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Aim We previously revealed that Ca＋＋-activated calmodulin binds to ABCA1 by the region near the PEST sequence and retards its calpain-mediated degradation to increase HDL biogenesis. Calmodulin activity is reportedly modulated also by other nutritional divalent cations; thus, we attempted to determine whether Zn＋＋ is involved in the regulation of ABCA1 stability through the modulation of calmodulin activity. Methods The effects of Zn＋＋ on ABCA1 expression was investigated in J774 mouse macrophage cell-line cells and HepG2 human hepatoma cell-line cells. Results Zn＋＋ increased ABCA1 expression, not by increasing the mRNA but by attenuating its decay rate, more prominently in the presence of cAMP. Accordingly, it enhanced cell cholesterol release with extracellular apolipoprotein A-I. Calmodulin binding to ABCA1 was increased by Zn＋＋ and Ca＋＋. Zn＋＋ suppressed calpain-mediated hydrolysis of the peptide of ABCA1 cytosolic loop, including the PEST sequence and the calmodulin-binding site, in a calmodulin-dependent fashion, in the presence of the minimum amount of Ca＋＋ to activate calpain, but not calmodulin. Calpain activity was not directly inhibited by Zn＋＋ at the concentration for enhancing calmodulin binding to ABCA1. Conclusion Nutritional divalent cation Zn＋＋ is involved in the regulation of ABCA1 activity and biogenesis of HDL through the modulation of calmodulin activity. The results were consistent with previous clinical findings that Zn＋＋ increased plasma HDL in the conditions of sympathetic activation, such as type 2 diabetes and chronic hemodialysis.

Introduction To compare the effects of ipragliflozin, a sodium-glucose transporter 2 inhibitor, with those of metformin on visceral fat (as well as muscles and bones) in Japanese elderly patients with type 2 diabetes (T2D), we conducted a sub-analysis of a prospective, multicenter, blinded-endpoint randomized-controlled study. Methods In total, 103 patients with T2D (body mass index ≥ 22 kg/m2; glycated hemoglobin, 7–10%) and being treated with sitagliptin (a dipeptidyl peptidase-4 inhibitor) were included and randomized to receive ipragliflozin or metformin. The primary outcome was the change in visceral fat area measured using computed tomography 24 weeks following treatment. The secondary outcomes included changes in subcutaneous and total fat area, muscle volume, bone density measured using computed tomography, handgrip strength, bone markers, plasma glucose, insulin, homeostasis model assessment (HOMA)2-beta, HOMA2-R, glycated hemoglobin, lipid panel, uric acid, blood pressure, adiponectin, and high-sensitivity C-reactive protein. All patients aged 65–74 years were selected for sub-analysis. Results The sub-analysis included 15 and 14 patients in the ipragliflozin and metformin groups, respectively. The patients’ backgrounds were well balanced. Visceral fat area reduction was greater in the ipragliflozin group than in the metformin group (− 10.58% vs. − 6.93%; P = 0.034). There were significant differences in the changes in bone absorption markers, uric acid, and total cholesterol levels between the groups. Conclusion Ipragliflozin significantly reduced the visceral fat area compared with metformin when added to sitagliptin in elderly patients with T2D. Long-term and large-scale studies are required to elucidate whether ipragliflozin is suitable for elderly patients. Trial Registration The study was registered at https://www.umin.ac.jp/ctr/ (UMIN-ID: UMIN 000015170). Electronic supplementary material The online version of this article (10.1007/s13300-020-00949-0) contains supplementary material, which is available to authorized users.

Carotenoids are photosynthetic pigments and hydrophobic antioxidants that are necessary for the survival of photosynthetic organisms, including the microalga Euglena gracilis. In the present study, we identified an uncharacterized gene encoding the E. gracilis β-carotene synthetic enzyme lycopene cyclase (EgLCY) and discovered a relationship between EgLCY-mediated carotenoid synthesis and the reactive oxygen species (ROS) scavenging system ascorbate-glutathione cycle. The EgLCY cDNA sequence was obtained via homology searching E. gracilis transcriptome data. An enzyme assay using Escherichia coli demonstrated that EgLCY converts lycopene to β-carotene. E. gracilis treated with EgLCY double-stranded RNA (dsRNA) produced colorless cells with hypertrophic appearance, inhibited growth, and marked decrease in carotenoid and chlorophyll content, suggesting that EgLCY is essential for the synthesis of β-carotene and downstream carotenoids, which are abundant and physiologically functional. In EgLCY dsRNA-treated cells, the ascorbate-glutathione cycle, composed of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR), was unusually modulated; APX and GR activities significantly decreased, whereas DHAR and MDAR activities increased. Ascorbate content was significantly increased and glutathione content significantly decreased in EgLCY dsRNA-treated cells and was correlated with their recycling enzyme activities. Fluorescent imaging demonstrated that EgLCY dsRNA-treated cells accumulated higher levels of H2O2 compared to wild-type cells. Taken together, this study revealed that EgLCY-mediated synthesis of β-carotene and downstream carotenoid species upregulates APX activity and increases glutathione pool size for H2O2 scavenging. Our study suggests a possible relationship between carotenoid synthesis and the ascorbate-glutathione cycle for ROS scavenging in E. gracilis.

Monodehydroascorbate reductase (MDAR) is an enzyme involved in ascorbate recycling. Arabidopsis thaliana has five MDAR genes that encode two cytosolic, one cytosolic/peroxisomal, one peroxisomal membrane-attached, and one chloroplastic/mitochondrial isoform. In contrast, tomato plants possess only three enzymes, lacking the cytosol-specific enzymes. Thus, the number and distribution of MDAR isoforms differ according to plant species. Moreover, the physiological significance of MDARs remains poorly understood. In this study, we classify plant MDARs into three classes: class I, chloroplastic/mitochondrial enzymes, class II, peroxisomal membrane-attached enzymes, and class III, cytosolic/peroxisomal enzymes. The cytosol-specific isoforms form a subclass of class III and are conserved only in Brassicaceae plants. With some exceptions, all land plants and a charophyte algae, Klebsormidium flaccidum, contain all three classes. Using reverse genetic analysis of Arabidopsis thaliana mutants lacking one or more isoforms, we provide new insight into the roles of MDARs, for example, (1) the lack of two isoforms in a specific combination results in lethality, and (2) the role of MDARs in ascorbate redox regulation in leaves can be largely compensated by other systems. Based on these findings, we discuss the distribution and function of MDAR isoforms in land plants and their cooperation with other recycling systems.

X-ray diffraction and Raman scattering measurements, and first-principles calculations are performed to search for the formation of NaCl-hydrogen compound. When NaCl and H$_{2}$ mixture is laser-heated to above 1500 K at pressures exceeding 40 GPa, we observed the formation of NaClH$_{\textit{x}}$ with $\textit{P}$6$_{3}$/$\textit{mmc}$ structure which accommodates H$_{2}$ molecules in the interstitial sites of NaCl lattice forming ABAC stacking. Upon the decrease of pressure at 300 K, NaClH$_\textit{x}$ remains stable down to 17 GPa. Our calculations suggest the observed NaClH$_{\textit{x}}$ is NaCl(H$_{2}$). Besides, a hydrogen-richer phase NaCl(H$_{2}$)$_{4}$ is predicted to become stable at pressures above 40 GPa.

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 13(2),17(3)-cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.

