Publications by authors named "Kazunori Sasaki"

73 Publications

GLO 1 and PKCλ Regulate ALDH1-positive Breast Cancer Stem Cell Survival.

Anticancer Res 2021 Dec;41(12):5959-5971

Department of Medicinal and Life Sciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan;

Background/aim: We examined the inhibitory effects of both glyoxalase 1 (GLO 1) and protein kinase C (PKC)λ in aldehyde dehydrogenase 1 (ALDH1)-positive breast cancer stem cells (CSCs).

Materials And Methods: Breast cancer genomics datasets (TCGA, n=593; METABRIC, n=1904) were downloaded and statistically analyzed. The effects of GLO 1 and PKCλ on trypan blue staining and tumor-sphere formation by ALDH1 cells derived from triple negative breast cancer (TNBC) and basal-like breast cancer were examined.

Results: GLO 1, PKCλ, and ALDH1A3 tumors were enriched in stage I/II/III/IV samples, associated with the HER2 and TNBC subtypes according to receptor status, and associated with the HER2-enriched and basal-like subtypes according to PAM50. Inhibition of either GLO 1 (TLSC702) or PKCλ (ANF) suppressed tumor-sphere formation and enhanced death in ALDH1 cells. TLSC702 also effectively inhibited tumor-sphere formation and induced death in PKCλ knockout ALDH1 cells.

Conclusion: GLO 1 and PKCλ are important for the survival of ALDH1-positive breast CSCs, and may represent potential therapeutic targets for the treatment of ALDH1-positive breast CSCs.
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http://dx.doi.org/10.21873/anticanres.15415DOI Listing
December 2021

Luteolin Modulates Neural Stem Cells Fate Determination: Study on Human Neural Stem Cells, and Study on LPS-Induced Depression Mice Model.

Front Cell Dev Biol 2021 1;9:753279. Epub 2021 Nov 1.

Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Tsukuba, Japan.

Luteolin is a natural flavone with neurotrophic effects observed on different neuronal cell lines. In the present study, we aimed to assess the effect of luteolin on hNSCs fate determination and the LPS-induced neuroinflammation in a mouse model of depression with astrocytogenesis defect. hNSCs were cultured in basal cell culture medium (control) or medium supplemented with luteolin or AICAR, a known inducer of astrogenesis. A whole-genome transcriptomic analysis showed that luteolin upregulated the expressions of genes related to neurotrophin, dopaminergic, hippo, and Wnt signaling pathways, and downregulated the genes involved in p53, TNF, FOXO, and Notch signaling pathways. We also found that astrocyte-specific gene GFAP, as well as other genes of the key signaling pathways involved in astrogenesis such as Wnt, BMP, and JAK-STAT pathways were upregulated in luteolin-treated hNSCs. On the other hand, neurogenesis and oligodendrogenesis-related genes, , , and , were downregulated in luteolin-treated hNSCs. Furthermore, immunostaining showed that percentages of GFAP+ cells were significantly higher in luteolin- and AICAR-treated hNSCs compared to control hNSCs. Additionally, RT-qPCR results showed that luteolin upregulated the expressions of , , and , whereas the expression of remained unchanged. Next, we evaluated the effects of luteolin in LPS-induced mice model of depression that represents defects in astrocytogenesis. We found that oral administration of luteolin (10 mg/Kg) for eight consecutive days could decrease the immobility time on tail suspension test, a mouse behavioral test measuring depression-like behavior, and attenuate LPS-induced inflammatory responses by significantly decreasing IL-6 production in mice brain-derived astrocytes and serum, and TNFα and corticosterone levels in serum. Luteolin treatment also significantly increased mature BDNF, dopamine, and noradrenaline levels in the hypothalamus of LPS-induced depression mice. Though the behavioral effects of luteolin did not reach statistical significance, global gene expression analyses of mice hippocampus and brain-derived NSCs highlighted the modulatory effects of luteolin on different signaling pathways involved in the pathophysiology of depression. Altogether, our findings suggest an astrocytogenic potential of luteolin and its possible therapeutic benefits in neuroinflammatory and neurodegenerative diseases. However, further studies are required to identify the specific mechanism of action of luteolin.
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http://dx.doi.org/10.3389/fcell.2021.753279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8591246PMC
November 2021

Nontargeted Serum Lipid Profiling of Nonalcoholic Steatohepatitis by Multisegment Injection-Nonaqueous Capillary Electrophoresis-Mass Spectrometry: A Multiplexed Separation Platform for Resolving Ionic Lipids.

J Proteome Res 2021 Oct 22. Epub 2021 Oct 22.

Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4M1, Canada.

New methods are needed for global lipid profiling due to the complex chemical structures and diverse physicochemical properties of lipids. Herein we introduce a robust data workflow to unambiguously select lipid features from serum ether extracts by multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS). An iterative three-stage screening strategy is developed for nontargeted lipid analyses when using multiplexed electrophoretic separations coupled to an Orbitrap mass analyzer under negative ion mode. This approach enables the credentialing of 270 serum lipid features annotated based on their accurate mass and relative migration time, including 128 ionic lipids reliably measured (median CV ≈ 13%) in most serum samples (>75%) from nonalcoholic steatohepatitis (NASH) patients ( = 85). A mobility map is introduced to classify charged lipid classes over a wide polarity range with selectivity complementary to chromatographic separations, including lysophosphatidic acids, phosphatidylcholines, phosphatidylinositols, phosphatidylethanolamines, and nonesterified fatty acids (NEFAs). Serum lipidome profiles were also used to differentiate high- from low-risk NASH patients using a -means clustering algorithm, where elevated circulating NEFAs (e.g., palmitic acid) were associated with increased glucose intolerance, more severe liver fibrosis, and greater disease burden. MSI-NACE-MS greatly expands the metabolome coverage of conventional aqueous-based CE-MS protocols and is a promising platform for large-scale lipidomic studies.
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http://dx.doi.org/10.1021/acs.jproteome.1c00682DOI Listing
October 2021

Antidepressant- and anxiolytic-like activities of Rosmarinus officinalis extract in rodent models: Involvement of oxytocinergic system.

