Published papers

© 2018 American Association for Clinical Chemistry BACKGROUND: Although circulating exosomes in blood play crucial roles in cancer development and progression, difficulties in quantifying exosomes hamper their application for reliable clinical testing. By combining the properties of nanobeads with optical disc technology, we have developed a novel device named the ExoCounter to determine the exact number of exosomes in the sera of patients with various types of cancer. METHOD: In this system, individual exosomes were captured in the groove of an optical disc coated with antibodies against exosome surface antigens. The captured exosomes were labeled with antibody-conjugated magnetic nanobeads, and the number of the labeled exosomes was counted with an optical disc drive. RESULTS: We showed that the ExoCounter could detect specific exosomes derived from cells or human serum without any enrichment procedures. The detection sensitivity and linearity with this system were higher than those with conventional detection methods such as ELISA or flow cytometry. In addition to the ubiquitous exosome markers CD9 and CD63, the cancer-related antigens CD147, carcinoembryonic antigen, and human epidermal growth factor receptor 2 (HER2) were also used to quantify cancer cell line-derived exosomes. Furthermore, analyses of a cross-sectional cohort of sera samples revealed that HER2-positive exosomes were significantly increased in patients with breast cancer or ovarian cancer compared with healthy individuals and those with noncancer diseases. CONCLUSIONS: The ExoCounter system exhibits high performance in the direct detection of exosomes in cell culture and human sera. This method may enable reliable analysis of liquid biopsies.

© 2019 The Authors Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our culture system provides a means to evaluate properties of steady-state HSCs and test effects of defined factors in vitro under near-physiological conditions. Maintaining quiescent HSCs under physiological conditions facilitates evaluation of the properties of steady-state HSCs. Kobayashi et al. report that low cytokine concentrations, hypoxia, and high fatty acid levels mimicking the bone marrow microenvironment enable maintenance of engraftable quiescent HSCs for 1 month in culture.

© 2019 International Institute of Anticancer Research. All Rights Reserved. Background/Aim: The present study assessed whether and how tumor cells undergoing hypoxia contribute to disease progression after moving to areas with different oxygen conditions. Materials and Methods: Human colorectal carcinoma HCT116 cells cultured under mild hypoxia were subjected to in vivo experiments using transfer to immunodeficient murine recipients and to in vitro experiments using pharmacological inhibition of fatty acid β-oxidation (FAO). Results: Bone involvement and hepatic metastases were accelerated in transfer models of hypoxically cultured HCT116 cells. Hypoxic HCT116 cells exhibited FAO-dependent glycogen synthesis. FAO-dependent and -independent induction of gene expression also occurred under hypoxia. The distribution of glucose transporter 1 expression compared with heme oxygenase 1 expression in HCT116 cell spheroids seemed consistent with differential dependence of hypoxic expression of these molecules on FAO. Conclusion: These results provide insights into the contribution of hypoxia to tumor progression and the relevance of FAO.

© 2018 JCBN. Progesterone receptor membrance associated component 1 is a multifunctional heme-binding protein that plays a role in several biological processes such as tumor progression, metabolic regulation, and viability control of nerve cells. Notably, progesterone receptor membrane associated component 1 is highly expressed in various types of cancer cells, and facilitates cancer proliferation and chemoresistance. Recently, progesterone receptor membrane associated component 1 structure has been explored by X-ray crystallographic analysis. Interestingly, whereas apoprogesterone receptor membrane associated component 1 exists as a monomer, the heme-bound progesterone receptor membrane associated component 1 converts into a stable dimer by forming a unique heme-heme stacking structure, leading to activation of epidermal growth factor receptor signaling and chemoresistance in cancer cells. Furthermore, the gas mediator carbon monoxide inhibits progesterone receptor membrane associated component 1-mediated activation in cancer cells by dissociating the heme-stacking dimer of progesterone receptor membrane associated component 1. The dynamic structural regulation of progesterone receptor membrane associated component 1 will provide new insights for understanding the mechanisms underlying its various functions.