A prospective, multicentre, open‐label, blinded‐endpoint, randomized controlled study was conducted to evaluate the efficacy of treatment with ipragliflozin (sodium‐dependent glucose transporter‐2 inhibitor) versus metformin for visceral fat reduction and glycaemic control among Japanese patients with type 2 diabetes treated with sitagliptin, HbA1c levels of 7%‐10%, and body mass index (BMI) ≥ 22 kg/m2. Patients were randomly assigned (1:1) to receive ipragliflozin 50 mg or metformin 1000‐1500 mg daily. The primary outcome was change in visceral fat area as measured by computed tomography after 24 weeks of therapy. The secondary outcomes were effects on glucose metabolism and lipid metabolism. Mean percentage reduction in visceral fat area was significantly greater in the ipragliflozin group than in the metformin group (−12.06% vs. −3.65%, P = 0.040). Ipragliflozin also significantly reduced BMI, subcutaneous fat area, waist circumference, fasting insulin, and homeostatic model assessment (HOMA)‐resistance, and increased HDL‐cholesterol levels. Metformin significantly reduced HbA1c and LDL‐cholesterol levels and increased HOMA‐beta. There were no severe adverse events. The use of ipragliflozin or metformin in combination with dipeptidyl peptidase‐4 inhibitors, widely used in Japan, may have beneficial effects in ameliorating multiple cardiovascular risk factors.

Purpose Viral vector-mediated gene therapies for cancers have been eagerly studied in recent years. However, lentiviral vectors are not widely used despite their practical characteristics such as low cytotoxicity and prolonged expression of the transgenes. Renal cell carcinoma (RCC) is highly refractory to known cancer treatment drugs, and surgical resection is used as the most common treatment of RCC. Therefore, we thought that lentiviral vector-mediated gene therapy would become a promising strategy of the treatment of RCC. In this study, we examine the lentiviral vector-mediated transfection of tumor suppressor genes against RCC cell lines. Methods Lentiviral vectors that contained the tumor suppressor genes, p53, p16, and PTEN, were transfected into human RCC cell lines, RCC4/VHL, 786-O and ACHN. Results Significant growth inhibition was observed in RCC cells on transfection with the tumor suppression genes. Especially, transfection of p16 showed remarkable effect on all experimental cell lines. Conclusions The results of this study demonstrate that lentivirus-mediated transfection of tumor suppressor genes exerts promising anti-tumor effects on RCC cell lines.

Objectives Prurigo is a treatment‐resistant inflammatory disease of unknown etiology. Persistent and severe itch is a major and important clinical symptom, but pathological mechanisms and/or actual mediators for itch in prurigo remain to be defined. Methods We investigated blood levels of β‐endorphin, dynorphin A, and autotaxin in adult patients with prurigo (n = 18), including prurigo nodularis, prurigo chronica multiformis, and other forms. Patients with hemodialysis, hepatic dysfunction, and pregnancy were excluded. Immunofluorescence staining for β‐endorphin and autotaxin in lesional skin was also performed. In addition, β‐endorphin and autotaxin synthesis of cultured epidermal keratinocytes in response to several cytokine stimulation was assessed in vitro. Results Blood levels of β‐endorphin were higher in patients with prurigo than in control subjects (n = 20), while dynorphin A levels were comparable in the two groups. Blood autotaxin levels also were increased in patients with prurigo compared to control subjects. On the one hand, epidermal expression of β‐endorphin was increased in skin lesions of prurigo chronica multiformis, but not in prurigo nodularis. On the other hand, enhanced expression of autotaxin was observed in the epidermis in both prurigo nodularis and prurigo chronica multiformis. In vitro studies showed that β‐endorphin‐expressing epidermal keratinocytes increased following stimulation with TNF‐α, interleukin‐31, and/or IFN‐γ. Autotaxin was also detected in the cytoplasm of cultured epidermal keratinocytes. The fraction of autotaxin‐expressing cells increased when stimulated with TNF‐α and/or interleukin‐31. Conclusions Imbalance of opioid receptor signals and/or the autotaxin‐lysophosphatidic acid axis may contribute, at least in part, to the pathogenesis and intractable pruritus in some of the patients with prurigo.

Some carotenoids are found in the Euglena gracilis, including β-carotene, diadinoxanthin, diatoxanthins, and neoxanthin as the major species; however, the molecular mechanism underlying carotenoid biosynthesis in E. gracilis is not well understood. To clarify the pathway and regulation of carotenoid biosynthesis in this alga, we functionally characterized the cytochrome P450 (CYP)-type carotene hydroxylase gene EgCYP97H1. Heterologous in vivo enzyme assay in E. coli indicated that EgCYP97H1 hydroxylated β-carotene to β-cryptoxanthin. E. gracilis cells suppressing EgCYP97H1 resulted in marked growth inhibition and reductions in total carotenoid and chlorophyll contents. Analysis of carotenoid composition revealed that suppression of EgCYP97H1 resulted in higher level of β-carotene, suggesting that EgCYP97H1 is physiologically essential for carotenoid biosynthesis and thus normal cell growth. To our knowledge, this is the first time EgCYP97H1 has been suggested to be β-carotene monohydroxylase, but not β-carotene dihydroxylase. Moreover, during light adaptation of dark-grown E. gracilis, transcript levels of the carotenoid biosynthetic genes (EgCYP97H1, geranylgeranyl pyrophosphate synthase EgcrtE, and phytoene synthase EgcrtB) remained virtually unchanged. In contrast, carotenoid accumulation in E. gracilis grown under the same conditions was inhibited by treatment with a translational inhibitor but not a transcriptional inhibitor, indicating that photo-responsive carotenoid biosynthesis is regulated post-transcriptionally in this alga.

Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d-mannose/l-galactose (d-Man/l-Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d-galacturonate (d-GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d-Man/l-Gal pathway and an orthologous gene encoding aldonolactonase (ALase) - a key enzyme for the d-GalUA pathway - Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l-galactonic acid to the final precursor l-galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encode the rate-limiting enzyme GDP-l-Gal phosphorylase in the d-Man/l-Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P. patens is the d-Man/l-Gal pathway, not the d-GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis.

Photosensitivity is a skin reaction disorder mediated by phototoxic and/or photoallergic mechanisms. The accumulation of porphyrins is generally considered to induce phototoxicity. ATP-binding cassette subfamily G member 2 (ABCG2) has been identified as a transporter of porphyrins and its common variants—p.Gln126Ter (rs72552713) and p.Gln141Lys (rs2231142)—reportedly decrease the function of porphyrin transport in vitro; however, the physiological importance of ABCG2 as a porphyrin transporter remains to be fully elucidated. We herein investigated whether ABCG2 dysfunction could lead to porphyrin accumulation and photosensitivity in Japanese subjects, and found it to be significantly correlated with erythrocyte protoporphyrin levels (P = 0.012). This appears to be the first clinical finding of ABCG2 dysfunction-associated protoporphyrin accumulation in humans. We divided the patients into a chronic actinic dermatosis (CAD) group and a non-CAD group. CAD was diagnosed based on the criteria of reduced minimal erythema doses to ultraviolet B (UVB) and/or ultraviolet A (UVA). The non-CAD group was composed of patients who exhibited normal reactions to UVB and UVA on phototesting, but had histories of recurrent erythema/papules on sun-exposed areas. Estimated ABCG2 function according to ABCG2 genotypes in the non-CAD group was significantly lower than in the general Japanese population (P = 0.045). In contrast, no difference was found in ABCG2 function between the CAD group and the general population, suggesting that ABCG2 dysfunction might be a genetic factor in non-CAD patients with clinical photosensitivity. In this context, genetic dysfunction of ABCG2 might be an overlooked pathological etiology of “photosensitivity of unknown cause.”

