuppressive therapy. Pulmonary alveolar proteinosis is a rare lung complication of BD and MDS. Herein, we present an extremely rare case of intestinal BD presenting with MDS and several chromosomal abnormalities, followed by secondary pulmonary proteinosis. CASE PRESENTATION: A 58-year-old Japanese woman with a 3-year history of genital ulcers and oral aphthae was admitted to our hospital. The patient developed abdominal pain and persistent diarrhea. Colonoscopy revealed multiple, round, punched-out ulcers from the terminal ileum to the descending colon. Intestinal BD was diagnosed and the patient was treated with colchicine, prednisolone, and adalimumab. However, her symptoms were unstable. Bone marrow examination to investigate the persistent macrocytic anemia revealed the presence of trisomy 8, trisomy 9, and X chromosome abnormalities (48, + 8, + 9, X, i(X) (q10) in 12 out of the examined 20 cells). Based on her hypoplastic bone marrow, the patient was diagnosed with low-risk MDS (refractory anemia). At the age of 61, the patient developed pneumonia with fever and diffuse ground-glass opacities on the lung computed tomography (CT). Chest high-resolution CT and histopathology via transbronchial lung biopsy revealed the presence of pulmonary alveolar proteinosis (PAP). These findings combined with the underlying disease led to the diagnosis of secondary PAP. CONCLUSIONS: Secondary pulmonary proteinosis may accompany intestinal BD with MDS and several chromosomal abnormalities. Physicians should pay attention to lung complications, such as PAP, in patients with intestinal BD complicated by MDS. Genetic abnormalities may be associated with the development of such diseases.

Genetic sequence analysis and characterization of bioactive compounds in mushroom-forming fungi.

Mushroom-forming fungi produce unique bioactive compounds that have potential applications as medicines, supplements, and agrochemicals. Thus, it is necessary to clarify the biosynthetic pathways of these compounds using genome and transcriptome analyses. This review introduces some of our research on bioactive compounds isolated from mushrooms, as well as genetic analysis with next-generation sequencing.

An efficient heterologous Escherichia coli-based expression system for lectin production from Pleurocybella porrigens.

In this study, we report a more efficient heterologous expression of lectin from Pleurocybella porrigens (PPL) using an Escherichia coli-based expression system. The yield (9.3 mg/L culture broth) of recombinant PPL (rPPL) using this expression system was increased approximately 9-fold compared to our previous study. The rPPL obtained in this study exhibited the same biochemical properties as the native PPL.

Measurement of the nuclear concentration of α-ketoglutarate during adipocyte differentiation by using a fluorescence resonance energy transfer-based biosensor with nuclear localization signals.

α-Ketoglutarate (α-KG) also known as 2-oxoglutarate (2-OG) is an intermediate metabolite in the tricarboxylic acid (TCA) cycle and is also produced by the deamination of glutamate. It is an indispensable cofactor for a series of 2-oxoglutarate-dependent oxygenases including epigenetic modifiers such as ten-eleven translocation DNA demethylases (TETs) and JmjC domain-containing histone demethylases (JMJDs). Since these epigenetic enzymes target genomic DNA and histone in the nucleus, the nuclear concentration of α-KG would affect the levels of transcription by modulating the activity of the epigenetic enzymes. Thus, it is of great interest to measure the nuclear concentration of α-KG to elucidate the regulatory mechanism of these enzymes. Here, we report a novel fluorescence resonance energy transfer (FRET)-based biosensor with multiple nuclear localization signals (NLSs) to measure the nuclear concentration of α-KG. The probe contains the α-KG-binding GAF domain of NifA protein from Azotobacter vinelandii fused with EYFP and ECFP. Treatment of 3T3-L1 preadipocytes expressing this probe with either dimethyl-2-oxoglutarate (dimethyl-2-OG), a cell-permeable 2-OG derivative, or citrate elicited time- and dose-dependent changes in the FRET ratio, proving that this probe functions as an α-KG sensor. Measurement of the nuclear α-KG levels in the 3T3-L1 cells stably expressing the probe during adipocyte differentiation revealed that the nuclear concentration of α-KG increased in the early stage of differentiation and remained high thereafter. Thus, this nuclear-localized α-KG probe is a powerful tool for real-time monitoring of α-KG concentrations with subcellular resolution in living cells and is useful for elucidating the regulatory mechanisms of epigenetic enzymes.

Immunoglobulin A Vasculitis in a Japanese Patient with Complete Familial Mediterranean Fever Carrying MEFV Exon 10 Mutation.

Immunoglobulin A (IgA) vasculitis is a systemic small-vessel vasculitis involving the skin, kidney, joints, and gastrointestinal tract. Familial Mediterranean fever (FMF) is the most common autoinflammatory disease characterized by periodic fever, peritonitis, pleuritis, or arthritis. It is well known that FMF may coexist with vasculitis, especially small and medium vessel vasculitis. Here we present a Japanese male patient with FMF who later developed IgA vasculitis and a relapsing disease course. A 51-year-old Japanese male was referred because of upper abdominal pain, arthralgia, and bilateral purpura of the lower limbs. He fulfilled the criteria for IgA vasculitis, which was successfully treated by corticosteroid and immunosuppressive therapy. He had a medical history of periodic fever since the age of 10 years old. The Mediterranean fever (MEFV) gene analysis revealed that he was heterozygous for M694I and E148Q mutations. Colchicine therapy resolved his periodic febrile attacks. To our knowledge, coexistence of FMF with IgA vasculitis has not been reported in East Asia, including Japan. Our case suggests that MEFV gene exon 10 mutations could be related to the development of IgA vasculitis and affects its clinical course.