© The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Cardiology. Aims The metabolism of the failing heart is characterized by an increase in glucose uptake with reduced fatty acid (FA) oxidation. We previously found that the genetic deletion of FA-binding protein-4 and-5 [double knockout (DKO)] induces an increased myocardial reliance on glucose with decreased FA uptake in mice. However, whether this fuel switch confers functional benefit during the hypertrophic response remains open to debate. To address this question, we investigated the contractile function and metabolic profile of DKO hearts subjected to pressure overload. Methods and results Transverse aortic constriction (TAC) significantly reduced cardiac contraction in DKO mice (DKO-TAC), although an increase in cardiac mass and interstitial fibrosis was comparable with wild-Type TAC (WT-TAC). DKO-TAC hearts exhibited enhanced glucose uptake by 8-fold compared with WT-TAC. Metabolic profiling and isotopomer analysis revealed that the pool size in the TCA cycle and the level of phosphocreatine were significantly reduced in DKO-TAC hearts, despite a marked increase in glycolytic flux. The ingestion of a diet enriched in medium-chain FAs restored cardiac contractile dysfunction in DKO-TAC hearts. The de novo synthesis of amino acids as well as FA from glycolytic flux was unlikely to be suppressed, despite a reduction in each precursor. The pentose phosphate pathway was also facilitated, which led to the increased production of a coenzyme for lipogenesis and a precursor for nucleotide synthesis. These findings suggest that reduced FA utilization is not sufficiently compensated by a robust increase in glucose uptake when the energy demand is elevated. Glucose utilization for sustained biomass synthesis further enhances diminishment of the pool size in the TCA cycle. Conclusions Our data suggest that glucose is preferentially utilized for biomass synthesis rather than ATP production during pressure-overload-induced cardiac hypertrophy and that the efficient supplementation of energy substrates may restore cardiac dysfunction caused by energy insufficiency.

© 2018 The Author(s). Gold deposition with diagonal angle towards boehmite-based nanostructure creates random arrays of horse-bean-shaped nanostructures named gold-nanofève (GNF). GNF generates many electromagnetic hotspots as surface-enhanced Raman spectroscopy (SERS) excitation sources, and enables large-area visualization of molecular vibration fingerprints of metabolites in human cancer xenografts in livers of immunodeficient mice with sufficient sensitivity and uniformity. Differential screening of GNF-SERS signals in tumours and those in parenchyma demarcated tumour boundaries in liver tissues. Furthermore, GNF-SERS combined with quantum chemical calculation identified cysteine-derived glutathione and hypotaurine (HT) as tumour-dominant and parenchyma-dominant metabolites, respectively. CD44 knockdown in cancer diminished glutathione, but not HT in tumours. Mechanisms whereby tumours sustained HT under CD44-knockdown conditions include upregulation of PHGDH, PSAT1 and PSPH that drove glycolysis-dependent activation of serine/glycine-cleavage systems to provide one-methyl group for HT synthesis. HT was rapidly converted into taurine in cancer cells, suggesting that HT is a robust anti-oxidant for their survival under glutathione-suppressed conditions.

© 2018 Matsui et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Discovery of unidentified protein functions is of biological importance because it often provides new paradigms for many research areas. Mammalian heme oxygenase (HO) enzyme catalyzes the O2-dependent degradation of heme into carbon monoxide (CO), iron, and biliverdin through numerous reaction intermediates. Here, we report that H2S, a gaseous signaling molecule, is part of a novel reaction pathway that drastically alters HO's products, reaction mechanism, and catalytic properties. Our prediction of this interplay is based on the unique reactivity of H2S with one of the HO intermediates. We found that in the presence of H2S, HO produces new linear tetrapyr-roles, which we identified as isomers of sulfur-containing biliverdin (SBV), and that only H2S, but not GSH, cysteine, and polysulfides, induces SBV formation. As BV is converted to bilirubin (BR), SBV is enzymatically reduced to sulfur-containing bilirubin (SBR), which shares similar properties such as antioxidative effects with normal BR. SBR was detected in culture media of mouse macrophages, confirming the existence of this H2S-induced reaction in mammalian cells. H2S reacted specifically with a ferric verdoheme intermediate of HO, and verdoheme cleavage proceeded through an O2-independent hydrolysis-like mechanism. This change in activation mode diminished O2 dependence of the overall HO activity, circumventing the rate-limiting O2 activation of HO. We propose that H2S could largely affect O2 sensing by mammalian HO, which is supposed to relay hypoxic signals by decreasing CO output to regulate cellular functions. Moreover, the novel H2S-induced reaction identified here helps sustain HO's heme-degrading and antioxidant-generating capacity under highly hypoxic conditions.