Modern high-performance permanent magnets are made from alloys of rare earth and transition metal elements, and large magnetization is achieved in the alloys with high concentration of transition metals. We applied evolutionary search scheme based on first-principles calculations to the Y-Co-B system and predicted 37 cobalt-rich compounds with high probability of being stable. Focusing on remarkably cobalt-rich compounds, YCo$_{16}$ and YCo$_{20}$, we found that, although they are metastable phases, the phase stability is increased with increase of temperature due to the contribution of vibrational entropy. The magnetization and Curie temperature are higher by 0.22 T and 204 K in YCo$_{16}$ and by 0.29 T and 204 K in YCo$_{20}$ than those of Y$_{2}$Co$_{17}$ which has been well studied as strong magnetic compounds., 5 pages, 4 figures

We report x-ray diffraction and Raman scattering measurements in combination with density functional theory calculations that reveal the formation of beryllium polyhydride ${\mathrm{Be}}_{4}{\mathrm{H}}_{8}{({\mathrm{H}}_{2})}_{2}$ by the laser heating of a Be-${\mathrm{H}}_{2}$ mixture to above 1700 K at pressures between 5 and 8 GPa. ${\mathrm{Be}}_{4}{\mathrm{H}}_{8}{({\mathrm{H}}_{2})}_{2}$ crystallizes in a $P{6}_{3}/mmc$ structure and consists of the corner-sharing ${\mathrm{BeH}}_{4}$ tetrahedrons and the ${\mathrm{H}}_{2}$ molecules that occupy an interstitial site. ${\mathrm{Be}}_{4}{\mathrm{H}}_{8}{({\mathrm{H}}_{2})}_{2}$ is stable at least to 14 GPa upon compression and stable down to 4 GPa at room temperature. Our ab initio calculations suggest that ${\mathrm{Be}}_{4}{\mathrm{H}}_{8}{({\mathrm{H}}_{2})}_{2}$ is a metastable phase of the Be-H system.

Aims/Introduction Diabetic polyneuropathy (DPN) and diabetic retinopathy (DR) are traditionally regarded as microvascular complications. However, these complications may share similar neurodegenerative pathologies. Here we evaluate the correlations in the severity of DPN and changes in the thickness of neuroretinal layers to elucidate whether these complications exist at similar stages of progression. Materials and Methods A total of 43 patients with type 2 diabetes underwent a nerve conduction study (NCS), a macular optical coherence tomography, and a carotid artery ultrasound scan. Diabetic polyneuropathy was classified according to Baba’s classification using NCS. The retina was automatically segmented into four layers; ganglion cell complex (GCC), inner nuclear layer/outer plexiform layer (INL/OPL), outer nuclear layer/photoreceptor inner and outer segments, and retinal pigment epithelium (RPE). The thickness of each retinal layer was separately analyzed for the fovea and the parafovea. Results Fourteen patients were classified as having moderate to severe diabetic polyneuropathy. The thicknesses of the foveal and parafoveal INL/OPL increased in patients with diabetic polyneuropathy compared with patients without. The thickness of the parafoveal retinal pigment epithelium decreased in patients with diabetic polyneuropathy. The thinning of parafoveal ganglion cell complex and foveal and parafoveal retinal pigment epithelium were positively correlated with deterioration of nerve functions in the nerve conduction study, but the thickening of INL/OPL was positively correlated with the nerve function deterioration. The thinning of parafoveal ganglion cell complex and foveal retinal pigment epithelium were positively correlated with the thickening of the carotid intima‐media. Conclusions Depending on the progression of diabetic polyneuropathy, the ganglion cell complex and retinal pigment epithelium became thinner and the INL/OPL became thicker. These retinal changes might be noteworthy for pathological investigations and for the assessment of diabetic polyneuropathy and diabetic retinopathy., (1) The thickness of the foveal and parafoveal inner nuclear layer/outer plexiform layer increased in patients with clinical diabetic polyneuropathy compared with patients without clinical diabetic polyneuropathy. (2) A decrease in the thickness of the parafoveal ganglion cell complex correlated with deterioration of nerve functions in the nerve conduction study. (3) A decrease in the thickness of the parafoveal ganglion cell complex was positively correlated with an increase in the parameters of atherosclerosis and cardiovascular risk factors.

We present an evolutionary construction technique of formation-energy convex hull to search for thermodynamically stable compounds. In this technique, candidates with a wide variety of chemical compositions and crystal structures are created by systematically applying evolutionary operators, ``mating,'' ``mutation,'' and ``adaptive mutation,'' to two target compounds, and the convex hull is directly updated through the evolution. We applied the technique to carbon-hydrogen binary system at 10 GPa and obtained 15 hydrocarbons within the convex hull distance less than 0.5 mRy/atom: graphane, polybutadiene, polyethylene, butane, ethane, methane, three molecular compounds of ethane and methane, and six molecular compounds of methane and hydrogen. These results suggest that our evolutionary construction technique is useful for the exploration of stable phases under extreme conditions and the synthesis of new compounds.

Not only in kidney glomerular physiological function but also glomerular pathology especially in diabetic condition, glomerular podocytes play pivotal roles. Therefore, it is important to increase our knowledge about the genes and proteins expressed in podocytes. Recently, we have identified a novel podocyte-expressed gene, R3h domain containing-like (R3hdml) and analyzed its function in vivo as well as in vitro. Transforming growth factor-β (TGF-β) signaling regulated the expression of R3hdml. And R3hdml inhibited p38 mitogen-activated protein kinase phosphorylation, which was induced by TGF-β, leading to the amelioration of podocyte apoptosis. Furthermore, a lack of R3hdml in mice significantly worsened glomerular function in streptozotocin (STZ)-induced diabetes, while overexpression of R3hdml ameliorated albuminuria in STZ-induced diabetes. Our results surmise that the functional analyses of R3hdml may lead to the development of novel therapeutic strategies for diabetic nephropathy in the future. KEY MESSAGES: • A novel podocyte expressed protein R3h domain containing-like was identified. • R3HDML inhibits podocyte apoptosis by inhibiting TGF-β-mediated p38 MAPK signaling. • Overexpression of R3HDML ameliorates albuminuria in STZ-induced diabetes mice. • R3HDML may prove to be a novel therapeutic strategy for diabetic nephropathy.

Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabidopsis (Arabidopsis thaliana) triple mutant lacking all three DHAR genes (herein called ∆dhar) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated ∆dhar Arabidopsis plants as well as a quadruple mutant line combining ∆dhar with an additional vtc2 mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a nonnegligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Because GSH itself can reduce DHA nonenzymatically, we used the pad2 mutant that contains ∼30% of the wild-type GSH level. The pad2 mutant accumulated ascorbate at a wild-type level under high light; however, when the pad2 mutation was combined with ∆dhar, there was near-complete inhibition of high-light-dependent ascorbate accumulation. The lack of ascorbate accumulation was consistent with a marked increase in the ascorbate degradation product threonate. These findings indicate that ascorbate recycling capacity is limited in ∆dhar pad2 plants, and that both DHAR activity and GSH content set a threshold for high-light-induced ascorbate accumulation.

