Publications by authors named "Seiji Okada"

316 Publications

Autoreactivity of Peripheral Helper T Cells in the Joints of Rheumatoid Arthritis.

J Immunol 2021 Apr 12. Epub 2021 Apr 12.

Department of Orthopedic Surgery, Kyushu University, Fukuoka, Japan.

Autoreactive CD4 T cells are thought to play pivotal roles in the pathogenesis of rheumatoid arthritis (RA). Recently, a subset of CD4 T cells that express high levels of programmed death-1 (PD-1) but are distinct from follicular helper T cells have been identified in the joints of RA patients and named peripheral helper T (Tph) cells. Because PD-1 is expressed on T cells chronically stimulated with the Ags, we tested a hypothesis that Tph cells are the pathogenic autoreactive CD4 T cells in RA. We found that human Tph cells in RA joints produce proinflammatory effector cytokines, including IFN-γ, TNF-α, and GM-CSF, in addition to B cell-helping cytokines, such as IL-21 and CXCL13. Flow cytometric analysis showed different bias of TCR Vβ usage between PD-1 Tph cells and PD-1 CD4 T cells, including Th1 cells, in the joint or memory CD4 T cells in the peripheral blood, whereas there was little difference between the latter two subsets. In line with this, deep sequencing of TCR demonstrated an overlap of expanded clones between peripheral blood memory CD4 T cells and PD-1 CD4 T cells but not Tph cells in the joint. Interestingly, Tph cells preferentially exhibited autologous MLR in vitro, which required recognition of self-MHC class II and was pronounced by blocking PD-1 signaling. Taken together, these results suggest that Tph cells are the pathogenic autoreactive CD4 T cells in RA, which expand locally in the joints and are regulated by PD-1 signaling.
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http://dx.doi.org/10.4049/jimmunol.2000783DOI Listing
April 2021

Improved safety of induced pluripotent stem cell-derived antigen-presenting cell-based cancer immunotherapy.

Mol Ther Methods Clin Dev 2021 Jun 5;21:171-179. Epub 2021 Mar 5.

Division of Cancer Immunotherapy, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa 277-8577, Japan.

The tumorigenicity and toxicity of induced pluripotent stem cells (iPSCs) and their derivatives are major safety concerns in their clinical application. Recently, we developed granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing proliferating myeloid cells (GM-pMCs) from mouse iPSCs as a source of unlimited antigen-presenting cells for use in cancer immunotherapy. As GM-pMCs are generated by introducing and into iPSC-derived MCs and are dependent on self-produced GM-CSF for proliferation, methods to control their proliferation after administration should be introduced to improve safety. In this study, we compared the efficacy of two promising suicide gene systems, herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and inducible caspase-9 (iCasp9)/AP1903, for safeguarding GM-pMCs in cancer immunotherapy. The expression of HSV-TK or iCasp9 did not impair the fundamental properties of GM-pMCs. Both of these suicide gene-expressing cells selectively underwent apoptosis after treatment with the corresponding apoptosis-inducing drug, and they were promptly eliminated . iCasp9/AP1903 induced apoptosis more efficiently than HSV-TK/GCV. Furthermore, high concentrations of GCV were toxic to cells not expressing HSV-TK, whereas AP1903 was bioinert. These results suggest that iCasp9/AP1903 is superior to HSV-TK/GCV in terms of both safety and efficacy when controlling the fate of GM-pMCs after priming antitumor immunity.
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http://dx.doi.org/10.1016/j.omtm.2021.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994724PMC
June 2021

A Therapeutic Strategy to Combat HIV-1 Latently Infected Cells With a Combination of Latency-Reversing Agents Containing DAG-Lactone PKC Activators.

Front Microbiol 2021 17;12:636276. Epub 2021 Mar 17.

National Center for Global Health and Medicine Research Institute, Tokyo, Japan.

