Publications by authors named "Etsuro Ito"

334 Publications

Ultrasensitive ELISA detection of proteins in separated lumen and membrane fractions of cancer cell exosomes.

Anal Biochem 2022 Jul 31;654:114831. Epub 2022 Jul 31.

Department of Biology, Waseda University, Shinjuku, Tokyo, 162-8480, Japan; Waseda Research Institute for Science and Engineering, Waseda University, Shinjuku, Tokyo, 169-8555, Japan; Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan. Electronic address:

Exosomes transfer molecules horizontally to surrounding cells and therefore have a key role in cancer progression. To clarify the role of exosomes in cancer progression, trace amounts of proteins in their lumen and membrane fractions should be analyzed separately. For this purpose, an adequate and easy-to-use method of separating the lumen and membrane fractions of exosomes must be developed. Further, because exosomes contain only trace amounts of proteins, an ultrasensitive protein detection method is necessary. To develop an adequate and easy-to-use lumen and membrane fraction separation method, we applied a commercially available kit originally developed for cells to exosomes and examined the validity of the results compared with those obtained using a conventional, complicated NaCO method. To develop an ultrasensitive protein detection method, we designated GRP78, which is upregulated in cancer cells and contributes to cancer progression, as the target protein and detected it at the subattomolar level using an ultrasensitive ELISA combined with thio-NAD cycling. By applying these methods together, GRP78 was successfully quantified in both the lumen and membrane fractions of exosomes obtained from cultured cancer cells. The present results will facilitate studies to broaden our understanding of the tumor microenvironment.
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http://dx.doi.org/10.1016/j.ab.2022.114831DOI Listing
July 2022

Dyserythropoietic anaemia with an intronic splicing mutation in patients suspected to have Diamond-Blackfan anaemia.

EJHaem 2022 Feb 10;3(1):163-167. Epub 2022 Jan 10.

Department of Pediatrics Hirosaki University Graduate School of Medicine Hirosaki Japan.

Diamond-Blackfan anaemia (DBA) shares clinical features with two recently reported sporadic cases of dyserythropoietic anaemia with a cryptic splicing mutation (c.871-24 C>T). We hypothesized that some patients clinically diagnosed with DBA but whose causative genes were unknown may carry the intronic mutation. Here, we examined 79 patients in our DBA cohort, who had no detectable causative genes. The intronic mutation was identified in two male patients sharing the same pedigree that included multiple cases with anaemia. Cosegregation of this mutation and disease in multiple family members provide evidence to support the pathogenicity of the intronic mutation.
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http://dx.doi.org/10.1002/jha2.374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9175706PMC
February 2022

Exercise and the Brain: Lessons From Invertebrate Studies.

Front Behav Neurosci 2022 28;16:928093. Epub 2022 Jun 28.

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, Russia.

Benefits of physical exercise for brain functions are well documented in mammals, including humans. In this review, we will summarize recent research on the effects of species-specific intense locomotion on behavior and brain functions of different invertebrates. Special emphasis is made on understanding the biological significance of these effects as well as underlying cellular and molecular mechanisms. The results obtained in three distantly related clades of protostomes, Nematodes, Molluscs and Artropods, suggest that influence of intense locomotion on the brain could have deep roots in evolution and wide adaptive significance. In , improved learning, nerve regeneration, resistance to neurodegenerative processes were detected after physical activity; in -facilitation of decision making in the novel environment, in -increased endurance, improved sleep and feeding behavior, in -improved orientation in conspecific phonotaxis, enhanced aggressiveness, higher mating success, resistance to some disturbing stimuli. Many of these effects have previously been described in mammals as beneficial results of running, suggesting certain similarity between distantly-related species. Our hypothesis posits that the above modulation of cognitive functions results from changes in the organism's predictive model. Intense movement is interpreted by the organism as predictive of change, in anticipation of which adjustments need to be made. Identifying the physiological and molecular mechanisms behind these adjustments is easier in experiments in invertebrates and may lead to the discovery of novel neurobiological mechanisms for regulation and correction of cognitive and emotional status.
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http://dx.doi.org/10.3389/fnbeh.2022.928093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9275788PMC
June 2022

Dorsolateral prefrontal cortex sensing analgesia.

Biophys Physicobiol 2022 8;19:1-10. Epub 2022 Apr 8.

Department of Psychology, Waseda University, Shinjuku, Tokyo 162-8644, Japan.