Aims/Introduction Recent randomized clinical trials have suggested that sodium–glucose cotransporter 2 inhibitors might reduce cardiovascular events and heart failure, and have renal protective effects. Despite these remarkable benefits, the effects of sodium–glucose cotransporter 2 inhibitors on bone and muscle are unclear. Materials and Methods A subanalysis of a randomized controlled study was carried out to evaluate the effects of the sodium–glucose cotransporter 2 inhibitor, ipragliflozin, versus metformin on bone and muscle in Japanese patients with type 2 diabetes mellitus (baseline body mass index ≥22 kg/m2 and hemoglobin A1c 7–10%) who were already receiving sitagliptin. These patients were randomly administered ipragliflozin 50 mg or metformin 1,000–1,500 mg daily. The effects of these medications on the bone formation marker, bone alkali phosphatase; the bone resorption marker, tartrate‐resistant acid phosphatase 5b (TRACP‐5b); handgrip strength; abdominal cross‐sectional muscle area; and bone density of the fourth lumbar vertebra were evaluated. Results After 24 weeks of treatment, the changes in bone density of the fourth lumbar vertebra, handgrip strength and abdominal cross‐sectional muscle area were not significantly different between the two groups. However, TRACP‐5b levels increased in patients treated with ipragliflozin compared with patients treated with metformin (median 11.94 vs −10.30%, P, Ipragliflozin in combination with sitagliptin did not affect bones or muscles adversely compared to metformin. However, ipragliflozin increased the levels of the bone resorption marker, tartrate‐resistant acid phosphatase 5b.

Objective Common reed (Phragmites communis Trin.) could potentially provide an alternative resource for silage; however, its silage quality is poor. The aim of this study was to investigate the factors in reed that contribute to poor quality and determine how the use of additives at ensiling could improve fermentation quality. Methods In Experiment 1, we determined the chemical composition and the presence of indigenous lactic acid bacteria (LAB) in reed. We further examined fermentation quality of reed silage under conditions without additives (NA) and treated glucose (G), lactic acid bacteria (L), and their combination (G+L). In Experiment 2, silage of NA, and with an addition of cellulase and lactic acid bacteria (CL) were prepared from harvested reed. The harvested reeds were fertilized at nitrogen concentrations of 0, 4, 8, and 12 g N/m2 and were harvested thrice within one year. Results The indigenous LAB and fermentable carbohydrates are at extremely low concentrations in reed. Reed silage, to which we added G+L, provided the highest quality silage among treatments in Experiment 1. In Experiment 2, N fertilization had no negative effect on silage quality of reed. The harvest times decreased fermentable carbohydrate content in reed. The CL treatment provided a higher lactic acid content compared to the NA treatment. However, the quality of CL treated silage at the second and third harvests was significantly lower than at the first harvest, due to a reduction in carbohydrates caused by frequent harvesting. Conclusion The causes of poor quality in reed silage are its lack of indigenous LAB and fermentable carbohydrates and its high moisture content. In addition, reed managed by frequent harvesting reduces carbohydrate content. Although the silage quality could be improved by adding CL, higher-quality silage could be prepared by adding fermentable carbohydrates, such as glucose (rather than adding cellulases).

In Euglena gracilis, wax ester fermentation produces ATP during anaerobiosis. Here, we report that anaerobic wax ester production is suppressed when the mitochondrial electron transport chain complex I is inhibited by rotenone, whereas it is increased by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The ADP/ATP ratio in anaerobic cells is elevated by treatment with either rotenone or CCCP. Gene silencing experiments indicate that acyl-CoA dehydrogenase, electron transfer flavoprotein (ETF), and rhodoquinone (RQ) participate in wax ester production. These results suggest that fatty acids are synthesized in mitochondria by the reversal of β-oxidation, where trans-2-enoyl-CoA is reduced mainly by acyl-CoA dehydrogenase using the electrons provided by NADH via the electron transport chain complex I, RQ, and ETF, and that ATP production is highly supported by anaerobic respiration utilizing trans-2-enoyl-CoA as a terminal electron acceptor.

A low 134 Cs/ 137 Cs ratio anomaly in the north-northwest (NNW) direction from the Fukushima Dai-ichi Nuclear Power Station (FDNPS) is identified by a new analysis of the 134 Cs/ 137 Cs ratio dataset which we had obtained in 2011–2015 by a series of car-borne surveys that employed a germanium gamma-ray spectrometer. We found that the 134 Cs/ 137 Cs ratio is slightly lower (0.95, decay-corrected to March 11, 2011) in an area with a length of about 15 km and a width of about 3 km in the NNW direction from the FDNPS than in other directions from the station. Furthermore, the area of this lower 134 Cs/ 137 Cs ratio anomaly corresponds to a narrow contamination band that runs NNW from the FDNPS and it is nearly parallel with the major and heaviest contamination band in the west-northwest. The plume trace with a low 134 Cs/ 137 Cs ratio previously found by other researchers within the 3-km radius of the FDNPS is in a part of the area with the lower 134 Cs/ 137 Cs ratio anomaly that we found. Our result suggests that this lower 134 Cs/ 137 Cs ratio anomaly is the area which was contaminated before March 13, 2011 (UTC) in association with the hydrogen explosion of Unit 1 on March 12, 2011 at 06:36 (UTC) and it was less influenced by later subsequent plumes.

We report the fabrication of novel nanofibers using naturally occurring antimicrobial honey incorporated in poly(1,4-cyclohexane dimethylene isosorbide trephthalate) (PICT) for the potential wound dressing applications. We fabricated PICT/honey using three blend ratios 90:10, 85:15 and 80:20 respectively. Morphology of PICT nanofibers and PICT/honey nanofibers was observed under Scanning Electron Microscope and it showed bead-free nanofibers. Fourier Transform Infrared Spectroscope was used to confirm the presence of honey in PICT electrospun nanofibers. Tensile strength of PICT/honey nanofibers was slightly reduced with variation in effect of elongation. Water contact angle measurements were done with the static contact angle by a contact angle meter, which showed that hydrophobicity was decreased by adding the honey. The XPS spectra showed that honey was present in the PICT/honey nanofibers. The release behavior of honey was investigated by UV-visible Spectrophotometer. The release was complete in 15min and the maximum release of honey was 72mg/L in 10min. Therefore, PICT/honey nanofibers having 15% concentration of honey are suitable for good elastic behavior and tensile strength as compared to other concentrations of honey.

In the microalga Euglena gracilis, the storage polysaccharide paramylon (β-1,3-D-glucan) is degraded to glucose, and finally converted to wax esters under anaerobic conditions. The wax esters and paramylon are now considered to be valuable materials for applications in biofuel production and in medicine. Genetic improvements of wax esters and paramylon accumulation in Euglena would facilitate their large-scale industrial applications; however, such improvements have thus far been difficult to realize because the regulatory factors involved in the wax ester fermentation pathway remain mostly unknown. Recently, two of dual-specificity Tyr phosphorylation-regulated kinases, starch degradation 1 (STD1) and triacylglycerol accumulation regulator 1 (TAR1), have been reported to regulate triacylglycerol metabolism in Chlamydomonas reinhardtii. In this study, we identified the Euglena DYRK orthologous sequences, and gene-silencing of EgSTD1 and EgSTD2 showed an increase in accumulation of paramylon and the following anaerobic wax ester production. This result indicated that EgSTD1 and EgSTD2 play a significant role in regulation of the wax ester fermentation pathway.