© 2020, The Author(s). Physiological regulation of blood flow in bone marrow is important to maintain oxygen and glucose supplies but also the physiological hypoxic state of the hematopoietic stem cell (HSC) niche. However, regulatory mechanisms underlying microcirculation in the bone marrow (BM) niche remain unclear. Here, we identify vessels functioning in control of blood flow in bone marrow and assess their contractility. To evaluate contractile potential of Alexa Fluor 633 (AF633; an arterial marker)-positive vessels, we performed immunohistochemistry for α-smooth muscle actin (α-SMA) and found it expressed around AF633+ vessels in the femoral and calvarial marrow. To validate AF633+ vessel contractility, we developed a simple system to locally administer vasoactive agents that penetrate BM through transcalvarial vessels. After exposure of the calvarial surface to FITC-dextran (70 kDa), FITC intensity in calvarial bone marrow gradually increased. When we evaluated the effect of transcalvarial administration (TCA) of norepinephrine (NE) on vascular tone of AF633+ arteries and behavior of transplanted blood cells, NE administration decreased artery diameter and transendothelial migration of transplanted cells, suggesting that adrenergic signaling regulates the HSC niche microcirculation and blood cell migration into the BM via effects on BMarteries. We conclude that TCA is a useful tool for bone marrow research.

© 2019, The Author(s). Sesamin is a furofuran-type lignan that is found abundantly in seeds of Sesamum indicum (sesame) and has been widely accepted as a dietary supplement with positive effects on human health. The biological activity of sesamin in human cells and organs has been analysed extensively, although comparatively few studies show biological functions for sesamin in planta. Herein we screened sesamin-binding proteins (SBP) from sesame seedling extracts using sesamin-immobilized nano-beads. In subsequent peptide mass fingerprinting analyses, we identified a SBP, Steroleosin B, which is one of the membrane proteins found in oil bodies. In addition, pull-down assays and saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments demonstrated that sesamin binds directly to recombinant Steroleosin B in vitro. Finally, ectopic accumulations of sesamin and Steroleosin B in transgenic Arabidopsis thaliana plants induced severe growth defects including suppression of leaf expansion and root elongation. Collectively, these results indicate that sesamin influences tissue development in the presence of Steroleosin B.

© 2020, The Author(s). Under hypoxic conditions, nitroimidazoles can replace oxygen as electron acceptors, thereby enhancing the effects of radiation on malignant cells. These compounds also accumulate in hypoxic cells, where they can act as cytotoxins or imaging agents. However, whether these effects apply to cancer stem cells has not been sufficiently explored. Here we show that the 2-nitroimidazole doranidazole potentiates radiation-induced DNA damage in hypoxic glioma stem cells (GSCs) and confers a significant survival benefit in mice harboring GSC-derived tumors in radiotherapy settings. Furthermore, doranidazole and misonidazole, but not metronidazole, manifested radiation-independent cytotoxicity for hypoxic GSCs that was mediated by ferroptosis induced partially through blockade of mitochondrial complexes I and II and resultant metabolic alterations in oxidative stress responses. Doranidazole also limited the growth of GSC-derived subcutaneous tumors and that of tumors in orthotopic brain slices. Our results thus reveal the theranostic potential of 2-nitroimidazoles as ferroptosis inducers that enable targeting GSCs in their hypoxic niche.