Chronic pain often has an unknown cause, and many patients with chronic pain learn to accept that their pain is incurable and pharmacologic treatments are only temporarily effective. Complementary and integrative health approaches for pain are thus in high demand. One such approach is soft touch, e.g., adhesion of pyramidal thorn patches in a pain region. The effects of patch adhesion on pain relief have been confirmed in patients with various types of pain. A recent study using near-infrared spectroscopy revealed that the dorsolateral prefrontal cortex (DLPFC), especially the left side, is likely to be inactivated in patients experiencing pain relief during patch treatment. Mindfulness meditation is another well-known complementary and integrative approach for achieving pain relief. The relation between pain relief due to mindfulness meditation and changes in brain regions, including the DLPFC, has long been examined. In the present review article, we survey the literature describing the effects of the above-mentioned complementary and integrative treatments on pain relief, and outline the important brain regions, including the DLPFC, that are involved in analgesia. We hope that the present article will provide clues to researchers who hope to advance neurosensory treatments for pain relief without medication.
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http://dx.doi.org/10.2142/biophysico.bppb-v19.0014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9173858PMC
April 2022

Genetic analysis of pheochromocytoma and paraganglioma complicating cyanotic congenital heart disease.

J Clin Endocrinol Metab 2022 Jun 22. Epub 2022 Jun 22.

Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Context: Pheochromocytoma and paraganglioma (PPGL) may appear as a complication of cyanotic congenital heart disease (CCHD-PPGL) with frequent EPAS1 mutations, suggesting a close link between EPAS1 mutations and tissue hypoxia in CCHD-PPGL pathogenesis.

Objective: Our aim is to further investigate the role of EPAS1 mutations in the hypoxia-driven mechanism of CCHD-PPGL pathogenesis, particularly focusing on metachronous and/or multifocal CCHD-PPGL tumors.

Methods: We performed whole exome sequencing (WES) for somatic and germline mutations in 15 PPGL samples from 7 CCHD patients, including 3 patients with metachronous and/or multifocal tumors, together with an adrenal medullary hyperplasia (AMH) sample.

Results: We detected EPAS1 mutations in 15 out of 16 PPGL/AMH samples from 7 cases. Conspicuously, all EPAS1 mutations in each of three cases with multifocal or metachronous tumors were mutually independent and typical examples of parallel evolution, which is suggestive of strong positive selection of EPAS1-mutated clones. Compared to 165 TCGA non-CCHD-PPGL samples, CCHD-PPGL/AMH samples were enriched for 11p deletions (13/16) and 2p amplifications (4/16). Of particular note, the multiple metachronous PPGL tumors with additional copy number abnormalities developed 18-23 years after the resolution of hypoxemia, suggesting that CCHD-induced hypoxic environments are critical for positive selection of EPAS1 mutants in early life, but may no longer be required for development of PPGL in later life.

Conclusions: Our results highlight a key role of activated HIF2α due to mutated EPAS1 in positive selection under hypoxic environments, although hypoxemia itself may not necessarily be required for the EPAS1-mutated clones to progress to PPGL.
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http://dx.doi.org/10.1210/clinem/dgac362DOI Listing
June 2022

Insulin and Memory in Invertebrates.

Front Behav Neurosci 2022 26;16:882932. Epub 2022 Apr 26.

Department of Biology, Waseda University, Tokyo, Japan.

Insulin and insulin-like peptides (ILP) help to maintain glucose homeostasis, whereas insulin-like growth factor (IGF) promotes the growth and differentiation of cells in both vertebrates and invertebrates. It is sometimes difficult to distinguish between ILP and IGF in invertebrates, however, because in some cases ILP has the same function as IGF. In the present review, therefore, we refer to these peptides as ILP/IGF signaling (IIS) in invertebrates, and discuss the role of IIS in memory formation after classical conditioning in invertebrates. In the arthropod , IIS is involved in aversive olfactory memory, and in the nematode , IIS controls appetitive/aversive response to NaCl depending on the duration of starvation. In the mollusk , IIS has a critical role in conditioned taste aversion. Insulin in mammals is also known to play an important role in cognitive function, and many studies in humans have focused on insulin as a potential treatment for Alzheimer's disease. Although analyses of tissue and cellular levels have progressed in mammals, the molecular mechanisms, such as transcriptional and translational levels, of IIS function in cognition have been far advanced in studies using invertebrates. We anticipate that the present review will help to pave the way for studying the effects of insulin, ILPs, and IGFs in cognitive function across phyla.
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http://dx.doi.org/10.3389/fnbeh.2022.882932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9087806PMC
April 2022

Profile of dorsal root ganglion neurons: study of oxytocin expression.