Advances in antiviral therapy have dramatically improved the therapeutic effects on HIV type 1 (HIV-1) infection. However, even with potent combined antiretroviral therapy, HIV-1 latently infected cells cannot be fully eradicated. Latency-reversing agents (LRAs) are considered a potential tool for eliminating such cells; however, recent and studies have raised serious concerns regarding the efficacy and safety of the "shock and kill" strategy using LRAs. In the present study, we examined the activity and safety of a panel of protein kinase C (PKC) activators with a diacylglycerol (DAG)-lactone structure that mimics DAG, an endogenous ligand for PKC isozymes. YSE028, a DAG-lactone derivative, reversed HIV-1 latency when tested using HIV-1 latently infected cells (e.g., ACH2 and J-Lat cells) and primary cells from HIV-1-infected individuals. The activity of YSE028 in reversing HIV-1 latency was synergistically enhanced when combined with JQ1, a bromodomain and extra-terminal inhibitor LRA. DAG-lactone PKC activators also induced caspase-mediated apoptosis, specifically in HIV-1 latently infected cells. In addition, these DAG-lactone PKC activators showed minimal toxicity and . These data suggest that DAG-lactone PKC activators may serve as potential candidates for combination therapy against HIV-1 latently infected cells, especially when combined with other LRAs with a different mechanism, to minimize side effects and achieve maximum efficacy in various reservoir cells of the whole body.
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http://dx.doi.org/10.3389/fmicb.2021.636276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010149PMC
March 2021

Induction of Apoptotic Cell Death in Human Leukemia U937 Cells by C18 Hydroxy Unsaturated Fatty Acid Isolated from Red Alga .

Mar Drugs 2021 Mar 2;19(3). Epub 2021 Mar 2.

Graduate School of Fisheries and Environmental Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.

Our previous studies have found that (±)-()-12-hydroxyoctadec-10-enoic acid (HOEA) isolated from the red alga showed cytotoxic effects on various living organisms including harmful microalgae, Gram-positive bacteria, and mammalian tumor cells. Since natural products with apoptosis-inducing ability can be promising anti-cancer agents, in this study, we investigated the cytotoxic mechanism of HOEA on U937 cells focusing on apoptosis induction. HOEA showed much stronger cytotoxic and cytolytic effects on U937 cells than elaidic acid, which has similar structure but no 12-hydroxy group, suggesting that hydroxy group is important for the cytotoxicity of HOEA. HOEA induced apoptotic nuclear morphological changes, DNA fragmentation, and decrease in mitochondrial membrane potential. Furthermore, time-dependent increase in annexin V+/PI+ cell population in HOEA-treated U937 cells was detected. Among the apoptosis-related reagents, caspase-family inhibitor almost completely inhibited HOEA-induced DNA fragmentation. In the analyses using specific caspase-substrates, extremely high cleavage activity toward caspase-3/7/8 substrate was observed in HOEA-treated U937 cells, and weak activities of caspase-1 and -3 were detected. Analyses using specific caspase inhibitors suggested that caspase-3 and caspase-8 might be predominantly responsible for the cleavage activity. Activation of these caspases were also confirmed by western blotting in which significant levels of cleaved forms of caspase 3, caspase 8, and PARP were detected in HOEA-treated U937 cells. Our results suggest that HOEA is capable of inducing apoptosis in U937 cells in which caspase-3 and caspase-8 might play important roles. Since the cytotoxic effect of HOEA is not strictly specific to tumor cells, development of appropriate drug delivery system for selective tumor targeting is necessary for the clinical applications to reduce the possible side effects.
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http://dx.doi.org/10.3390/md19030138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001238PMC
March 2021

Cucurbitacin B induces apoptosis of primary effusion lymphoma via disruption of cytoskeletal organization.

Phytomedicine 2021 Mar 10;85:153545. Epub 2021 Mar 10.

Division of Hematopoiesis, Joint Research Center for Human Retrovirus Infection, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan; Division of Hematopoiesis, Graduate School of Medical Sciences, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Electronic address:

Background: Primary effusion lymphoma (PEL) is an aggressive B cell non-Hodgkin lymphoma that develops especially in AIDS patients and immunocompromised patients infected with human herpes virus-8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus (KSHV). PEL has a poor prognosis in patients despite conventional chemotherapeutic treatment, and a safe and efficient therapy is required.

Purpose: To examine the effects on PEL of cucurbitacin B (CuB), a triterpene found in plants of the Cucurbitaceae family that has several anti-cancer activities.