© 2019, Springer Nature Limited. There is a growing appreciation for the importance of the gut microbiota as a therapeutic target in various diseases. However, there are only a handful of known commensal strains that can potentially be used to manipulate host physiological functions. Here we isolate a consortium of 11 bacterial strains from healthy human donor faeces that is capable of robustly inducing interferon-γ-producing CD8 T cells in the intestine. These 11 strains act together to mediate the induction without causing inflammation in a manner that is dependent on CD103 + dendritic cells and major histocompatibility (MHC) class Ia molecules. Colonization of mice with the 11-strain mixture enhances both host resistance against Listeria monocytogenes infection and the therapeutic efficacy of immune checkpoint inhibitors in syngeneic tumour models. The 11 strains primarily represent rare, low-abundance components of the human microbiome, and thus have great potential as broadly effective biotherapeutics.

© 2018 Elsevier B.V. The quantification of aldosterone (ALD) in adrenal tributary venous blood serum/plasma combined with the super-selective adrenal venous sampling (ssAVS) technique is recognized as a definitive procedure for differentiation of the forms of primary aldosteronism (PA), identification of the affected segment(s) and operating decision-making. In this study, an enhanced throughput and sensitive method was developed and validated for the quantification of ALD in ssAVS serum samples by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using the Girard P reagent (GP) isotopologues (2H0- and 2H5-GP). The right and left adrenal serum samples were separately pretreated and derivatized with either isotopologue. The two samples were then combined and injected together for LC/ESI-MS/MS analysis. Based on the mass differences between the isotopologues, the derivatized ALD in the two samples were quantified within a single run. This method enabled the reproducible (intra- and inter-assay relative standard deviations, 6.6% or lower) and accurate (98.2–107.0%) quantification of the serum ALD using a 25-μL sample, and the lower limit of quantification was 1.0 ng/mL. The developed method was used for the analysis of 11 pairs of ssAVS serum samples (total of 22 samples) of patients with ALD-producing adenoma and proven to have a satisfactory applicability; this method enabled the identification of the affected adrenal and the determination of the laterality of PA, and reduced the analysis time to about 2/3 compared to the previous method for 22 samples.

© 2019, The Author(s). Sucralfate is effective for the treatment of gastric and duodenal ulcers owing to its protective gel-forming ability. However, the mechanism by which sucralfate protects the oesophageal mucosa against reflux oesophagitis has not been clarified. We aimed to investigate the mechanisms of action of sucralfate and sucrose octasulfate (SOS), a component of sucralfate. SOS and sucralfate were administered to oesophagitis-induced rats, and the ulcer lesion size was macroscopically examined and scored. Effective pepsin activity in the gastric juices obtained from the animal model was evaluated by a casein digestion test. Sucralfate and SOS improved the pathology scores in a dose-dependent manner, whereas gastric juice pepsin activity was not impaired by therapeutic doses of SOS. As SOS lacks the ability to form a thick gel layer by polymerisation, we examined the distribution of SOS in the mucosal lumen by imaging mass spectrometry (IMS) to determine whether SOS directly adheres to the mucosal surface. A clear homogeneous thin-layer SOS film (>100 μm thick) was visualized on the oesophageal mucosal surface. Moreover, this SOS film formation was enhanced at ulcer lesion sites. Taken together, SOS appears to protect oesophageal mucosa against reflux oesophagitis via thin-layer formation on the mucosal surface.

© 2018 American Heart Association, Inc. Primary aldosteronism is a secondary hypertensive disease caused by autonomous aldosterone production that often caused by an aldosterone-producing adenoma (APA). Immunohistochemistry of aldosterone synthase (CYP11B2) shows the presence of aldosterone-producing cell clusters (APCCs) even in non-primary aldosteronism adult adrenal cortex. An APCC-like structure also exists as possible APCC-to-APA transitional lesions (a speculative designation) in primary aldosteronism adrenals. However, whether APCCs produce aldosterone or 18-oxocortisol, a potential serum marker of APA, remains unknown because of lack of technology to visualize adrenocorticosteroids on tissue sections. To address this obstacle, in this study, we used highly sensitive Fourier transform ion cyclotron resonance mass spectrometry to image various adrenocorticosteroids, including 18-oxocortisol, in adrenal tissue sections from 8 primary aldosteronism patients with APCC (cases 1-4), possible APCC-to-APA transitional lesions (case 5), and APA (cases 6-8). Further analyses by tandem mass spectrometry imaging allowed us to differentially visualize aldosterone from cortisone, which share identical mass-to-charge ratio value (m/z). In conclusion, these advanced imaging techniques revealed that aldosterone and 18-oxocortisol coaccumulated within CYP11B2-expressing lesions. These imaging outcomes along with a growing body of aldosterone research led us to build a progressive development hypothesis of an aldosterone-producing pathology in the adrenal glands.