Mol Brain 2022 05 9;15(1):44. Epub 2022 May 9.

Department of Biology, Waseda University, Tokyo, 162-8480, Japan.

Although dorsal root ganglion (DRG) neurons have been so far classified according to the difference in their fibers (Aβ, Aδ, and C), this classification should be further subdivided according to gene expression patterns. We focused on oxytocin (OXT) and its related receptors, because OXT plays a local role in DRG neurons. We measured the mRNA levels of OXT, OXT receptor (OXTR), vasopressin V1a receptor (V1aR), transient receptor potential cation channel subfamily V member 1 (TRPV1), and piezo-type mechanosensitive ion channel component 2 (Piezo2) in single DRG neurons by using real-time PCR, and then performed a cluster analysis. According to the gene expression patterns, DRG neurons were classified into 4 clusters: Cluster 1 was characterized mainly by Piezo2, Cluster 2 by TRPV1, Cluster 4 by OXTR, and neurons in Cluster 3 did not express any of the target genes. The cell body diameter of OXT-expressing neurons was significantly larger in Cluster 1 than in Cluster 2. These results suggest that OXT-expressing DRG neurons with small cell bodies (Cluster 2) and large cell bodies (Cluster 1) probably correspond to C-fiber neurons and Aβ-fiber neurons, respectively. Furthermore, the OXT-expressing neurons contained not only TRPV1 but also Piezo2, suggesting that OXT may be released by mechanical stimulation regardless of nociception. Thus, mechanoreception and nociception themselves may induce the autocrine/paracrine function of OXT in the DRG, contributing to alleviation of pain.
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http://dx.doi.org/10.1186/s13041-022-00927-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9082903PMC
May 2022

Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly.

Life Sci Alliance 2022 07 23;5(7). Epub 2022 Mar 23.

Cancer Institute of Japanese Foundation for Cancer Research, Tokyo, Japan

The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.
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http://dx.doi.org/10.26508/lsa.202101045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942980PMC
July 2022

CNS serotonin content mediating food deprivation-enhanced learning is regulated by hemolymph tryptophan concentration and autophagic flux in the pond snail.

Nutr Neurosci 2022 Feb 14:1-11. Epub 2022 Feb 14.

Department of Biology, Waseda University, Tokyo, Japan.

Nutritional status affects cognitive function in many types of organisms. In the pond snail , 1 day of food deprivation enhances taste aversion learning ability by decreasing the serotonin (5-hydroxytryptamin; 5-HT) content in the central nervous system (CNS). On the other hand, after 5 days of food deprivation, learning ability and the CNS 5-HT concentration return to basal levels. How food deprivation leads to alterations of 5-HT levels in the CNS, however, is unknown. Here, we measured the concentration of the 5-HT precursor tryptophan in the hemolymph and CNS, and demonstrated that the CNS tryptophan concentration was higher in 5-day food-deprived snails than in non-food-deprived or 1-day food-deprived snails, whereas the hemolymph tryptophan concentration was not affected by the duration of food deprivation. This finding suggests the existence of a mediator of the CNS tryptophan concentration independent of food deprivation. To identify the mediator, we investigated autophagic flux in the CNS under different food deprivation conditions. We found that autophagic flux was significantly upregulated by inhibition of the tropomyosin receptor kinase (Trk)-Akt-mechanistic target of rapamycin complex 1 (MTORC1) pathway in the CNS of 5-day food-deprived snails. Moreover, when autophagy was inhibited, the CNS 5-HT content was significantly downregulated in 5-day food-deprived snails. Our results suggest that the hemolymph tryptophan concentration and autophagic flux in the CNS cooperatively regulate learning ability affected by different durations of food deprivation. This mechanism may underlie the selection of behaviors appropriate for animal survival depending on the degree of nutrition.
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http://dx.doi.org/10.1080/1028415X.2022.2033045DOI Listing
February 2022

Clinical and genetic diagnosis of thirteen Japanese patients with hereditary spherocytosis.

Hum Genome Var 2022 Jan 12;9(1). Epub 2022 Jan 12.

Department of Transfusion Medicine and Cell Processing, Tokyo Women's Medical University, Tokyo, Japan.