Biomed Pharmacother 2021 Dec 12;144:112291. Epub 2021 Oct 12.

Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan; Open Innovation Laboratory for Food and Medicinal Resource Engineering, National Institute of Advanced Industrial Science and Technology (AIST) and University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan; Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan; Tsukuba Life Science Innovation Program (T-LSI), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8577 Ibaraki, Japan. Electronic address:

Background: Oxytocin (OXT), a neuropeptide involved in mammal reproductive and prosocial behaviors, has been reported to interact with various stressor-provoked neurobiological changes, including neuroendocrine, neurotransmitter, and inflammatory processes. In view of disturbances in psychosocial relationships due to social isolation and physical distancing measures amid the COVID-19 pandemic, being one of the triggering factors for the recent rise in depression and anxiety, OXT is a potential candidate for a new antidepressant.

Methods: In this present study, we have aimed to investigate the effects of oral administration of Rosmarinus officinalis extract (RE), extracted from distillation residue of rosemary essential oil, on central OXT level in the context of other stress biomarkers and neurotransmitter levels in mice models. Tail suspension test (TST) and elevated plus maze test (EPMT) following LPS injection were employed to assess depressive- and anxiety-like behavior in mice, respectively.

Findings: Pretreatment with RE for seven days significantly improved behavior in TST and EPMT. Whole-genome microarray analysis reveals that RE significantly reversed TST stress-induced alterations in gene expressions related to oxytocinergic and neurotransmitter pathways and inflammatory processes. In both models, RE significantly increased central Oxt and Oxtr expressions, as well as OXT protein levels. RE also significantly attenuated stress-induced changes in serum corticosterone, brain and serum BDNF levels, and brain neurotransmitters levels in both models.

Interpretation: Altogether, our study is the first to report antidepressant- and anxiolytic-like activities of RE through modulating oxytocinergic system in mice brain and thus highlights the prospects of RE in the treatment of depressive disorders of psychosocial nature.
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http://dx.doi.org/10.1016/j.biopha.2021.112291DOI Listing
December 2021

Preventive effect of a Kampo medicine, kososan, on recurrent depression in a mouse model of repeated social defeat stress.

Gene 2022 Jan 26;806:145920. Epub 2021 Aug 26.

Oriental Medicine Research Center, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan.

Depression is deemed a mood disorder characterized by a high rate of relapse. Therefore, overcoming of the recurrent depression is globally expecting. Kososan, a traditional Japanese herbal medicine, has been clinically used for mild depressive mood, and our previous studies have shown some evidence for its antidepressive-like efficacy in experimental animal models of depression. However, it remains unclear whether kososan has beneficial effects on recurrent depression. Here, we examined its effect using a mouse model of modified repeated social defeat stress (SDS) paradigm. Male BALB/c mice were exposed to a 5-min SDS from unfamiliar aggressive CD-1 mice for 5 days. Kososan extract (1.0 kg/kg/day) or an antidepressant milnacipran (60 mg/kg/day) was administered orally for 26 days (days 7-32) to depression-like mice with social avoidant behaviors on day 6. Single 5 min of SDS was subjected to mice recovered from the social avoidance on day 31, and then the recurrence of depression-like behaviors was evaluated on day 32. Hippocampal gene expression patterns were also assayed by DNA microarray analysis. Water- or milnacipran-administered mice resulted in a recurrence of depression-like behaviors by re-exposure of single SDS, whereas kososan-administered mice did not recur depression-like behaviors. Distinct gene expression patterns were also found for treating kososan and milnacipran. Collectively, this finding suggests that kososan exerts a preventive effect on recurrent depression-like behaviors in mice. Pretreatment of kososan is more useful for recurrent depression than that of milnacipran.
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http://dx.doi.org/10.1016/j.gene.2021.145920DOI Listing
January 2022

De novo ARF3 variants cause neurodevelopmental disorder with brain abnormality.

Hum Mol Genet 2021 Aug 4. Epub 2021 Aug 4.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, 236-0004, Japan.

An optimal Golgi transport system is important for mammalian cells. The adenosine diphosphate (ADP) ribosylation factors (ARF) are key proteins for regulating cargo sorting at the Golgi network. In this family, ARF3 mainly works at the trans-Golgi network (TGN), and no ARF3-related phenotypes have yet been described in humans. We here report the clinical and genetic evaluations of two unrelated children with de novo pathogenic variants in the ARF3 gene: c.200A > T (p.Asp67Val) and c.296G > T (p.Arg99Leu). Although the affected individuals presented commonly with developmental delay, epilepsy, and brain abnormalities, there were differences in severity, clinical course, and brain lesions. In vitro subcellular localization assays revealed that the p.Arg99Leu mutant localized to Golgi apparatus, similar to the wild-type, whereas the p.Asp67Val mutant tended to show a disperse cytosolic pattern together with abnormally dispersed Golgi localization, similar to that observed in a known dominant negative variant (p.Thr31Asn). Pull-down assays revealed that the p.Asp67Val had a loss-of-function effect and the p.Arg99Leu variant had increased binding of the adaptor protein, Golgi-localized, γ-adaptin ear-containing, ARF-binding protein 1 (GGA1), supporting the gain of function. Furthermore, in vivo studies revealed that p.Asp67Val transfection led to lethality in flies. In contrast, flies expressing p.Arg99Leu had abnormal rough eye, as observed in the gain-of-function variant p.Gln71Leu. These data indicate that two ARF3 variants, the possibly loss-of-function p.Asp67Val and the gain-of-function p.Arg99Leu, both impair the Golgi transport system. Therefore, it may not be unreasonable that they showed different clinical features like diffuse brain atrophy (p.Asp67Val) and cerebellar hypoplasia (p.Arg99Leu).
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http://dx.doi.org/10.1093/hmg/ddab224DOI Listing
August 2021

Grape skin extract modulates neuronal stem cell proliferation and improves spatial learning in senescence-accelerated prone 8 mice.

Aging (Albany NY) 2021 07 28;13(14):18131-18149. Epub 2021 Jul 28.

Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.