© 2020, The Author(s). Sesamin [(7α,7′α,8α,8′α)-3,4:3′,4′-bis(methylenedioxy)-7,9′:7′,9-diepoxylignane] is a major lignan in sesame seeds. Sesamin is converted to the catechol metabolite, SC1 [(7α,7′α,8α,8′α)-3′,4′-methylenedioxy-7,9′:7′,9-diepoxylignane-3,4-diol] with anti-inflammatory effects after oral administration. However, its molecular target remains unknown. Analysis using high-performance affinity nanobeads led to the identification of annexin A1 (ANX A1) as an SC1-binding protein. SC1 was found to bind to the annexin repeat 3 region of ANX A1 with a high-affinity constant (Kd = 2.77 μmol L−1). In U937 cells, SC1 exhibited an anti-inflammatory effect dependent on ANX A1. Furthermore, administration of sesamin or SC1 attenuated carbon tetrachloride-induced liver damage in mice and concurrently suppressed inflammatory responses dependent on ANX A1. The mechanism involved SC1-induced ANX A1 phosphorylation at serine 27 that facilitates extracellular ANX A1 release. Consequently, the ANX A1 released into the extracellular space suppressed the production of tumor necrosis factor α. This study demonstrates that ANX A1 acts as a pivotal target of sesamin metabolites to attenuate inflammatory responses.

© 2019, The Author(s). Nanomaterials have extensive applications in the life sciences and in clinical diagnosis. We have developed magnetic nanoparticles with high dispersibility and extremely low nonspecific binding to biomolecules and have demonstrated their application in chemical biology (e.g., for the screening of drug receptor proteins). Recently, the excellent properties of nanobeads have made possible the development of novel rapid immunoassay systems and high-precision technologies for exosome detection. For immunoassays, we developed a technology to encapsulate a fluorescent substance in magnetic nanobeads. The fluorescent nanobeads allow the rapid detection of a specific antigen in solution or in tissue specimens. Exosomes, which are released into the blood, are expected to become markers for several diseases, including cancer, but techniques for measuring the absolute quantity of exosomes in biological fluids are lacking. By integrating magnetic nanobead technology with an optical disc system, we developed a novel method for precisely quantifying exosomes in human serum with high sensitivity and high linearity without requiring enrichment procedures. This review focuses on the properties of our magnetic nanobeads, the development of novel biosensors using these nanobeads, and their broad practical applications. [Figure not available: see fulltext.].

© 2019, The Author(s). Selective autophagy ensures the removal of specific soluble proteins, protein aggregates, damaged mitochondria, and invasive bacteria from cells. Defective autophagy has been directly linked to metabolic disorders. However how selective autophagy regulates metabolism remains largely uncharacterized. Here we show that a deficiency in selective autophagy is associated with suppression of lipid oxidation. Hepatic loss of Atg7 or Atg5 significantly impairs the production of ketone bodies upon fasting, due to decreased expression of enzymes involved in β-oxidation following suppression of transactivation by PPARα. Mechanistically, nuclear receptor co-repressor 1 (NCoR1), which interacts with PPARα to suppress its transactivation, binds to the autophagosomal GABARAP family proteins and is degraded by autophagy. Consequently, loss of autophagy causes accumulation of NCoR1, suppressing PPARα activity and resulting in impaired lipid oxidation. These results suggest that autophagy contributes to PPARα activation upon fasting by promoting degradation of NCoR1 and thus regulates β-oxidation and ketone bodies production.