Hereditary spherocytosis is the most frequent cause of hereditary hemolytic anemia and is classified into five subtypes (SPH1-5) according to OMIM. Because the clinical and laboratory features of patients with SPH1-5 are variable, it is difficult to classify these patients into the five subtypes based only on these features. We performed target capture sequencing in 51 patients with hemolytic anemia associated with/without morphological abnormalities in red blood cells. Thirteen variants were identified in five hereditary spherocytosis-related genes (six in ANK1 [SPH1]; four in SPTB [SPH2]; and one in each of SPTA1 [SPH3], SLC4A1 [SPH4], and EPB42 [SPH5]). Among these variants, seven were novel. The distribution pattern of the variants was different from that reported previously in Japan but similar to those reported in other Asian countries. Comprehensive genomic analysis would be useful and recommended, especially for patients without a detailed family history and those receiving frequent blood transfusions due to chronic hemolytic anemia.
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http://dx.doi.org/10.1038/s41439-021-00179-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8755803PMC
January 2022

Ultrasensitive Detection of SARS-CoV-2 Spike Proteins Using the Thio-NAD Cycling Reaction: A Preliminary Study before Clinical Trials.

Microorganisms 2021 Oct 25;9(11). Epub 2021 Oct 25.

Department of Biology, Waseda University, Tokyo 162-8480, Japan.

To help control the global pandemic of coronavirus disease 2019 (COVID-19), we developed a diagnostic method targeting the spike protein of the virus that causes the infection, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We applied an ultrasensitive method by combining a sandwich enzyme-linked immunosorbent assay (ELISA) and the thio-nicotinamide adenine dinucleotide (thio-NAD) cycling reaction to quantify spike S1 proteins. The limit of detection (LOD) was 2.62 × 10 moles/assay for recombinant S1 proteins and 2.6 × 10 RNA copies/assay for ultraviolet B-inactivated viruses. We have already shown that the ultrasensitive ELISA for nucleocapsid proteins can detect ultraviolet B-inactivated viruses at the 10 RNA copies/assay level, whereas the nucleocapsid proteins of SARS-CoV-2 are difficult to distinguish from those in conventional coronaviruses and SARS-CoV. Thus, an antigen test for only the nucleocapsid proteins is insufficient for virus specificity. Therefore, the use of a combination of tests against both spike and nucleocapsid proteins is recommended to increase both the detection sensitivity and testing accuracy of the COVID-19 antigen test. Taken together, our present study, in which we incorporate S1 detection by combining the ultrasensitive ELISA for nucleocapsid proteins, offers an ultrasensitive, antigen-specific test for COVID-19.
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http://dx.doi.org/10.3390/microorganisms9112214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8619787PMC
October 2021

Modified ELISA for Ultrasensitive Diagnosis.

J Clin Med 2021 Nov 7;10(21). Epub 2021 Nov 7.

Department of Biology, Waseda University, Tokyo 162-8480, Japan.

An enzyme-linked immunosorbent assay (ELISA) can be used for quantitative measurement of proteins, and improving the detection sensitivity to the ultrasensitive level would facilitate the diagnosis of various diseases. In the present review article, we first define the term 'ultrasensitive'. We follow this with a survey and discussion of the current literature regarding modified ELISA methods with ultrasensitive detection and their application for diagnosis. Finally, we introduce our own newly devised system for ultrasensitive ELISA combined with thionicotinamide adenine dinucleotide cycling and its application for the diagnosis of infectious diseases and lifestyle-related diseases. The aim of the present article is to expand the application of ultrasensitive ELISAs in the medical and biological fields.
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http://dx.doi.org/10.3390/jcm10215197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8585087PMC
November 2021

[New insights into inherited bone marrow failure syndrome].

Rinsho Ketsueki 2021 ;62(10):1455-1464

Department of Pediatrics, Hirosaki University Graduate School of Medicine.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by bone marrow failure, congenital anomalies, and increased risk of malignant disease. Next generation sequencing methods have greatly facilitated the discovery of genetic etiology in IBMFS. Recently, de novo mutations activating TP53 were detected in patients with BMFS, mimicking Diamond-Blackfan anemia (DBA), using whole exome sequencing, and these patients were recognized as having a novel disorder. This discovery provides important insights into the previously postulated connection between p53 activation and IBMFS. Furthermore, a novel IBMFS, aldehyde degradation deficiency syndrome, was found in patients with aplastic anemia resembling Fanconi anemia (FA). This disorder is caused by combined inactivating mutations in ADH5 and ALDH2 coding formaldehyde-detoxifying enzymes. In this review, we highlight recent studies on DBA, FA, and their related diseases in Japan.
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http://dx.doi.org/10.11406/rinketsu.62.1455DOI Listing
November 2021

Enzymatic Changes in Red Blood Cells of Diamond-Blackfan Anemia.