In recent years, the number of patients with neurodegenerative illness such as Alzheimer's disease (AD) has increased with the aging of the population. In this study, we evaluated the effect of Grape skin extract (GSE) on neurotypic SH-SY5Y cells as an AD model, murine neurospheres as an neurogenesis model and SAMP8 mice as an AD model. Our result showed that pre-treatment of SH-SY5Y cells with GSE ameliorated Aβ-induced cytotoxicity. Moreover, GSE treatment significantly decreased the number of neurospheres, but increased their size suggesting reduced stem cell self-renewal but increased proliferation. Our Morris water maze test indicated that GSE improves learning and memory in SAMP8 mice. To detect proliferation and newborn neurons, we measured BrdU+ cells in the dentate gyrus (DG). GSE treatment increased the number of BrdU+ cells in the DG of SAMP8 mice. Finally, we showed that GSE induced a decrease in inflammatory cytokines and an increase in neurotransmitters in the cerebral cortex of SAMP8 mice. These results suggested that GSE increased neurogenic zone proliferation and memory but decreased oxidative stress associated with pro-inflammatory cytokines in aging, thus protecting neurons.
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http://dx.doi.org/10.18632/aging.203373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8351719PMC
July 2021

Glyoxalase 1 and protein kinase Cλ as potential therapeutic targets for late-stage breast cancer.

Oncol Lett 2021 Jul 24;22(1):547. Epub 2021 May 24.

Department of Medicinal and Life Sciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan.

Cancer cells upregulate the expression levels of glycolytic enzymes in order to reach the increased glycolysis required. One such upregulated glycolytic enzyme is glyoxalase 1 (GLO 1), which catalyzes the conversion of toxic methylglyoxal to nontoxic S-D-lactoylglutathione. Protein kinase Cλ (PKCλ) is also upregulated in various types of cancer and is involved in cancer progression. In the present study, the association between enhanced glycolysis and PKCλ in breast cancer was investigated. In human breast cancer, high GLO 1 expression was associated with high PKCλ expression at the protein (P<0.01) and mRNA levels (P<0.01). Furthermore, Wilcoxon and Cox regression model analysis revealed that patients with stage III-IV tumors with high GLO 1 and PKCλ expression had poor overall survival compared with patients expressing lower levels of these genes [P=0.040 (Gehan-Breslow generalized Wilcoxon test) and P=0.031 (hazard ratio, 2.36; 95% confidence interval, 1.08-5.16), respectively]. Treatment of MDA-MB-157 and MDA-MB-468 human basal-like breast cancer cells with TLSC702 (a GLO 1 inhibitor) and/or aurothiomalate (a PKCλ inhibitor) reduced both cell viability and tumor-sphere formation. These results suggested that GLO 1 and PKCλ were cooperatively involved in cancer progression and contributed to a poor prognosis in breast cancer. In conclusion, GLO 1 and PKCλ serve as potentially effective therapeutic targets for treatment of late-stage human breast cancer.
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http://dx.doi.org/10.3892/ol.2021.12808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8170180PMC
July 2021

Capillary Electrophoresis Mass Spectrometry-Based Metabolomics of Plasma Samples from Healthy Subjects in a Cross-Sectional Japanese Population Study.

Metabolites 2021 May 13;11(5). Epub 2021 May 13.

Department of Metabolomics Innovation, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.

For large-scale metabolomics, such as in cohort studies, normalization protocols using quality control (QC) samples have been established when using data from gas chromatography and liquid chromatography coupled to mass spectrometry. However, normalization protocols have not been established for capillary electrophoresis-mass spectrometry metabolomics. In this study, we performed metabolome analysis of 314 human plasma samples using capillary electrophoresis-mass spectrometry. QC samples were analyzed every 10 samples. The results of principal component analysis for the metabolome data from only the QC samples showed variations caused by capillary replacement in the first principal component score and linear variation with continuous measurement in the second principal component score. Correlation analysis between diagnostic blood tests and plasma metabolites normalized by the QC samples was performed for samples from 188 healthy subjects who participated in a Japanese population study. Five highly correlated pairs were identified, including two previously unidentified pairs in normal healthy subjects of blood urea nitrogen and guanidinosuccinic acid, and gamma-glutamyl transferase and cysteine glutathione disulfide. These results confirmed the validity of normalization protocols in capillary electrophoresis-mass spectrometry using large-scale metabolomics and comprehensive analysis.
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http://dx.doi.org/10.3390/metabo11050314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153282PMC
May 2021

De novo ATP1A3 variants cause polymicrogyria.

Sci Adv 2021 Mar 24;7(13). Epub 2021 Mar 24.

Department of Pediatrics, Tottori Prefectural Central Hospital, Tottori 680-0901, Japan.

Polymicrogyria is a common malformation of cortical development whose etiology remains elusive. We conducted whole-exome sequencing for 124 patients with polymicrogyria and identified de novo variants in eight patients. Mutated causes functional brain diseases, including alternating hemiplegia of childhood (AHC), rapid-onset dystonia parkinsonism (RDP), and cerebellar ataxia, areflexia, pes cavus, optic nerve atrophy, and sensorineural deafness (CAPOS). However, our patients showed no clinical features of AHC, RDP, or CAPOS and had a completely different phenotype: a severe form of polymicrogyria with epilepsy and developmental delay. Detected variants had different locations in and different functional properties compared with AHC-, RDP-, or CAPOS-associated variants. In the developing cerebral cortex of mice, radial neuronal migration was impaired in neurons overexpressing the variant of the most severe patients, suggesting that this variant is involved in cortical malformation pathogenesis. We propose a previously unidentified category of polymicrogyria associated with abnormalities.
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http://dx.doi.org/10.1126/sciadv.abd2368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990330PMC
March 2021

Microalgae Sp. Increases Neurogenesis and Improves Spatial Learning and Memory in Senescence-Accelerated Mouse-Prone 8 Mice.

Front Cell Dev Biol 2020 9;8:600575. Epub 2021 Feb 9.

Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Tsukuba, Japan.