Study Design: We evaluated the anti-cancer activities of CuB in vitro and in vivo.

Methods: Cell proliferation of PEL cell lines was measured by MTT assay. Cleaved caspases and signaling transduction associated proteins were analyzed by western blotting. Wright and Giemsa staining and immunofluorescence staining were carried out to observe cell morphology. Cell cycles were analyzed by flow cytometry. RT-PCR was performed to detect viral gene expressions. A xenograft mouse model was employed to evaluate the anti-cancer activity of CuB in vivo.

Results: CuB inhibited cell proliferation of PEL cell lines (BCBL-1, BC-1, GTO and TY-1) in a dose-dependent manner (0-50 nM) and induced apoptosis of BCBL-1 cells via caspase activation in a dose- and time-dependent manner. In addition, CuB caused cell-shape disruption by inducing actin aggregation and suppressing the p-cofilin level, resulting in BCBL-1 cell arrest at the G2/M phase. In contrast, CuB showed almost no suppression of p-STAT3 and p-Akt activation, which were constitutively activated by KSHV-derived proteins. Furthermore, CuB (0.5 mg/kg) via intraperitoneal injection significantly (p < 0.05) suppressed solid tumor growth in the xenograft mouse model.

Conclusion: This study suggests that CuB is a promising agent for PEL treatment.
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http://dx.doi.org/10.1016/j.phymed.2021.153545DOI Listing
March 2021

Bioinformatics analysis identified CDC20 as a potential drug target for cholangiocarcinoma.

PeerJ 2021 17;9:e11067. Epub 2021 Mar 17.

Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand.

Background: Cholangiocarcinoma (CCA) is a malignancy that originates from bile duct cells. The incidence and mortality of CCA are very high especially in Southeast Asian countries. Moreover, most CCA patients have a very poor outcome. Presently, there are still no effective treatment regimens for CCA. The resistance to several standard chemotherapy drugs occurs frequently; thus, searching for a novel effective treatment for CCA is urgently needed.

Methods: In this study, comprehensive bioinformatics analyses for identification of novel target genes for CCA therapy based on three microarray gene expression profiles (GSE26566, GSE32225 and GSE76297) from the Gene Expression Omnibus (GEO) database were performed. Based on differentially expressed genes (DEGs), gene ontology and pathway enrichment analyses were performed. Protein-protein interactions (PPI) and hub gene identifications were analyzed using STRING and Cytoscape software. Then, the expression of candidate genes from bioinformatics analysis was measured in CCA cell lines using real time PCR. Finally, the anti-tumor activity of specific inhibitor against candidate genes were investigated in CCA cell lines cultured under 2-dimensional and 3-dimensional cell culture models.

Results: The three microarray datasets exhibited an intersection consisting of 226 DEGs (124 up-regulated and 102 down-regulated genes) in CCA. DEGs were significantly enriched in cell cycle, hemostasis and metabolism pathways according to Reactome pathway analysis. In addition, 20 potential hub genes in CCA were identified using the protein-protein interaction (PPI) network and sub-PPI network analysis. Subsequently, CDC20 was identified as a potential novel targeted drug for CCA based on a drug prioritizing program. In addition, the anti-tumor activity of a potential CDC20 inhibitor, namely dinaciclib, was investigated in CCA cell lines. Dinaciclib demonstrated huge anti-tumor activity better than gemcitabine, the standard chemotherapeutic drug for CCA.

Conclusion: Using integrated bioinformatics analysis, CDC20 was identified as a novel candidate therapeutic target for CCA.
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http://dx.doi.org/10.7717/peerj.11067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980698PMC
March 2021

Metabolic Alteration in Hepatocellular Carcinoma: Mechanism of Lipid Accumulation in Well-Differentiated Hepatocellular Carcinoma.

Can J Gastroenterol Hepatol 2021 18;2021:8813410. Epub 2021 Feb 18.

Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

Objective: Metabolic alteration is widely considered as one of the hallmarks of cancer. Hepatocellular carcinoma (HCC) presents a unique pathological feature in which lipid accumulation is common in well-differentiated HCC and rare in poorly differentiated HCC; however, the underlying mechanism remains unclear.