Tohoku J Exp Med 2021 09;255(1):49-55

Department of Transfusion Medicine and Cell Processing, Faculty of Medicine, Tokyo Women's Medical University.

Diamond-Blackfan anemia is a congenital bone marrow failure syndrome characterized by red blood cell (RBC) aplasia with varied malformations in infants. Elevated activity of adenosine deaminase (ADA) has been considered as a useful biomarker of Diamond-Blackfan anemia, and ADA assay has been shown to be more sensitive than genetic diagnosis. Approximately, 80% of the examined patients showed elevated ADA activity, whereas genetic tests of ribosome subunit genes identified mutations in approximately 60% of the patients. We previously reported that reduced glutathione (GSH) levels in RBCs may serve as a biomarker of Diamond-Blackfan anemia. In this study, to confirm the universality of our data, we extended the analysis to seven RBC enzymes and GSH of 14 patients with Diamond-Blackfan anemia and performed a cross-analysis study using enzyme activity assay and recently reported proteome data. Statistical analysis revealed that both data exhibited high similarity, upregulation in the hexokinase and pentose-phosphate pathway, and downregulation in glycolytic enzymes such as phosphofructokinase and pyruvate kinase, in the RBCs obtained from the subjects with Diamond-Blackfan anemia. The only discrepancy between enzyme activity and proteome data was observed in glucose-6-phosphate dehydrogenase (G6PD), as increased G6PD activity showed no relation with the significant elevation in protein levels. These results suggest that our enzymatic activity data of Diamond-Blackfan anemia are universal and that the enzymatic activation of G6PD via a hitherto-unveiled mechanism is another metabolic feature of RBCs of Diamond-Blackfan anemia.
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http://dx.doi.org/10.1620/tjem.255.49DOI Listing
September 2021

Pain relief associated with decreased oxyhemoglobin level in left dorsolateral prefrontal cortex.

PLoS One 2021 23;16(8):e0256626. Epub 2021 Aug 23.

Department of Biology, Waseda University, Tokyo, Japan.

Pain in the elbow, shoulder, knee, lower back, and various other joints is relieved by adhesion of pyramidal thorn patches. To elucidate the pain relief mechanism induced by the patches, we established a quantitative method for estimating the pain reduction and investigated the brain regions that change in association with pain relief. We first attempted to quantify the pain relief using transcutaneous electric stimulation (TCES) and a visual analog scale (VAS), and then applied near-infrared spectroscopy (NIRS) to the prefrontal cortex, including the dorsolateral prefrontal cortex (DLPFC) and the orbitofrontal cortex (OFC). We also examined the salivary oxytocin levels, which are thought to reflect oxytocin secretion levels from the posterior pituitary in the brain. Application of pyramidal thorn patches to pain regions decreased the pain degree estimated using TCES and VAS. Oxyhemoglobin levels were likely to be decreased in the left DLPFC on the basis of NIRS measurements during patch treatment, suggesting that the left DLPFC is involved in pain relief. On the other hand, the salivary oxytocin levels varied widely. A potential reason for the varying salivary oxytocin levels is its utilization in the pain region as an analgesic agent. Our results suggest that the left DLPFC will become a target brain region for pain therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0256626PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8382195PMC
December 2021

Improved Detection Sensitivity of an Antigen Test for SARS-CoV-2 Nucleocapsid Proteins with Thio-NAD Cycling.

Biol Pharm Bull 2021 Sep 19;44(9):1332-1336. Epub 2021 Jun 19.

Department of Biology, Waseda University.