Much attention has recently been focused on nutraceuticals, with minimal adverse effects, developed for preventing or treating neurological diseases such as Alzheimer's disease (AD). The present study was conducted to investigate the potential effect on neural development and function of the microalgae sp. as a nutraceutical. To test neuroprotection by the ethanol extract of (EEA) and a derivative, the n-Hexane layer of EEA (HEEA), amyloid-β-stimulated SH-SY5Y cells, was used as an AD model. We then assessed the potential enhancement of neurogenesis by EEA and HEEA using murine neurospheres. We also administered EEA or HEEA to senescence-accelerated mouse-prone 8 (SAMP8) mice, a non-transgenic strain with accelerated aging and AD-like memory loss for evaluation of spatial learning and memory using the Morris water maze test. Finally, we performed immunohistochemical analysis for assessment of neurogenesis in mice administered EEA. Pretreatment of SH-SY5Y cells with EEA or the squalene-rich fraction of EEA, HEEA, ameliorated amyloid-β-induced cytotoxicity. Interestingly, only EEA-treated cells showed a significant increase in cell metabolism and intracellular adenosine triphosphate production. Moreover, EEA treatment significantly increased the number of neurospheres, whereas HEEA treatment significantly increased the number of β-III-tubulin+ young neurons and GFAP+ astrocytes. SAMP8 mice were given 50 mg/kg EEA or HEEA orally for 30 days. EEA and HEEA decreased escape latency in the Morris water maze in SAMP8 mice, indicating improved memory. To detect stem cells and newborn neurons, we administered BrdU for 9 days and measured BrdU+ cells in the dentate gyrus, a neurogenic stem cell niche of the hippocampus. In SAMP8 mice, EEA rapidly and significantly increased the number of BrdU+GFAP+ stem cells and their progeny, BrdU+NeuN+ mature neurons. In conclusion, our data in aggregate indicate that EEA and its constituents could be developed into a nutraceutical for promoting brain health and function against several age-related diseases, particularly AD.
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http://dx.doi.org/10.3389/fcell.2020.600575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900145PMC
February 2021

Tryptophan Metabolism Regulates Proliferative Capacity of Human Pluripotent Stem Cells.

iScience 2021 Feb 26;24(2):102090. Epub 2021 Jan 26.

Department of Cardiology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan.

Human pluripotent stem cells (hPSCs) have a unique metabolic signature for maintenance of pluripotency, self-renewal, and survival. Although hPSCs could be potentially used in regenerative medicine, the prohibitive cost associated with large-scale cell culture presents a major barrier to the clinical application of hPSC. Moreover, without a fully characterized metabolic signature, hPSC culture conditions are not optimized. Here, we performed detailed amino acid profiling and found that tryptophan (TRP) plays a key role in the proliferation with maintenance of pluripotency. In addition, metabolome analyses revealed that intra- and extracellular kynurenine (KYN) is decreased under TRP-supplemented conditions, whereas N-formylkynurenine (NFK), the upstream metabolite of KYN, is increased thereby contributing to proliferation promotion. Taken together, we demonstrate that TRP is indispensable for survival and proliferation of hPSCs. A deeper understanding of TRP metabolism will enable cost-effective large-scale production of hPSCs, leading to advances in regenerative medicine.
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http://dx.doi.org/10.1016/j.isci.2021.102090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878994PMC
February 2021

Dietary administration of cumin-derived cuminaldehyde induce neuroprotective and learning and memory enhancement effects to aging mice.

Aging (Albany NY) 2021 01 20;13(2):1671-1685. Epub 2021 Jan 20.

Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.

Cuminaldehyde (CA) is one of the major compounds of the essential oil of . The aim of this study was to evaluate the effects of CA on aging, specifically on spatial learning and memory. To achieve our objective, an study on SH-SY5Y cells was performed to analyze the neuroprotective effect of CA against dexamethasone using the MTT assay. An study was performed for evaluation of the spatial learning and memory using Morris water maze (MWM). RT-PCR was performed to quantify the expression of specific genes ( and ) in the mice brain. The results obtained showed a neuroprotective effect of CA against dexamethasone-induced neuronal toxicity. The escape latency of CA-treated aged mice was significantly decreased as compared to the water-treated aged mice after 4 days of training in MWM. Moreover, CA treatment up-regulated the gene expression of , and , while it down-regulated the gene expression of . These findings suggest that CA has a neuroprotective effect, as well as a spatial learning and memory enhancement potential through the modulation of genes coding for neurotrophic factors and/or those implicated in the imbalance of neural circuitry and impairment of synaptic plasticity. Cuminaldehyde (CA) is one of the major compound of the essential oil of . The aim of this study was to evaluate the effects of CA on aging, specifically on spatial learning and memory. To achieve our objective, an study on SH-SY5Y cells was performed to analyze the neuroprotective effect of CA against dexamethasone using the MTT assay. An study was performed for evaluation of the spatial learning and memory using Morris water maze (MWM). RT-PCR was performed to quantify the expression of specific genes ( and ) in the mice brain. The results obtained showed a neuroprotective effect of CA against dexamethasone-induced neuronal toxicity. The escape latency of CA-treated aged mice was significantly decreased as compared to the water-treated aged mice after 4 days of training in MWM. Moreover, CA treatment up-regulated the gene expression of , and , while it down-regulated the gene expression of . These findings suggest that CA has a neuroprotective effect, as well as a spatial learning and memory enhancement potential through the modulation of genes coding for neurotrophic factors and/or those implicated in the imbalance of neural circuitry and impairment of synaptic plasticity.
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http://dx.doi.org/10.18632/aging.202516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880363PMC
January 2021

Effects of Continuous Intake of Rosemary Extracts on Mental Health in Working Generation Healthy Japanese Men: Post-Hoc Testing of a Randomized Controlled Trial.

Nutrients 2020 Nov 20;12(11). Epub 2020 Nov 20.

Department of Clinical and Translational Research Methodology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan.