The phytoflagellate Euglena gracilis produces a large amount of paramylon (PM), a conglomerate of liner β-1,3-glucan chains, as a storage polysaccharide. PM is synthesized from uridine diphosphate-glucose, but its mechanism of formation is largely unknown. Two enzymes, glucan synthase-like (EgGSL) 1 and EgGSL2 were previously identified as candidates for PM synthesis in a Euglena transcriptome analysis. Here, we performed a reverse genetic analysis on these enzymes. Knockdown of EgGSL2, but not EgGSL1, significantly inhibits PM accumulation in Euglena cells. Additionally, β-1,3-glucan synthesis is detected in a PM-associated membrane fraction extracted from Euglena cells. Our findings indicate that EgGSL2 is the predominant enzyme for PM biosynthesis.

Oxidative stress is implicated in various human diseases and conditions, such as a neurodegeneration, which is the major symptom of vitamin B12 deficiency, although the underlying disease mechanisms associated with vitamin B12 deficiency are poorly understood. Vitamin B12 deficiency was found to significantly increase cellular H2O2 and NO content in Caenorhabditis elegans and significantly decrease low molecular antioxidant [reduced glutathione (GSH) and L-ascorbic acid] levels and antioxidant enzyme (superoxide dismutase and catalase) activities, indicating that vitamin B12 deficiency induces severe oxidative stress leading to oxidative damage of various cellular components in worms. An NaCl chemotaxis associative learning assay indicated that vitamin B12 deficiency did not affect learning ability but impaired memory retention ability, which decreased to approximately 58% of the control value. When worms were treated with 1 mmol/L GSH, L-ascorbic acid, or vitamin E for three generations during vitamin B12 deficiency, cellular malondialdehyde content as an index of oxidative stress decreased to the control level, but the impairment of memory retention ability was not completely reversed (up to approximately 50%). These results suggest that memory retention impairment formed during vitamin B12 deficiency is partially attributable to oxidative stress., Graphical abstract fx1

Skeletal muscle secretes biologically active proteins that contribute to muscle hypertrophy in response to either exercise or dietary intake. The identification of skeletal muscle-secreted proteins that induces hypertrophy can provide critical information regarding skeletal muscle health. Dietary provitamin A, β-carotene, induces hypertrophy of the soleus muscle in mice. Here, we hypothesized that skeletal muscle produces hypertrophy-inducible secretory proteins via dietary β-carotene. Knockdown of retinoic acid receptor (RAR) γ inhibited the β-carotene-induced increase soleus muscle mass in mice. Using RNA sequencing, bioinformatic analyses, and literature searching, we predicted transglutaminase 2 (TG2) to be an all-trans retinoic acid (ATRA)-induced secretory protein in cultured C2C12 myotubes. Tg2 mRNA expression increased in ATRA- or β-carotene-stimulated myotubes and in the soleus muscle of β-carotene-treated mice. Knockdown of RARγ inhibited β-carotene-increased mRNA expression of Tg2 in the soleus muscle. ATRA increased endogenous TG2 levels in conditioned medium from myotubes. Extracellular TG2 promoted the phosphorylation of Akt, mechanistic target of rapamycin (mTOR), and ribosomal p70 S6 kinase (p70S6K), and inhibitors of mTOR, phosphatidylinositol 3-kinase, and Src (rapamycin, LY294002, and Src I1, respectively) inhibited TG2-increased phosphorylation of mTOR and p70S6K. Furthermore, extracellular TG2 promoted protein synthesis and hypertrophy in myotubes. TG2 mutant lacking transglutaminase activity exerted the same effects as wild-type TG2. Knockdown of G protein-coupled receptor 56 (GPR56) inhibited the effects of TG2 on mTOR signaling, protein synthesis, and hypertrophy. These results indicated that TG2 expression was upregulated through ATRA-mediated RARγ and that extracellular TG2 induced myotube hypertrophy by activating mTOR signaling-mediated protein synthesis through GPR56, independent of transglutaminase activity.

Background Global demand for energy is on the rise at a time when limited natural resources are fast depleting. To address this issue, microalgal biofuels are being recommended as a renewable and eco-friendly substitute for fossil fuels. Euglena gracilis is one such candidate that has received special interest due to their ability to synthesize wax esters that serve as precursors for production of drop-in jet fuel. However, to realize economic viability and achieve industrial-scale production, development of novel methods to characterize algal cells, evaluate its culture conditions, and construct appropriate genetically modified strains is necessary. Here, we report a Raman microspectroscopy-based method to visualize important metabolites such as paramylon and ester during wax ester fermentation in single Euglena gracilis cells in a label-free manner. Results We measured Raman spectra to obtain intracellular biomolecular information in Euglena under anaerobic condition. First, by univariate approach, we identified Raman markers corresponding to paramylon/esters and constructed their time-lapse chemical images. However, univariate analysis is severely limited in its ability to obtain detailed information as several molecules can contribute to a Raman band. Therefore, we further employed multivariate curve resolution analysis to obtain chain length-specific information and their abundance images of the produced esters. Accumulated esters in Euglena were particularly identified to be myristyl myristate (C28), a wax ester candidate suitable to prepare drop-in jet fuel. Interestingly, we found accumulation of two different forms of myristyl myristate for the first time in Euglena through our exploratory multivariate analysis. Conclusions We succeeded in visualizing molecular-specific information in Euglena during wax ester fermentation by Raman microspectroscopy. It is obvious from our results that simple univariate approach is insufficient and that multivariate curve resolution analysis is crucial to extract hidden information from Raman spectra. Even though we have not measured any mutants in this study, our approach is directly applicable to other systems and is expected to deepen the knowledge on lipid metabolism in microalgae, which eventually leads to new strategies that will help to enhance biofuel production efficiency in the future.

Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short chain length CoA-substrates (such as crotonyl-CoA). A TER enzyme (EgTER1) has been previously identified and enzymologically characterized; however, its physiological significance remained to be evaluated by genetic analysis. We herein generated EgTER1-knockdown Euglena cells, in which total crotonyl-CoA reductase activity was decreased to 10% of control value. Notably, the knockdown cells showed a severe bleaching phenotype with deficiencies in chlorophylls and glycolipids, but grew normally under heterotrophic conditions (with glucose supplementation). Moreover, the knockdown cells accumulated much greater quantities of wax ester than control cells before and after transfer to anaerobic conditions, which was accompanied by a large metabolomic change. Furthermore, we failed to find any contribution of other potential TER genes in wax ester production. Our findings propose a novel role of EgTER1 in the greening process and demonstrate that this enzyme is dispensable for wax ester production under anaerobic conditions.

High-pressure components are generally designed with safety factors based on the tensile strength (TS) of the material; accordingly, materials with higher TS permit designed components with thinner walls, which reduce the weight and cost of the parts. However, many high-strength metals are severely degraded by hydrogen. To this point, efforts to develop a high-strength metal with a TS far beyond 1000 MPa and excellent resistance to hydrogen embrittlement (HE) have failed. This study introduces a high-strength metal with an excellent HE resistance, composed of a precipitation-hardened copper-beryllium alloy with the TS of 1400 MPa. Slow strain rate tensile (SSRT) tests of both smooth and notched specimens were performed in 115-MPa hydrogen gas at room temperature (RT). The alloy had a relative reduction in area RRA ≈ 1 and a relative notch tensile strength RNTS ≈ 1, without degradation in either characteristic.