Antigen tests for infectious diseases are inexpensive and easy-to-use, but the limit of detection (LOD) is generally higher than that of PCR tests, which are considered the gold standard. In the present study, we combined a sandwich enzyme-linked immunosorbent assay (ELISA) with thionicotinamide-adenine dinucleotide (thio-NAD) cycling to improve the LOD of antigen tests for coronavirus disease 2019 (COVID-19). For recombinant nucleocapsid proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the LOD of our ELISA with thio-NAD cycling was 2.95 × 10 moles/assay. When UV-irradiated inactive SARS-CoV-2 was used, the minimum detectable virions corresponding to 2.6 × 10 RNA copies/assay were obtained using our ELISA with thio-NAD cycling. The assay volume for each test was 100 µL. The minimum detectable value was smaller than that of the latest antigen test using a fluorescent immunoassay for SARS-CoV-2, indicating the validity of our detection system for COVID-19 diagnosis.
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http://dx.doi.org/10.1248/bpb.b21-00387DOI Listing
September 2021

Isolated Bone Recurrence of Medulloblastoma With MYCN Amplification and TP53 Loss: A Case Report.

J Pediatr Hematol Oncol 2022 03;44(2):e593-e596

Departments of Pediatrics.

Extraneural recurrence of a medulloblastoma is rare with dismal prognosis. A 9-year-old girl with medulloblastoma was treated with gross total resection followed by a combination of chemotherapy and radiotherapy. Fourteen months after treatment completion, she developed multifocal bone metastases. Despite chemotherapy combined with irradiation, she died 18 months after recurrence due to progressive disease. Fluorescence in situ hybridization on formalin-fixed paraffin-embedded tissue sections revealed MYCN amplification and TP53 loss, consistent with the genetic alterations of a rapidly progressive subgroup of recurrent medulloblastomas. In clinical practice, dismal biologic features can be determined using fluorescence in situ hybridization in defective materials.
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http://dx.doi.org/10.1097/MPH.0000000000002234DOI Listing
March 2022

Peripheral-neuron-like properties of differentiated human dental pulp stem cells (hDPSCs).

PLoS One 2021 6;16(5):e0251356. Epub 2021 May 6.

Faculty of Science and Technology, Department of Bioscience and Informatics, Keio University, Kanagawa, Japan.

Elucidating the mechanisms underlying human pain sensation requires the establishment of an in vitro model of pain reception comprising human cells expressing pain-sensing receptors and function properly as neurons. Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells and a promising candidate for producing human neuronal cells, however, the functional properties of differentiated hDPSCs have not yet been fully characterized. In this study, we demonstrated neuronal differentiation of hDPSCs via both their expression of neuronal marker proteins and their neuronal function examined using Ca2+ imaging. Moreover, to confirm the ability of nociception, Ca2+ responses in differentiated hDPSCs were compared to those of rat dorsal root ganglion (DRG) neurons. Those cells showed similar responses to glutamate, ATP and agonists of transient receptor potential (TRP) channels. Since TRP channels are implicated in nociception, differentiated hDPSCs provide a useful in vitro model of human peripheral neuron response to stimuli interpreted as pain.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0251356PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101759PMC
October 2021

Antigen tests for COVID-19.

Biophys Physicobiol 2021 10;18:28-39. Epub 2021 Feb 10.

Department of Biology, Waseda University, Shinjuku, Tokyo 162-8480, Japan.

PCR diagnosis has been considered as the gold standard for coronavirus disease 2019 (COVID-19) and other many diseases. However, there are many problems in using PCR, such as non-specific (i.e., false-positive) and false-negative amplifications, the limits of a target sample volume, deactivation of the enzymes used, complicated techniques, difficulty in designing probe sequences, and the expense. We, thus, need an alternative to PCR, for example an ultrasensitive antigen test. In the present review, we summarize the following three topics. (1) The problems of PCR are outlined. (2) The antigen tests are surveyed in the literature that was published in 2020, and their pros and cons are discussed for commercially available antigen tests. (3) Our own antigen test on the basis of an ultrasensitive enzyme-linked immunosorbent assay (ELISA) is introduced. Finally, we discuss the possibility that our antigen test by an ultrasensitive ELISA technique will become the gold standard for diagnosis of COVID-19 and other diseases.
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http://dx.doi.org/10.2142/biophysico.bppb-v18.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049777PMC
February 2021

Zeptomole Detection of an Enzyme by a Simple Colorimetric Method.

Anal Sci 2021 Oct 19;37(10):1469-1472. Epub 2021 Mar 19.

Department of Biology, Waseda University.

An enzyme immunoassay, in which an enzyme (e.g., alkaline phosphatase, ALP) is conjugated with an antibody, is a precise and simple protein detection method. Precise measurements of enzymes at low concentrations allow for ultrasensitive protein detection. The application of a phosphorylated substrate to ALP, followed by using a dephosphorylated substrate in thionicotinamide-adenine dinucleotide cycling, provides a simple and precise quantification of ALP. We describe a protocol for detecting ALP at the zeptomole level using a simple colorimetric method.
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http://dx.doi.org/10.2116/analsci.21N009DOI Listing
October 2021

Early diagnosis with ultrasensitive ELISA.