We previously performed a 4 week interventional trial that suggested that continuous intake of rosemary extract improves the mood states, fatigue, and cognitive function of working generation healthy adult Japanese men. However, the severity of depression in participants in our previous study was relatively mild. Therefore, in the present study, a post-hoc analysis of our previous study was conducted, limited to participants whose total mood disturbance (TMD) scores, which indicate greater mood disturbance, were above the median at baseline, to evaluate whether rosemary extract was effective for individuals with poor mental health. Following the intervention, the scores of TMD and "Confusion-Bewilderment" were significantly decreased (both < 0.05), and scores of "Vigor-Activity" were significantly increased in the rosemary group ( = 8) compared with those in the control group ( = 13; < 0.01). When comparing the scores from pre- and post-intervention, significant improvements in "Tension-Anxiety", "Vigor-Activity", "Fatigue on awakening", "Daytime sleepiness", and "Psychomotor speed" were observed in the rosemary group only (all < 0.05). Based on these results, it was expected that rosemary extracts were effective for improving the mental energy and sleep quality of work-age men with poor mental health.
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http://dx.doi.org/10.3390/nu12113551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699484PMC
November 2020

Magnesium oxide induces immunity against Fusarium wilt by triggering the jasmonic acid signaling pathway in tomato.

J Biotechnol 2021 Jan 10;325:100-108. Epub 2020 Nov 10.

Department of Biological and Environmental Sciences, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan; Research Center for Thermotolerant Microbial Resources (RCTMR), Yamaguchi University, Yamaguchi, 753-8515, Japan. Electronic address:

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is a worldwide tomato disease. Although Fusarium wilt management remains unsuccessful, enhancing host FOL resistance using magnesium oxide to activate plant immunity may enable effective control. We demonstrated that MgO-pretreatment of roots induced FOL resistance in susceptible tomato plants. Resistance was not induced in tomato mutants deficient in the jasmonic acid (JA) signaling pathway, whereas the opposite trend was observed in mutants deficient in the salicylic acid and ethylene signaling pathways, suggesting that JA signaling activation is essential for MgO-induced FOL immunity. Quantitative real-time polymerase chain reaction analysis of MgO-pretreated tomato plants, and challenge-inoculated with FOL, revealed that MYELOCYTOMATOSIS ONCOGENE HOMOLOG 2 (MYC2), the master regulator of JA signaling, as well as MYC2-targeted transcription factors that directly regulate the JA-induced transcription of late defense genes and their downstream wound-responsive genes were preferentially upregulated in both roots and stems. Moreover, in MgO-pretreated tomato plants challenge-inoculated with FOL, the late wound-responsive THREONINE DEAMINASE 2 (TD) gene was expressed earlier than its upstream genes, including MYC2, suggesting that a primed state for defense was established in MgO-pretreated plants. We conclude that MgO is a promising agent for the control of Fusarium wilt.
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http://dx.doi.org/10.1016/j.jbiotec.2020.11.012DOI Listing
January 2021

Antihyperuricemic Effect of Urolithin A in Cultured Hepatocytes and Model Mice.

Molecules 2020 Nov 4;25(21). Epub 2020 Nov 4.

Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya 321-8505, Tochigi, Japan.

Hyperuricemia is defined as a disease with high uric acid (UA) levels in the blood and a strong risk factor for gout. Urolithin A (UroA) is a main microbial metabolite derived from ellagic acid (EA), which occurs in strawberries and pomegranates. In this study, we evaluated antihyperuricemic effect of UroA in both cultured hepatocytes and hyperuricemic model mice. In cultured hepatocytes, UroA significantly and dose-dependently reduced UA production. In model mice with purine bodies-induced hyperuricemia, oral administration of UroA significantly inhibited the increase in plasma UA levels and hepatic xanthine oxidase (XO) activity. In addition, DNA microarray results exhibited that UroA, as well as allopurinol, a strong XO inhibitor, induced downregulation of the expression of genes associated with hepatic purine metabolism. Thus, hypouricemic effect of UroA could be, at least partly, attributed to inhibition of purine metabolism and UA production by suppressing XO activity in the liver. These results indicate UroA possesses a potent antihyperuricemic effect and it could be a potential candidate for a molecule capable of preventing and improving hyperuricemia and gout.
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http://dx.doi.org/10.3390/molecules25215136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662530PMC
November 2020

De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy.

Hum Mutat 2021 Jan 10;42(1):66-76. Epub 2020 Nov 10.

Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.

We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1-4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA-binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole-exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense-mediated mRNA decay (NMD), and one canonical splice site variant, c.272-1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272-1G>C, were clustered within 20 amino acid residues of the C-terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.
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http://dx.doi.org/10.1002/humu.24130DOI Listing
January 2021

Sugarcane ( L.) Top Extract Ameliorates Cognitive Decline in Senescence Model SAMP8 Mice: Modulation of Neural Development and Energy Metabolism.

Front Cell Dev Biol 2020 6;8:573487. Epub 2020 Oct 6.

School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Japan.

Age-related biological alterations in brain function increase the risk of mild cognitive impairment and dementia, a global problem exacerbated by aging populations in developed nations. Limited pharmacological therapies have resulted in attention turning to the promising role of medicinal plants and dietary supplements in the treatment and prevention of dementia. Sugarcane ( L.) top, largely considered as a by-product because of its low sugar content, in fact contains the most abundant amounts of antioxidant polyphenols relative to the rest of the plant. Given the numerous epidemiological studies on the effects of polyphenols on cognitive function, in this study, we analyzed polyphenolic constituents of sugarcane top and examined the effect of sugarcane top ethanolic extract (STEE) on a range of central nervous system functions and . Orally administrated STEE rescued spatial learning and memory deficit in the senescence-accelerated mouse prone 8 (SAMP8) mice, a non-transgenic strain that spontaneously develops a multisystemic aging phenotype including pathological features of Alzheimer's disease. This could be correlated with an increased number of hippocampal newborn neurons and restoration of cortical monoamine levels in STEE-fed SAMP8 mice. Global genomic analysis by microarray in cerebral cortices showed multiple potential mechanisms for the cognitive improvement. Gene set enrichment analysis (GSEA) revealed biological processes such as neurogenesis, neuron differentiation, and neuron development were significantly enriched in STEE-fed mice brain compared to non-treated SAMP8 mice. Furthermore, STEE treatment significantly regulated genes involved in neurotrophin signaling, glucose metabolism, and neural development in mice brain. Our results suggest that STEE treatment enhances the metabolic activity of neuronal cells promoting glucose metabolism with significant upregulation of genes, namely , , , and . STEE also stimulated proliferation of human neural stem cells (hNSCs), regulated bHLH factor expression and induced neuronal differentiation and astrocytic process lengthening. Altogether, our findings suggest the potential of STEE as a dietary intervention, with promising implications as a novel nutraceutical for cognitive health.
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http://dx.doi.org/10.3389/fcell.2020.573487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573230PMC
October 2020

Emotional Impairments and Neuroinflammation are Induced in Male Mice Invulnerable to Repeated Social Defeat Stress.