Methods: Tissue samples were obtained from 103 HCC patients who had undergone hepatic resection and 12 living donors of liver transplantation. We evaluated metabolic gene expressions in cancer tissues as well as background noncancer tissues and compared the expressions by the degree of cancer differentiation and by liver disease states. Besides, the metabolomics was evaluated and integrated to gene expressions in nonalcoholic steatohepatitis (NASH)-HCC model mice.

Results: In cancer tissues, the expression levels of enzymes related to glycolysis, pentose phosphate pathway (PPP), and fatty acid (FA) synthesis were increased and that of tricarboxylic acid (TCA) cycle and -oxidation were suppressed. Same metabolic alterations were observed in noncancer tissue as the liver disease progresses from healthy liver to chronic hepatitis, cirrhosis, and HCC. Similar alterations of metabolic genes were detected in NASH-HCC mice, which were consistent with the results of metabolomics. As the degree of cancer differentiation decreased, glycolysis and PPP were accelerated; however, FA synthesis and uptake were diminished.

Conclusions: The metabolic alterations including glycolysis, PPP, TCA cycle, and -oxidation became more prominent as liver disease progresses from normal, chronic hepatitis, cirrhosis, well-, moderately, and poorly differentiated HCC. FA synthesis and uptake were highest in well-differentiated HCC, which could explain the lipid accumulation.
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http://dx.doi.org/10.1155/2021/8813410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910064PMC
February 2021

Ribosome induces transdifferentiation of A549 and H-111-TC cancer cell lines.

Biochem Biophys Rep 2021 Jul 12;26:100946. Epub 2021 Feb 12.

Department of Developmental Neurobiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, 860-8556, Japan.

Previously we reported that, lactic acid bacteria (LAB) can induce human dermal fibroblast (HDF) cells to form multipotent cell clusters which are able to transdifferentiate into three germ layer derived cell lineages. Later on, we confirmed that ribosome is responsible for the LAB-induced transdifferentiation and ribosomes from diverse organisms can mimic the LAB effect on HDF cells. In our present study we have shown that, upon incorporation of ribosomes, non-small cell lung cancer cell line A549 and gastric tubular adenocarcinoma cell line H-111-TC are transformed into spheroid like morphology those can be transdifferentiated into adipocytes and osteoblast. Our qPCR analysis has revealed that, during the formation of ribosome induced cancer cell spheroids, the expression of the cancer cell associated markers and cell cycle/proliferation markers were altered at different time point. Through our investigation, here we report a novel and a non-invasive approach for cancer cell reprogramming by incorporating ribosomes.
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http://dx.doi.org/10.1016/j.bbrep.2021.100946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887644PMC
July 2021

Modified Riceberry rice extract suppresses melanogenesis-associated cell differentiation through tyrosinase-mediated MITF downregulation on B16 cells and zebrafish embryos.

Res Pharm Sci 2020 Oct 19;15(5):491-502. Epub 2020 Oct 19.

Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

Background And Purpose: Excessive melanin production caused by overactive tyrosinase (TYR) enzyme results in several dermatological problems. The TYR inhibitor, derived from metabolite changes during fermentation, has been well recognized for pigmentation control.

Experimental Approach: This study is interested in alternative anti-melanogenic agents from bio-modified Riceberry rice through fermentation. Modified Riceberry rice extract (MRB) was evaluated for its cytotoxicity, melanin content, melanin excretion, and TYR activity in B16 cells. TYR and their melanogenesis-related molecules such as TYR-related proteins-1 and -2, and microphthalmia-associated transcription factor (MITF) were determined. The anti-melanogenic activity and toxicity were also tested using the embryonic zebrafish model. Furthermore, comprehensive genotoxicity testing was verified by cytokinesis-block micronucleus cytome assay.

Findings/results: The study found that non-cytotoxic concentrations of MRB at 20 and 40 mg/mL inhibited melanogenesis and melanin excretion by interfering B16 cell morphology. Cellular TYR enzymatic activity was also suppressed in the treated cells. The mRNA transcription and protein expression levels of TYR and MITF decreased by dose-dependent and time-dependent manners with MRB treatment. In the animal model, MRB was found to be safe and potent for melanogenesis-related TYR inhibition in embryonic zebrafish at 20 and 30 mg/mL. The toxicity of effective doses of MRB showed no genotoxicity and mutagenicity.