We explored the superconductivity in a oxygen-hydrogen binary system, H h O1 − h , under highpressure using an evolutionary algorithm-based materials informatics approach. We searched for metallic and superconducting phases in pressures up to 300 GPa using a crystal structure prediction technique based on a genetic algorithm and first-principles calculations, and found that hydrogen superoxide (HO2) with an orthorhombic Pnnm, which emerges as a metastable compound in pressure above 180 GPa, is the only compound showing the superconductivity. The superconducting critical temperature T c is 9.78 K at 200 GPa, which is higher than that of pure solid oxygen, 2.87 K, due to the effect of the hydrogenation. We confirmed that the T c value shows a good agreement with potential superconducting critical temperature T c p o t estimated by the superconductivity predictor, which we have previously developed by a genetic programming using 497 first-principles datasets of other binary hydrides. Moreover, supposing that all the H h O1 − h compounds are superconductors at pressures in 150–200 GPa, we investigated the potential superconductivity of the H h O1 − h system using the predictor and obtained that T c p o t shows 10 K for h = 0.033 3 and increases to 100 K with the increase of h to 0.966 7.

Euglena gracilis exhibits photomovements in response to various light stimuli, such as phototactic and photophobic responses. Our recent study revealed that carotenoids in the eyespot apparatus are required for triggering phototaxis in this alga. However, the role of chloroplasts in eyespot formation is not understood. Here, we isolated carotenoid-less (cl) strains of E. gracilis from cells silenced gene expression of phytoene synthase (EgcrtB). Unlike WT, the culture colors of cl1, cl3, and the non-photosynthetic mutant SM-ZK were orange, while that of cl4 was white. Electron microscope observations showed that SM-ZK, cl1, and cl3 had no developed chloroplast and formed a normal eyespot apparatus, similar to that of WT, but this was not the case for cl4. Carotenoids detected in WT were diadinoxanthin, neoxanthin, and β-carotene. However, the most abundant species of SM-ZK, cl1, and cl3 was zeaxanthin, and there was no diadinoxanthin or neoxanthin. Photomovement analysis showed that SM-ZK, cl1, and cl3 exhibited negative phototactic and photophobic responses, similar to those of WT, whereas cl4 lacked negative phototaxis. Taken together, the formation of the eyespot apparatus required for phototaxis is independent of chloroplast development in E. gracilis, suggesting that this property is different from other photosynthetic flagellates.

Objective: To determine the effects of SGLT2 inhibitor or metformin on reducing visceral fat in Asian patients with type 2 diabetes inadequately controlled with DPP-4 inhibitor, sitagliptin. Methods: A prospective, multicenter, blinded-endpoint, randomized controlled trial was conducted to evaluate the efficacy of treatment with either SGLT2 inhibitor (ipragliflozin) or metformin added on to sitagliptin for visceral fat reduction and glucose control in patients with type 2 diabetes. Patients who met the eligibility criteria were randomly selected (1:1) to receive ipragliflozin (Ipr; 50 mg daily) or metformin (Met; 1000 mg daily). The primary outcome is the change rate in visceral fat area, measured using computed tomography, after 24 weeks of therapy. Two radiologists, blinded to the information, analyzed the images. Results: A total of 51 patients were in enrolled in the Ipr group and 52 in the Met group. The reduction rate in visceral fat area was significantly greater in the Ipr group (-12.06% vs. -3.65%, group difference of -8.40%; 95% CI of -16.4 to -3.38, P Conclusion: Ipr significantly reduced the visceral fat area compared with Met as the secondary agent used in combination with DPP-4 inhibitors. As the fasting insulin level decreased, the reduction in visceral fat associated with Ipr administration may have contributed to the improvement of insulin resistance. Disclosure M. Koshizaka: None. K. Ishikawa: None. R. Ishibashi: None. Y. Maezawa: None. K. Sakamoto: None. D. Uchida: Speaker's Bureau; Self; Takeda Pharmaceuticals Japan, Tanabe Mitsubishi Pharmaceuticals Japan, Sanofi, MSD K.K., Novo Nordisk Inc. S. Nakamura: None. H. Yokoh: None. A. Kobayashi: None. S. Onishi: None. K. Kobayashi: None. J. Ogino: None. H. Tokuyama: None. F. Shimada: None. E. Ohara: None. T. Ishikawa: None. M. Shoji: None. K. Ide: None. S. Ide: None. Y. Baba: None. T. Horikoshi: None. R. Shimofusa: None. S. Takahashi: None. K. Nagashima: None. M. Takemoto: None. K. Yokote: Research Support; Self; Astellas Pharm Ltd, MSD K.K..

Euglenoids are unique protists that can grow photoautotrophically, photomixotrophically, and heterotrophically. Here we grew Euglena gracilis under these different growth conditions and determined cellular contents of seven membrane lipids and one storage lipid (triacylglycerol), which account for more than 94 mol% of total membrane lipids. We also describe the relationship among chloroplast and mitochondria developments with lipid contents, protein contents, and oxygen evolution/consumption rates. In photoautotrophic growth conditions, E. gracilis cells accumulated chlorophyll, photosynthetic proteins, and glycolipids typical to thylakoid membranes. The same occurred for the cells grown under photomixotrophic conditions with higher respiration rates. In heterotrophic conditions, E. gracilis cells had higher respiration rates compared to cells grown in other conditions with the accumulation of pyruvate: NADP+ oxidoreductase, a mitochondrial protein and phospholipid common in mitochondria. Cells were also observed using a confocal laser scanning microscope and found to show more chlorophyll autofluorescence when grown photoautotrophically and photomixotrophycally, and fluorescence of MitoTracker when grown photomixotrophically and heterotrophically. These results suggest that under illumination, E. gracilis develops functional thylakoid membranes with membrane lipids and proteins for photosynthesis. In the medium with glucose, the cells develop mitochondria with phospholipids and proteins for respiration. Possible application based on lipid analysis for the enhancement of wax ester or alkene synthesis is discussed.

Background/aim Currently, treatment of non-muscle invasive bladder cancer causes significant deterioration in a patient's quality of life (QOL). Therefore, development of novel therapeutic options without the deterioration of QOL is very important. In this study, we assessed the anti-tumor effect of lentivirus-mediated gene transfection of tumor-suppressor genes in human bladder cancer cells. Materials and methods Lentiviral vectors that contained the tumor suppressor genes, p53, p16, and PTEN, were transfected into human bladder cancer cell lines, 5637, T24, 253J, and UMUC3, and the normal human uroepithelial cell line, SV-HUC-1. Results Significant growth inhibition was observed in bladder cancer cells on transfection with the p16 and PTEN vectors. However, the effect of the p53 vector was limited. In normal cells, the lentiviral vectors did not exhibit a significant growth inhibitory effect. Conclusion Lentiviral vector-mediated gene transfection is useful for the application of gene therapy in bladder cancers.

L-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

Podocalyxin is a cell surface sialomucin, which is expressed in not only glomerular podocytes but also vascular endothelial cells. Urinary podocalyxin is used as a marker for glomerular disease. However, there are no reports describing serum podocalyxin (s-Podxl) levels. Therefore, the association between s-Podxl levels and clinical parameters were examined with 52 patients. s-Podxl level was evaluated using enzyme-linked immunosorbent assay. The median s-Podxl level was 14.2 ng/dL (interquartile range: 10.8–22.2 ng/dL). There were significant correlations (correlation coefficient: r > 0.2) of s-Podxl levels with carotid intima media thickness (IMT) (r = 0.30, p = 0.0307). Multiple logistic regression analysis showed that s-Podxl levels remained significantly associated with carotid IMT > 1 mm (OR: 1.15; 95% CI 1.02–1.31, p = 0.026) after adjustments for traditional cardiovascular risk factors such as age, sex, current smoking status, hypertension, dyslipidemias, and diabetes. In conclusion, s-Podxl is independently associated with carotid IMT and might be used as a novel biomarker for cardiovascular disease.