Neuroscience 2020 09 22;443:148-163. Epub 2020 Jul 22.

Department of Clinical Research, Oriental Medicine Research Center, Kitasato University, Minato-ku, Tokyo 108-8642, Japan.

Prolonged stress triggers neuroinflammation, which plays a significant role in the development of depression; however, stressed people do not always suffer from depression because of individual differences in stress vulnerability. Negative cognitive bias (NCB) toward pessimistic judgment often underlies depressive episodes. However, a relationship between stress vulnerability, neuroinflammation, and NCB remains elusive. In addition, an animal model with all the traits would be a powerful tool for studying the etiology of depression and its therapeutic approaches. Accordingly, this study evaluated the effect of stress vulnerability on neuroinflammation and depression-related behaviors, including NCB in males, using a modified version of repeated social defeat stress (mRSDS) paradigm, a validated animal model of psychosocial stress. Exposure to mRSDS, consisting of 5 min of social defeat by unfamiliar CD-1 aggressor mice for five consecutive days, caused NCB, which co-occurred with depressive- and anxiety-like behaviors, and neuroinflammation in male BALB/c mice. Treatment with minocycline, an antibiotic with anti-inflammatory property, blocked mRSDS-induced depressive-like behaviors and neuroinflammation, but not NCB, indicating the limited effect of an anti-inflammatory intervention. In addition, marked differences were found in neuroinflammatory profiles and hippocampal gene expression patterns between resilient and unstressed mice, as well as between susceptible and resilient mice. Therefore, mice resilient to mRSDS are indeed not intact. Our findings provide insights into the unique features of the mRSDS model in male BALB/c mice, which could be used to investigate the etiological mechanisms underlying depression as well as bridge the gap in the relationship between stress vulnerability, neuroinflammation, and NCB in males.
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http://dx.doi.org/10.1016/j.neuroscience.2020.07.023DOI Listing
September 2020

High PKCλ expression is required for ALDH1-positive cancer stem cell function and indicates a poor clinical outcome in late-stage breast cancer patients.

PLoS One 2020 13;15(7):e0235747. Epub 2020 Jul 13.

Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan.

Despite development of markers for identification of cancer stem cells, the mechanism underlying the survival and division of cancer stem cells in breast cancer remains unclear. Here we report that PKCλ expression was enriched in basal-like breast cancer, among breast cancer subtypes, and was correlated with ALDH1A3 expression (p = 0.016, χ2-test). Late stage breast cancer patients expressing PKCλhigh and ALDH1A3high had poorer disease-specific survival than those expressing PKCλlow and ALDH1A3low (p = 0.018, log rank test for Kaplan-Meier survival curves: hazard ratio 2.58, 95% CI 1.24-5.37, p = 0.011, multivariate Cox regression analysis). Functional inhibition of PKCλ through siRNA-mediated knockdown or CRISPR-Cas9-mediated knockout in ALDH1high MDA-MB 157 and MDA-MB 468 basal-like breast cancer cells led to increases in the numbers of trypan blue-positive and active-caspase 3-positive cells, as well as suppression of tumor-sphere formation and cell migration. Furthermore, the amount of CASP3 and PARP mRNA and the level of cleaved caspase-3 protein were enhanced in PKCλ-deficient ALDH1high cells. An Apoptosis inhibitor (z-VAD-FMK) suppressed the enhancement of cell death as well as the levels of cleaved caspase-3 protein in PKCλ deficient ALDH1high cells. It also altered the asymmetric/symmetric distribution ratio of ALDH1A3 protein. In addition, PKCλ knockdown led to increases in cellular ROS levels in ALDH1high cells. These results suggest that PKCλ is essential for cancer cell survival and migration, tumorigenesis, the asymmetric distribution of ALDH1A3 protein among cancer cells, and the maintenance of low ROS levels in ALDH1-positive breast cancer stem cells. This makes it a key contributor to the poorer prognosis seen in late-stage breast cancer patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235747PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357771PMC
September 2020

Acyl-CoA dehydrogenase long chain (ACADL) is a target protein of stylissatin A, an anti-inflammatory cyclic heptapeptide.

J Antibiot (Tokyo) 2020 08 21;73(8):589-592. Epub 2020 May 21.

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.

Stylissatin A (SA) is a cyclic heptapeptide isolated from the marine sponge Stylissa massa. SA shows anti-inflammatory activity against lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cells, but the detailed mechanism of action remains unclear. Here we report that D-Tyr-tBuSA, a more potent SA derivative, inhibited production of the proinflammatory cytokines Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in LPS-stimulated RAW264.7 cells (EC = 1.4 and 5.9 μM, respectively). This compound also inhibited the LPS-stimulated expression of inducible nitric oxide synthase (iNOS) at 20 μM. Using a biotin derivative of SA, acyl-CoA dehydrogenase long chain (ACADL) was identified as a target protein of SA and its derivatives. It is proposed that SA and its derivatives might suppress the β-oxidation of fatty acids by ACADL, and the accumulation of fatty acids on macrophages would inhibit the nuclear factor-kappa B (NF-κB) signaling pathway and iNOS expression to show anti-inflammatory activity. Our research might provide a new mechanism of inflammation in macrophages, and contribute to the development of treatments for inflammatory diseases.
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http://dx.doi.org/10.1038/s41429-020-0322-5DOI Listing
August 2020

Synthesis of 2-(2-Hydroxyethoxy)-3-hydroxysqualene and Characterization of Its Anti-Inflammatory Effects.

Biomed Res Int 2020 14;2020:9584567. Epub 2020 Apr 14.

Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.