Conclusion And Implications: This study suggests that MRB has anti-melanogenesis potential through TYR and its-related protein inhibitions. MRB is also safe for applications and maybe a promising anti-melanogenic agent for hyperpigmentation control.
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http://dx.doi.org/10.4103/1735-5362.297852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879784PMC
October 2020

Inflammation-driven senescence-associated secretory phenotype in cancer-associated fibroblasts enhances peritoneal dissemination.

Cell Rep 2021 Feb;34(8):108779

Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; Gastrointestinal Cancer Biology, International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan. Electronic address:

In the tumor microenvironment, senescent non-malignant cells, including cancer-associated fibroblasts (CAFs), exhibit a secretory profile under stress conditions; this senescence-associated secretory phenotype (SASP) leads to cancer progression and chemoresistance. However, the role of senescent CAFs in metastatic lesions and the molecular mechanism of inflammation-related SASP induction are not well understood. We show that pro-inflammatory cytokine-driven EZH2 downregulation maintains the SASP by demethylating H3K27me3 marks in CAFs and enhances peritoneal tumor formation of gastric cancer (GC) through JAK/STAT3 signaling in a mouse model. A JAK/STAT3 inhibitor blocks the increase in GC cell viability induced by senescent CAFs and peritoneal tumor formation. Single-cell mass cytometry revealed that fibroblasts exist in the ascites of GC patients with peritoneal dissemination, and the fibroblast population shows p16 expression and SASP factors at high levels. These findings provide insights into the inflammation-related SASP maintenance by histone modification and the role of senescent CAFs in GC peritoneal dissemination.
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http://dx.doi.org/10.1016/j.celrep.2021.108779DOI Listing
February 2021

Reactive astrocyte nomenclature, definitions, and future directions.

Nat Neurosci 2021 03 15;24(3):312-325. Epub 2021 Feb 15.

Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK.

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions.
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http://dx.doi.org/10.1038/s41593-020-00783-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007081PMC
March 2021

Microglial inflammation after chronic spinal cord injury is enhanced by reactive astrocytes via the fibronectin/β1 integrin pathway.

J Neuroinflammation 2021 Jan 6;18(1):12. Epub 2021 Jan 6.

Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.

Background: After spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration. Recently, we reported that anti-β1 integrin antibody (β1Ab) had a therapeutic effect on astrocytes by preventing the induction of glial scar formation. However, the cellular components within the glial scar are not only astrocytes but also microglia, and whether or not β1Ab treatment has any influence on microglia within the glial scar remains unclear.

Methods: To evaluate the effects of β1Ab treatment on microglia within the glial scar after SCI, we applied thoracic contusion SCI to C57BL/6N mice, administered β1Ab in the sub-acute phase, and analyzed the injured spinal cords with immunohistochemistry in the chronic phase. To examine the gene expression in microglia and glial scars, we selectively collected microglia with fluorescence-activated cell sorting and isolated the glial scars using laser-captured microdissection (LMD). To examine the interaction between microglia and astrocytes within the glial scar, we stimulated BV-2 microglia with conditioned medium of reactive astrocytes (RACM) in vitro, and the gene expression of TNFα (pro-inflammatory M1 marker) was analyzed via quantitative polymerase chain reaction. We also isolated both naïve astrocytes (NAs) and reactive astrocytes (RAs) with LMD and examined their expression of the ligands for β1 integrin receptors. Statistical analyses were performed using Wilcoxon's rank-sum test.

Results: After performing β1Ab treatment, the microglia were scattered within the glial scar and the expression of TNFα in both the microglia and the glial scar were significantly suppressed after SCI. This in vivo alteration was attributed to fibronectin, a ligand of β1 integrin receptors. Furthermore, the microglial expression of TNFα was shown to be regulated by RACM as well as fibronectin in vitro. We also confirmed that fibronectin was secreted by RAs both in vitro and in vivo. These results highlighted the interaction mediated by fibronectin between RAs and microglia within the glial scar.