Ascorbate is the most abundant water-soluble redox compound in plants and has multiple functions as a redox buffer. There is compelling genetic evidence that the biosynthesis of ascorbate proceeds via the d-mannose/l-galactose pathway and is the most significant source of ascorbate in plants. Alternative pathways, including those via d-galacturonate and d-glucuronate, have been proposed but there is no convincing genetic evidence. In order to carry out its physiological functions, cellular ascorbate contents need to be tightly controlled in response to various environmental conditions. In this chapter, we will describe the recent progression on the metabolic process of ascorbate biosynthesis and its regulation.

Background: Visceral fat accumulation is associated with insulin resistance and various metabolic complications. We aimed to determine the effect of ipragliflozin (sodium-dependent glucose transporter-2 inhibitor) versus metformin with a dipeptidyl peptidase-4 (DPP-4) inhibitor (sitagliptin) in reducing visceral fat in Asian patients with insufficiently controlled type 2 diabetes. Methods: A prospective, multicenter, blinded-endpoint, randomized controlled study was conducted to evaluate the efficacy of treatment with either ipragliflozin or metformin combined with sitagliptin on visceral fat reduction and glycemic control among Japanese patients with type 2 diabetes, HbA1c 7-10%, and BMI ≥22 kg/m2. Patients who met the eligibility criteria were randomly assigned (1:1) to receive ipragliflozin 50 mg (n=51) or metformin 1000-1500 mg daily (n=52). The primary outcome was rate change in visceral fat area measured by computed tomography at 24 weeks of therapy. Two radiologists, blinded to patients' clinical information and randomized treatment assignment, analyzed the images. Secondary outcomes included effects on blood glucose, fasting insulin level, and lipids. The full analysis set was used for analysis. Findings: Mean percent reduction in visceral fat area was significantly greater in the ipragliflozin group than in the metformin group (-12·06% vs. -3·65%, group difference [95% CI] -8·40%; [-16·4 to -3·38], P=0·040). The ipragliflozin group also showed significant lowering of fasting insulin (-20·73% vs. 0·85%, group difference [95% CI] -21·58%, [2·80 to 34·20], P=0·018), while HbA1c decreased in the metformin group compared with ipragliflozin group. Interpretation: Ipragliflozin significantly reduced the visceral fat area compared with metformin, as the secondary agent used in combination with DPP-4 inhibitor. Since the fasting insulin level decreased, the reduction in visceral fat associated with ipragliflozin administration might have contributed to the improvement in insulin resistance. Trial Registration Number: Trial registration: UMIN 000015170. Funding Statement: To conduct this study, an agreement was signed between Chiba University and Astellas Pharma Inc. (Tokyo, Japan). Astellas Pharma Inc. funded this work. Declaration of Interests: KY received research grants from Astellas Pharma Inc. and MSD K.K. (Tokyo, Japan), and received a lecture fee from Astellas Pharma Inc. and Sumitomo Dainippon Pharma (Tokyo, Japan). No conflicts of interest are declared for other authors. Ethics Approval Statement: The study protocol was approved by the Institutional Review Boards (IRBs), Ethics Review Committees, or Ethics Committees of the following institutions: Chiba University Hospital (ID number: G26009), Asahi General Hospital (ID number: 2014091602), National Hospital Organization Chiba Medical Center, Seirei Sakura Citizen Hospital, Chiba Rosai Hospital (ID number: 26-21), Toho University Sakura Medical Center (ID number: 2014-077), Tokyo Women’s Medical University Yachiyo Medical Center (ID number: 150303), Chiba Aoba Municipal Hospital, Kimitsu Chuo Hospital, Funabashi Central Hospital (ID number: H27-1), and Chiba Kaihin Municipal Hospital. In other facilities, approval was provided at Chiba University Hospital (which had the centralized IRB).

Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The Km and Vmax values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min−1 mg protein−1, respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells. EgFBPaseIII, a cytosolic FBPase, may play an important role in carbon partitioning between paramylon and wax ester biosynthesis in Euglena gracilis.

Background Patients with type 2 diabetes are at high risk for cardiovascular disease. Although hydroxymethylglutaryl-CoA reductase inhibitors (statins) can reduce cardiovascular events, residual risk remains even after target low-density lipoprotein cholesterol (LDL-C) levels have been achieved. Lipoprotein particle size and fraction changes are thought to contribute to such risks. The purpose of this study was to evaluate the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs), predominantly eicosapentaenoic acid and docosahexaenoic acid, on lipoprotein particle size, concentration, and glycemic control in Japanese patients with type 2 diabetes and hypertriglyceridemia. Methods This was a multicenter, prospective, open-label, single arm study. We enrolled 14 patients with type 2 diabetes and hypertriglyceridemia treated with statins and dipeptidyl peptidase-4 inhibitors with glycated hemoglobin (HbA1c)

Wax ester fermentation is a unique energy gaining pathway for a unicellular phytoflagellated protozoan, Euglena gracilis, to survive under anaerobiosis. Wax esters produced in E. gracilis are composed of saturated fatty acids and alcohols, which are the major constituents of myristic acid and myristyl alcohol. Thus, wax esters can be promising alternative biofuels. Here, we report the identification and characterization of wax ester synthase/diacylglycerol acyltrasferase (WSD) isoenzymes as the terminal enzymes of wax ester production in E. gracilis. Among six possible Euglena WSD orthologs predicted by BLASTX search, gene expression analysis and in vivo evaluation for enzyme activity with yeast expressing individual recombinant WSDs indicated that two of them (EgWSD2 and EgWSD5) predominantly function as wax ester synthase. Furthermore, experiments with gene silencing demonstrated a pivotal role of both EgWSD2 and EgWSD5 in wax ester synthesis, as evidenced by remarkably reduced wax ester contents in EgWSD2/5-double knockdown E. gracilis cells treated with anaerobic conditions. Interestingly, the decreased ability to produce wax ester did not affect adaptation of E. gracilis to anaerobiosis. Lipid profile analysis suggested allocation of metabolites to other compounds including triacylglycerol instead of wax esters.

Major intrinsic proteins (MIPs), commonly known as aquaporins, transport water and non-polar small solutes. Comparing the 3D models and the primary selectivity-related motifs (two Asn-Pro-Ala (NPA) regions, the aromatic/arginine (ar/R) selectivity filter, and Froger’s positions (FPs)) of all plant MIPs that have been experimentally proven to transport arsenic (As) and antimony (Sb), some substrate-specific signature sequences (SSSS) or specificity determining sites (SDPs) have been predicted. These SSSS or SDPs were determined in 543 MIPs found in the genomes of 12 crop plants; the As and Sb transporters were predicted to be distributed in noduline-26 like intrinsic proteins (NIPs), and every plant had one or several As and Sb transporter NIPs. Phylogenetic grouping of the NIP subfamily based on the ar/R selectivity filter and FPs were linked to As and Sb transport. We further determined the group-wise substrate selectivity profiles of the NIPs in the 12 crop plants. In addition to two NPA regions, the ar/R filter, and FPs, certain amino acids especially in the pore line, loop D, and termini contribute to the functional distinctiveness of the NIP groups. Expression analysis of transcripts in different organs indicated that most of the As and Sb transporter NIPs were expressed in roots.