Squalene (SQ), a natural precursor of many steroids, can inhibit tumor progression and decrease serum cholesterol levels. However, it is difficult to discern the effect of highly active molecules in the treatment of diseases because not enough active compounds reach the site of pathology in crowded biosystems. Therefore, it is necessary to design artificial probes that work effectively within crowded systems. In this study, to facilitate cell penetration, the ethylene glycol moiety (used as a probe) was chemically added to SQ to form 2-(2-hydroxyethoxy)-3-hydroxysqualene (HEHSQ). HEHSQ was prepared from 2,3-epoxysqualene and characterized by R, FT-IR, H NMR, C NMR, and high-resolution mass spectrometry. We then evaluated the anti-inflammatory effects of SQ and HEHSQ on lipopolysaccharide- (LPS-) stimulated RAW264.7 murine macrophages. To determine the effect of SQ and HEHSQ on the viability of RAW264.7 cells, an MTT assay was performed. To quantify the anti-inflammatory effect of SQ and HEHSQ, we measured nitric oxide (NO) production, gene expression, and secretion of the proinflammatory cytokine tumor necrosis factor (TNF-) and chemokine C-C motif chemokine 2 (CCL2) in LPS-stimulated RAW264.7 cells using an inflammatory model. 2,3-Epoxysqualene was prepared according to a reported methodology. The reaction of 2,3-epoxysqualene and ethylene glycol in 2-propanol produced 49% HEHSQ. MTT results showed that 10 and 100 g/mL HEHSQ treatment decreased cell viability, whereas SQ treatment (1-100 g/mL) did not have any effect on viability. SQ (100 g/mL) and HEHSQ (1 g/mL) treatment significantly reduced the production of LPS-stimulated NO and decreased the expression and secretion of proinflammatory TNF- and CCL2. Therefore, our results suggested that the anti-inflammatory effects of HEHSQ are 100 times higher than that of unmodified SQ. To the best of our knowledge, this study has demonstrated for the first time that HEHSQ can be potentially used as a safe alternative treatment to anti-inflammatory drugs.
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http://dx.doi.org/10.1155/2020/9584567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180416PMC
February 2021

Shank2 Binds to aPKC and Controls Tight Junction Formation with Rap1 Signaling during Establishment of Epithelial Cell Polarity.

Cell Rep 2020 04;31(1):107407

Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Electronic address:

Epithelial cells establish apicobasal polarity by forming tight junctions (TJs) at the apical-lateral boundary, which play fundamental roles in physiological functions. An evolutionarily conserved atypical protein kinase C (aPKC)-partitioning defective (PAR) complex functions as a platform for TJ assembly during cell polarity establishment. However, how this complex converts the spatial cues into a subsequent active unit is unclear. Here, we identify an epithelial isoform of Shank2 as a mediator of the aPKC-PAR complex. Shank2 binds to and colocalizes with aPKC at apical junctional regions of polarized epithelial cells. Shank2 knockdown results in defects in TJ formation. Mechanistically, we find that the N-terminal SPN domain is required for the junctional localization of Shank2 and binds to the active form of Rap1 small GTPase, which is involved in TJ formation. Our findings suggest that a close physical and functional relationship between aPKC and Shank2-active Rap1 signaling serves as the platform for TJ assembly to regulate epithelial cell polarity.
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http://dx.doi.org/10.1016/j.celrep.2020.02.088DOI Listing
April 2020

Microarray analysis of verbenalin-treated human amniotic epithelial cells reveals therapeutic potential for Alzheimer's Disease.

Aging (Albany NY) 2020 03 29;12(6):5516-5538. Epub 2020 Mar 29.

Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Tsukuba 305-8577, Ibaraki, Japan.

Alzheimer's disease (AD) has become a major world health problem as the population ages. There is still no available treatment that can stop or reverse the progression of AD. Human amnion epithelial cells (hAECs), an alternative source for stem cells, have shown neuroprotective and neurorestorative potentials when transplanted . Besides, studies have suggested that stem cell priming with plant-derived bioactive compounds can enhance stem cell proliferation and differentiation and improve the disease-treating capability of stem cells. Verbenalin is an iridoid glucoside found in medicinal herbs of Verbenaceae family. In the present study, we have conducted microarray gene expression profiling of verbenalin-treated hAECs to explore its therapeutic potential for AD. Gene set enrichment analysis revealed verbenalin treatment significantly enriched AD-associated gene sets. Genes associated with lysosomal dysfunction, pathologic angiogenesis, pathologic protein aggregation, circadian rhythm, age-related neurometabolism, and neurogenesis were differentially expressed in the verbenalin-treated hAECs compared to control cells. Additionally, the neuroprotective effect of verbenalin was confirmed against amyloid beta-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Our present study is the first to report the therapeutic potential of verbenalin for AD; however, further in-depth research in the and models are required to confirm our preliminary findings.
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http://dx.doi.org/10.18632/aging.102985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138585PMC
March 2020

The role of Mediator and Little Elongation Complex in transcription termination.

Nat Commun 2020 02 26;11(1):1063. Epub 2020 Feb 26.

Department of Biochemistry, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan.

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.
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http://dx.doi.org/10.1038/s41467-020-14849-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044329PMC
February 2020

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities.

Am J Hum Genet 2020 01 12;106(1):13-25. Epub 2019 Dec 12.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. Electronic address:

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.
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http://dx.doi.org/10.1016/j.ajhg.2019.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042485PMC
January 2020

High Throughput Screening of Serum γ-Glutamyl Dipeptides for Risk Assessment of Nonalcoholic Steatohepatitis with Impaired Glutathione Salvage Pathway.

J Proteome Res 2020 07 26;19(7):2689-2699. Epub 2019 Sep 26.

Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4M1, Canada.