Conclusion: Microglial inflammation was enhanced by RAs via the fibronectin/β1 integrin pathway within the glial scar after SCI. Our results suggested that β1Ab administration had therapeutic potential for ameliorating both glial scar formation and persistent neuroinflammation in the chronic phase after SCI.
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http://dx.doi.org/10.1186/s12974-020-02059-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789752PMC
January 2021

Shape Factor of the Spinal Cord: A Possible Predictor of Surgical Outcome for Intradural Extramedullary Spinal Tumors in the Thoracic Spine.

Global Spine J 2021 Jan 7:2192568220982571. Epub 2021 Jan 7.

Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan.

Study Design: Retrospective diagnostic analysis.

Objectives: To establish a new predictor of surgical outcome after surgery for intradural extramedullary spinal tumor (IDEMT) in the thoracic spine, we introduced shape factor (SF), a mathematical description of the morphology of the spinal cord. SF was calculated by dividing object area by the square of perimeter.

Materials And Methods: Forty-three consecutive patients with IDEMT, detected by magnetic resonance imaging at the thoracic level with myelopathic signs, were included. Preoperative transverse cross-sectional area (CSA) and perimeter of the spinal cord (perimeter) at the level of maximal compression were measured. SF was calculated as 4π × CSA/(perimeter). The association between clinicoradiological factors and surgical outcome of IDEMT was statistically analyzed.

Results: Mean CSA, perimeter, and SF were 27.8 ± 15.8 mm, 28.8 ± 6.1 mm, and 0.385 ± 0.14, respectively. A histogram distribution revealed that perimeter and SF, but not CSA, fit the normal distribution. The patients were subdivided into 2 groups according to postoperative modified Japanese Orthopedic Association Score (mJOA). [group F (favorable): n = 32, mJOA ≥ 9; group UF (unfavorable): n = 11, mJOA < 9). Group UF had significantly lower mean CSA and SF. In univariate analysis of possible predictive factors for IDEMT surgery, greater age, lower preoperative mJOA, and lower SF were significantly associated with unfavorable outcome. In multivariate analysis, lower SF was the only significant predictor of postoperative outcome (odds ratio = 2.66, 95% CI 1.10-6.39, p 0.0115).

Conclusion: Measurements of CSA and perimeter, followed by calculation of SF, may provide valuable quantitative information for the outcome of surgery for IDEMT.
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http://dx.doi.org/10.1177/2192568220982571DOI Listing
January 2021

Induction of apoptosis by Shikonin through ROS-mediated intrinsic and extrinsic apoptotic pathways in primary effusion lymphoma.

Transl Oncol 2021 Mar 2;14(3):101006. Epub 2021 Jan 2.

Division of Hematopoiesis, Joint Research Center for Human Retrovirus Infection, Kumamoto University, 2-2-1, Honjo, Kumamoto, 860-0811, Japan. Electronic address:

Primary effusion lymphoma (PEL) is an incurable non-Hodgkin's lymphoma and novel biology-based treatments are urgently needed in clinical settings. Shikonin (SHK), a napthoquinone derivative, has been used for the treatment of solid tumors. Here, we report that SHK is an effective agent for the treatment of PEL. Treatment with SHK results in significant reduction of proliferation in PEL cells and their rapid apoptosis in vitro. SHK-induced apoptosis of PEL cells is accompanied by the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (Δψm), an activation of c-Jun-N-terminal kinase (JNK), p38, as well as caspase-3, -8, and -9. Scavenging of ROS in the presence of N-acetylcysteine (NAC) almost blocks the loss of mitochondrial membrane Δψm, activation of JNK, cleavage of caspase-3, -9, and an induction of apoptosis in SHK treated PEL cells. SP600125, a specific inhibitor of JNK, also rescues a proportion of cells from the apoptotic effect of SHK. In addition, inhibition of caspase activation in the presence of pan-caspase inhibitor, Q-VD-OPh, blocks the SHK-inducing apoptosis, but doesn't completely inhibit SHK-mediated JNK activation. Therefore, ROS is an upstream trigger of SHK-induced caspase dependent apoptosis of PEL cells through disruption of mitochondrial membrane Δψm in an intrinsic pathway and an activation of JNK in an extrinsic pathway. In a PEL xenografted mouse model, SHK treatment suppresses PEL-mediated ascites formation without showing any significant adverse toxicity. These results suggested that SHK could be a potent anti-tumor agent for the treatment of PEL.
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http://dx.doi.org/10.1016/j.tranon.2020.101006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785961PMC
March 2021

Erratum to "Biofilm formation and transfer of a streptomycin resistance gene in enterococci from fermented pork" [J Glob Antimicrob Resist 22 (2020) 434-440].