Adv Clin Chem 2021 7;101:121-133. Epub 2020 Jul 7.

Waseda Research Institute for Science and Engineering, Waseda University, Tokyo, Japan; R&D Headquarters, TAUNS Laboratories, Inc., Izunokuni, Japan.

Accurate, rapid and simple detection methods are required to facilitate early diagnosis of various disorders including infectious and lifestyle diseases as well as cancer. These detection approaches reduce the window of infection, i.e., the period between infection and reliable detection. Optimally, these methods should target protein as an indicator of pathogenic microbes as well as other biomarkers. For example, although nucleic acid is easily detected by polymerase chain reaction (PCR), these markers are also present in dead microbes, and, in the case of mRNA, it is not known whether this target was successfully translated. Accordingly, early diagnostic approaches require the development of ultrasensitive protein detection methods. In this chapter, we introduce an ultrasensitive enzyme-linked immunosorbent assay (ELISA) which combines a traditional sandwich-based immunoassay with thionicotinamide adenine dinucleotide (thio-NAD) cycling. The performance characteristics of this unique approach are reviewed as well as its potential role in providing a novel and ultrasensitive diagnostic tool in the clinical laboratory.
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http://dx.doi.org/10.1016/bs.acc.2020.06.002DOI Listing
July 2021

A Case of Congenital Leukemia With MYB-GATA1 Fusion Gene in a Female Patient.

J Pediatr Hematol Oncol 2022 01;44(1):e250-e252

Department of Pediatrics, Hirosaki University Graduate School of Medicine, Aomori, Japan.

We report a female newborn with acute myelogenous leukemia (AML) associated with a MYB-GATA1 fusion gene. Morphologic findings of myeloid lineage were obtained using light microscopy. Cytogenetic analysis of peripheral blood showed a complex karyotype: 46,X,-X,add(3)(q21),der(6)add(6)(q21)del(6)(q?), +mar1[5]/46,XX[15]. Targeted RNA sequencing revealed a MYB-GATA1 fusion gene. Reduced-dose AML-type chemotherapy resulted in remission and survival for >3 years without relapse. The present case demonstrated the feasibility of carrying out targeted RNA sequencing for identifying MYB-GATA1 and supports the notion that neonatal AML with MYB-GATA1 with reduced chemotherapy may show better prognosis than other highly toxic therapies.
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http://dx.doi.org/10.1097/MPH.0000000000002119DOI Listing
January 2022

Association of Multiple Gene Polymorphisms Including Homozygous NUDT15 R139C With Thiopurine Intolerance During the Treatment of Acute Lymphoblastic Leukemia.

J Pediatr Hematol Oncol 2021 11;43(8):e1173-e1176

Community Medicine, Hirosaki University Graduate School of Medicine, Hirosaki.

Although thiopurine is a crucial drug for treating acute lymphoblastic leukemia, individual variations in intolerance are observed due to gene polymorphisms. A 3-year-old boy with B-cell precursor acute lymphoblastic leukemia who was administered thiopurine developed mucositis, sepsis, and hemophagocytic lymphohistiocytosis due to prolonged hematologic toxicity, chronic disseminated candidiasis, and infective endocarditis that triggered multiple brain infarctions. The patient was found to harbor 3 gene polymorphisms associated with thiopurine intolerance including homozygous NUDT15 R139C, heterozygous ITPA C94A, and homozygous MTHFR C677T and heterozygous RFC1 G80A. Thus, the combined effect of intolerance via multiple gene polymorphisms should be considered in case of unexpected adverse reactions.
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http://dx.doi.org/10.1097/MPH.0000000000002085DOI Listing
November 2021

Usefulness of functional splicing analysis to confirm precise disease pathogenesis in Diamond-Blackfan anemia caused by intronic variants in .

Pediatr Hematol Oncol 2021 Sep 24;38(6):515-527. Epub 2021 Feb 24.

Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.