Background Photosynthetic organisms utilize carotenoids for photoprotection as well as light harvesting. Our previous study revealed that high-intensity light increases the expression of the gene for phytoene synthase (EgcrtB) in Euglena gracilis (a unicellular phytoflagellate), the encoded enzyme catalyzes the first committed step of the carotenoid biosynthesis pathway. To examine carotenoid synthesis of E. gracilis in response to light stress, we analyzed carotenoid species and content in cells grown under various light intensities. In addition, we investigated the effect of suppressing EgcrtB with RNA interference (RNAi) on growth and carotenoid content. Results After cultivation for 7 days under continuous light at 920 μmol m−2 s−1, β-carotene, diadinoxanthin (Ddx), and diatoxanthin (Dtx) content in cells was significantly increased compared with standard light intensity (55 μmol m−2 s−1). The high-intensity light (920 μmol m−2 s−1) increased the pool size of diadinoxanthin cycle pigments (i.e., Ddx + Dtx) by 1.2-fold and the Dtx/Ddx ratio from 0.05 (control) to 0.09. In contrast, the higher-intensity light treatment caused a 58% decrease in chlorophyll (a + b) content and diminished the number of thylakoid membranes in chloroplasts by approximately half compared with control cells, suggesting that the high-intensity light-induced accumulation of carotenoids is associated with an increase in both the number and size of lipid globules in chloroplasts and the cytoplasm. Transient suppression of EgcrtB in this alga by RNAi resulted in significant decreases in cell number, chlorophyll, and total major carotenoid content by 82, 82 and 86%, respectively, relative to non-electroporated cells. Furthermore, suppression of EgcrtB decreased the number of chloroplasts and thylakoid membranes and increased the Dtx/Ddx ratio by 1.6-fold under continuous illumination even at the standard light intensity, indicating that blocking carotenoid synthesis increased the susceptibility of cells to light stress. Conclusions Our results indicate that suppression of EgcrtB causes a significant decrease in carotenoid and chlorophyll content in E. gracilis accompanied by changes in intracellular structures, suggesting that Dtx (de-epoxidized form of diadinoxanthin cycle pigments) contributes to photoprotection of this alga during the long-term acclimation to light-induced stress. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1066-7) contains supplementary material, which is available to authorized users.

The adaptation to a strong light is one of the essential characteristics of green algae, yet lacking relatively the information about the photophobic responses of Eukaryotic microalgae. We investigated the photophobic step-up responses of Euglena gracilis over a time course of several hours with alternated repetition of blue-light pulse illumination and spatially patterned blue-light illumination. Four distinctive photophobic motions in response to strong blue light were identified in a trace image analysis, namely on-site rotation, running and tumbling, continuous circular swimming, and unaffected straightforward swimming. The cells cultured in autotrophic conditions under weak light showed mainly the on-site rotation response at the beginning of blue-light illumination, but they acquired more blue-light tolerant responses of running and tumbling, circular swimming, or straightforward swimming. The efficiency of escaping from a blue-light illuminated area improved markedly with the development of these photophobic motions. Time constant of 3.0 h was deduced for the evolution of photophobic responses of E. gracilis. The nutrient-rich metabolic status of the cells resulting from photosynthesis during the experiments, i.e., the accumulation of photosynthesized nutrient products in balance between formation and consumption, was the main factor responsible for the development of photophobic responses. The reduction-oxidation status in and around E. gracilis cells did not affect their photophobic responses significantly, unlike the case of photophobic responses and phototaxis of Chlamydomonas reinhardtii cells. This study shows that the evolution of photophobic motion type of E. gracilis is dominated mainly by the nutrient metabolic status of the cells. The fact suggests that the nutrient-rich cells have a higher threshold for switching the flagellar motion from straightforward swimming to rotation under a strong light.

Ascorbate peroxidases (APXs) are, in general, photosynthetic eukaryote-specific enzymes, which catalyze the reduction of H2O2 using ascorbate as an electron donor. Considering the very low affinity of ascorbate with H2O2, the acquisition of APX was certainly an important event, allowing plants to use ascorbate for H2O2 metabolism. This also provides a plausible explanation for why plants accumulate a massive amount of ascorbate because this substrate is also required for stabilizing fragile APX enzymes (particularly chloroplastic isoforms). In higher plants, APXs are distributed in the cytosol, mitochondria, chloroplasts (both stroma and thylakoid membrane), and peroxisomes to modulate organellar and cellular levels of H2O2. Despite its potential toxicity, H2O2 is a relatively stable form of a reactive oxygen species, and consequently it can act as a key signaling molecule for plant stress responses. From this point of view, APXs also have a dual role, being antioxidant enzymes and H2O2 signaling regulators, and their balance is crucial for fine-tuning stress responses. In this chapter, we describe the physiological roles of APX isoforms in plants by overviewing the findings of biochemical, physiological, and genetic studies.

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are by-products of various metabolic processes in aerobic organisms including Euglena. Chloroplasts and mitochondria are the main sites of ROS generation by photosynthesis and respiration, respectively, through the active electron transport chain. An efficient antioxidant network is required to maintain intracellular ROS pools at optimal conditions for redox homeostasis. A comparison with the networks of plants and animals revealed that Euglena has acquired some aspects of ROS metabolic process. Euglena lacks catalase and a typical selenocysteine containing animal-type glutathione peroxidase for hydrogen peroxide scavenging, but contains enzymes involved in ascorbate-glutathione cycle solely in the cytosol. Ascorbate peroxidase in Euglena, which plays a central role in the ascorbate-glutathione cycle, forms a unique intra-molecular dimer structure that is related to the recognition of peroxides. We recently identified peroxiredoxin and NADPH-dependent thioredoxin reductase isoforms in cellular compartments including chloroplasts and mitochondria, indicating the physiological significance of the thioredoxin system in metabolism of ROS. Besides glutathione, Euglena contains the unusual thiol compound trypanothione, an unusual form of glutathione involving two molecules of glutathione joined by a spermidine linker, which has been identified in pathogenic protists such as Trypanosomatida and Schizopyrenida. Furthermore, in contrast to plants, photosynthesis by Euglena is not susceptible to hydrogen peroxide because of resistance of the Calvin cycle enzymes fructose-1,6-bisphosphatse, NADP+-glyceraldehyde-3-phosphatase, sedoheptulose-1,7-bisphosphatase, and phosphoribulokinase to hydrogen peroxide. Consequently, these characteristics of Euglena appear to exemplify a strategy for survival and adaptation to various environmental conditions during the evolutionary process of euglenoids.

In Euglena cells under anaerobic conditions, paramylon, the storage polysaccharide, is promptly degraded and converted to wax esters. The wax esters synthesized are composed of saturated fatty acids and alcohols with chain lengths of 10–18, and the major constituents are myristic acid and myristyl alcohol. Since the anaerobic cells gain ATP through the conversion of paramylon to wax esters, the phenomenon is named “wax ester fermentation”. The wax ester fermentation is quite unique in that the end products, i.e. wax esters, have relatively high molecular weights, are insoluble in water, and accumulate in the cells, in contrast to the common fermentation end products such as lactic acid and ethanol.