Nonalcoholic fatty liver disease (NAFLD) is the most common preventable chronic liver disorder in developed countries, the prevalence of which is increasing worldwide due to its association with obesity and type 2 diabetes. However, the exact mechanisms of NAFLD pathophysiology remain poorly understood including its progression to the more severe nonalcoholic steatohepatitis (NASH). New advances for early detection and monitoring of NASH progression are limited due to the lack of specific blood biomarkers, thus requiring invasive liver biopsies for histopathology. Herein, multisegment injection-capillary electrophoresis-tandem mass spectrometry (MSI-CE-MS/MS) is validated as a high throughput, robust, and quantitative platform for targeted analysis of a panel of 16 serum γ-glutamyl dipeptides from a cohort of NASH adult patients from Japan (median age = 53 years, median BMI = 27 kg/m, = 116). Multiplexed separations based on MSI-CE-MS/MS enable the design of unique data workflows that rely on customizable serial sample injection formats for accurate determination of γ-glutamyl dipeptides with quality control. Also, the introduction of a liquid coolant device to the capillary outlet improves long-term migration time stability in CE. Unsupervised pattern recognition methods revealed two distinctive NASH subgroups based on their contrasting γ-glutamyl dipeptide status despite patients having similar clinical phenotypes and NASH activity scores (median NAS ≈ 6.0). There was an inverse correlation between serum γ-glutamyl dipeptide concentrations and γ-glutamyltransferease (GGT) enzyme activity ( = -0.46; = 2.5 × 10), which was indicative of a low-risk ( = 64) as compared to a high-risk ( = 52) patient subgroup with impaired glutathione salvage pathway and likely poor clinical prognosis. Our findings highlight the key role of defects in the γ-glutamyl cycle for differentiation of NASH patients, which may enable better risk assessment of long-term survivorship as a complement to standard liver enzyme screens and histopathology.
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http://dx.doi.org/10.1021/acs.jproteome.9b00405DOI Listing
July 2020

Exploring the Potential Role of Rosmarinic Acid in Neuronal Differentiation of Human Amnion Epithelial Cells by Microarray Gene Expression Profiling.

Front Neurosci 2019 24;13:779. Epub 2019 Jul 24.

Alliance for Research on the Mediterranean and North Africa, University of Tsukuba, Tsukuba, Japan.

In the present study, we conducted microarray gene expression profiling to explore the direction of differentiation of human amnion epithelial cells (hAECs) treated with rosmarinic acid (RA). hAECs have several clinical advantages over other types of stem cells, including availability, low immunogenicity, low rejection rate, non-tumorigenicity, and less ethical constraint. On the other hand, RA is a phenolic compound having several health benefits, including neuroprotective and antidepressant-like activities. In this study, hAECs were isolated from discarded term placenta and were treated with 20 μM RA for 7 days. Microarray gene expression profiling was conducted for three biological replicates of RA-treated and untreated control cells on day 0 and day 7. Gene set enrichment analysis, and gene annotation and pathway analysis were conducted using online data mining tools GSEA and DAVID. Gene expression profiling showed that RA treatment biased hAECs toward ectodermal lineage progression, regulated transcription factors involved in neuronal differentiation, regulated neural specific epigenetic modifiers and several extracellular signaling pathways of neural induction, and significantly inhibited Notch signaling pathway. Gene expression profiling of RA-treated hAECs reveals for the first time a potential role of RA in neural induction and neuronal differentiation of hAECs. Having a naturally occurring compound as differentiation inducer as well as a readily available source of stem cells would have great advantages for the cell-based therapies. Findings from our genome-wide analysis could provide a foundation for further in-depth investigation.
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http://dx.doi.org/10.3389/fnins.2019.00779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667736PMC
July 2019

Antidepressant-Like Effect of Ferulic Acid via Promotion of Energy Metabolism Activity.

Mol Nutr Food Res 2019 10 22;63(19):e1900327. Epub 2019 Aug 22.

Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan.

Scope: Ferulic acid (FA), a natural phenolic phytochemical abundantly present in whole grains, herbs, and dried fruits, exhibits anti-inflammatory, antioxidant, and neuroprotective effects. In the present study, the antidepressant-like effects of FA in male ICR mice using tail suspension test (TST) are investigated and its molecular mechanisms are explored.

Methods And Results: Oral administration of FA at a dose of 5 mg kg for 7 days significantly reduces immobility of mice compared to vehicle-administered control group. Microarray and real-time PCR analyses reveal that FA upregulates the expression of several genes associated with cell survival and proliferation, energy metabolism, and dopamine synthesis in mice limbic system of brain. Interestingly, it is found that FA, unlike antidepressant drug bupropion, strongly promotes energy metabolism. Additionally, FA increases catecholamine (dopamine and noradrenaline), brain-derived neurotrophic factor, and ATP levels, and decreases glycogen levels in the limbic system of the mice brain.

Conclusion: The research provides the first evidence that FA enhances energy production, which can be the underlying mechanism of the antidepressant-like effects of FA observed in this study.
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http://dx.doi.org/10.1002/mnfr.201900327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790570PMC
October 2019

Elucidation of the Molecular Mechanism Underlying (Lim.)-Induced Relaxation and Anti-Depression.

Int J Mol Sci 2019 Jul 20;20(14). Epub 2019 Jul 20.

Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, 1-1-1 Tennodai, Tsukuba City 305-8572, Ibaraki, Japan.

ethanolic extract (VEE) and verbascoside (Vs), a phenypropanoid glycoside, have been demonstrated to exert relaxant and anxiolytic properties. However, the molecular mechanisms behind their effects are still unclear. In this work, we studied the effects and action mechanisms of VEE and Vs and , on human neurotypic SH-SY5Y cells.TST was conducted on mice treated orally with VEE (25, 50 and 100 mg/Kg), Vs (2.5 and 5 mg/Kg), Bupropion (20 mg/Kg) and Milli-Q water. Higher dose of VEE-treated mice showed an increase of immobility time compared to control groups, indicating an induction of relaxation. This effect was found to be induced by regulation of genes playing key roles in calcium homeostasis (calcium channels), cyclic AMP (cAMP) production and energy metabolism. On the other hand, low doses of VEE and Vs showed an antidepressant-like effect and was confirmed by serotonin, noradrenalin, dopamine and BDNF expressions. Finally, VEE and Vsenhancedcell viability, mitochondrial activity and calcium uptake confirming findings. Our results showed induction of relaxation and antidepressant-like effects depending on the administered dose of VEE and Vs, through modulation of cAMP and calcium.
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http://dx.doi.org/10.3390/ijms20143556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678442PMC
July 2019
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