Diamond-Blackfan anemia (DBA) is mainly caused by pathogenic variants in ribosomal proteins and 22 responsible genes have been identified to date. The most common causative gene of DBA is [NM_001022.4]. Nearly 180 variants have been reported, including three deep intronic variants outside the splicing consensus sequence (c.72-92A > G, c.356 + 18G > C, and c.411 + 6G > C). We also identified one case with a c.412-3C > G intronic variant. Without conducting transcript analysis, the pathogenicity of these variants is unknown. However, it is difficult to assess transcripts because of their fragility. In such cases, in functional splicing assays can be used to assess pathogenicity. Here, we report functional splicing analysis results of four deep intronic variants identified in our case and in previously reported cases. One splicing consensus variant (c.411 + 1G > A) was also examined as a positive control. Aberrant splicing with a 2-bp insertion between exons 5 and 6 was identified in the patient samples and minigene assay results also identified exon 6 skipping in our case. The exon 6 skipping transcript was confirmed by further evaluation using quantitative RT-PCR. Additionally, minigene assay analysis of three reported deep intronic variants revealed that none of them showed aberrant splicing and that these variants were not considered to be pathogenic. In conclusion, the minigene assay is a useful method for functional splicing analysis of inherited disease.
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http://dx.doi.org/10.1080/08880018.2021.1887984DOI Listing
September 2021

Activation of the orbitofrontal cortex by both meditation and exercise: A near-infrared spectroscopy study.

PLoS One 2021 23;16(2):e0247685. Epub 2021 Feb 23.

Department of Biology, Waseda University, Tokyo, Japan.

In some types of meditation, such as mindfulness and Zen, breathing is the focus of attention, whereas during an excessive, short-period of anaerobic exercise, the muscles become the focus of attention. Thus, during both efforts, one's attention is focused on a certain feature of the body. Both meditation and exercise generally provide mental refreshment to humans. We hypothesized that the same brain regions are activated by both efforts in humans. To examine this hypothesis, we engaged participants in 3 tasks: meditation, exercise, and a control task. After each task, the participants underwent a 2-back test to concentrate their thoughts, while changes in their blood hemoglobin levels were simultaneously monitored using near-infrared spectroscopy (NIRS). Seventeen participants (20-24 years of age; 11 men, 6 women) were enrolled. We applied a fast-Fourier transform (FFT) analysis to the NIRS wave data and calculated the correlation coefficients of the FFT data between (1) meditation and control, (2) exercise and control, and (3) meditation and exercise, at the orbitofrontal cortex (OFC) and dorsolateral prefrontal cortex (DLPFC), brain areas that are generally involved in mental refreshment. A significant difference in the correlation coefficients between the OFC and DLPFC was detected in the meditation and exercise analysis, and signal source analysis confirmed that the NIRS waves spread from the right and left OFC edges (i.e., right and left temples) toward the center. Our results suggest that both meditation and exercise activate the OFC, which is involved in emotional reactions and motivation behavior, resulting in mental refreshment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247685PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901739PMC
August 2021

Post-induction MRD by FCM and GATA1-PCR are significant prognostic factors for myeloid leukemia of Down syndrome.

Leukemia 2021 09 15;35(9):2508-2516. Epub 2021 Feb 15.

Division of Leukemia and Lymphoma, Children's Cancer Center, National Center for Child Health and Development, Tokyo, Japan.

Myeloid leukemia of Down syndrome (ML-DS) is associated with good response to chemotherapy, resulting in favorable outcomes. However, no universal prognostic factors have been identified to date. To clarify a subgroup with high risk of relapse, the role of minimal residual disease (MRD) was explored in the AML-D11 trial by the Japanese Pediatric Leukemia/Lymphoma Study Group. MRD was prospectively evaluated at after induction therapy and at the end of all chemotherapy, using flow cytometry (FCM-MRD) and GATA1-targeted deep sequencing (GATA1-MRD). A total of 78 patients were eligible and 76 patients were stratified to the standard risk (SR) group by morphology. In SR patients, FCM-MRD and GATA1-MRD after induction were positive in 5/65 and 7/59 patients, respectively. Three-year event-free survival (EFS) and overall survival (OS) rates were 95.0% and 96.7% in the FCM-MRD-negative population, and 60.0% and 80.0% in the positive population. Three-year EFS and OS rates were both 98.1% in the GATA1-MRD-negative population, and 57.1% and 71.4% in the positive population. Adjusted hazard ratios for associations of FCM-MRD with EFS were 14.67 (p = 0.01). Detection of MRD by either FCM or GATA1 after initial induction therapy represents a significant prognostic factor for predicting ML-DS relapse.
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http://dx.doi.org/10.1038/s41375-021-01157-wDOI Listing
September 2021
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