J Glob Antimicrob Resist 2020 Dec 23;23:473. Epub 2020 Oct 23.

Division of Hematopoiesis, Joint Research Center for Human Retrovirus Infection & Graduate School of Medical Sciences, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.jgar.2020.10.001DOI Listing
December 2020

Long-term outcomes of spinal meningioma resection with outer layer of dura preservation technique.

J Clin Neurosci 2021 Jan 13;83:68-70. Epub 2020 Dec 13.

Department of Orthopedic Surgery, Graduate School of Medical Sciences, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

Spinal meningioma is a common benign intradural spinal tumor. It has been reported that the local recurrence rate after surgical resection increases with longer follow-up duration. Simpson grade 1 resection could reduce the risk of recurrence, but this procedure needs dural reconstruction, which would cause cerebrospinal fluid (CSF) leakage or iatrogenic spinal cord injury. Saito et al. reported dura preservation technique to reduce the risk of CSF leakage, in which the meningioma together with the inner layer of the dura is removed and the outer layer is preserved for simple dural closure. The long-term outcomes with this technique have never been investigated. In this study, we retrospectively analyzed the data of 38 surgically treated patients (dura preservation technique, 12 patients; Simpson grade 2 resection, 26 patients) to assess the long-term recurrence rate (mean, 121.5 months; range, 60-228 months). The local recurrence rate in the dura preservation group was 8.3% (1 of 12 cases), which was similar to that in Simpson grade 2 resection group (2 of 26 cases [7.7%]). Although this case series did not indicate the significant difference in the recurrence rates between the dura preservation group and Simpson grade 2 group, we consider that this technique still has advantages for surgically less invasiveness in terms of dural reconstruction which is necessary for Simpson grade 1 and higher possibility of complete resection of tumors compared with Simpson grade 2 resection.
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http://dx.doi.org/10.1016/j.jocn.2020.11.026DOI Listing
January 2021

A novel synthetic acanthoic acid analogues and their cytotoxic activity in cholangiocarcinoma cells.

Bioorg Med Chem 2021 Jan 1;29:115886. Epub 2020 Dec 1.

Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Burapha University, Chonburi 20131, Thailand; The Research Unit in Synthetic Compounds and Synthetic Analogues from Natural Product for Drug Discovery (RSND), Burapha University, Chonburi 20131, Thailand. Electronic address:

A novel series of acanthoic acid analogues containing triazole moiety were synthesized through esterification and CuAAC reaction. Evaluation of their biological activities against four cell lines of cholangiocarcinoma cells showed that 3d exhibited the strongest activity with an IC value of 18 µM against KKU-213 cell line, which was 8 fold more potent than acanthoic acid. Interestingly, the triazole ring and nitro group on benzyl ring play very significant role in cytotoxic activity. The computational studies revealed that 3d occupies the binding energy of -12.7 and -10.8 kcal/mol with CDK-2 and EGFR protein kinases, respectively. This result might provide a beginning for the development of acanthoic acid analogues as an anticancer agent.
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http://dx.doi.org/10.1016/j.bmc.2020.115886DOI Listing
January 2021

Anti-human CD99 antibody exerts potent antitumor effects in mantle cell lymphoma.

Cancer Immunol Immunother 2020 Nov 19. Epub 2020 Nov 19.

Division of Hematopoiesis, Joint Research Center for Human Retrovirus Infection and Graduate School of Medical Sciences, Kumamoto University, Kumamoto, 860-0811, Japan.

CD99 is a surface molecule expressed on various cell types including cancer cells. Expression of CD99 on multiple myeloma is associated with CCND1-IGH fusion/t(11;14). This translocation has been reported to be a genetic hallmark of mantle cell lymphoma (MCL). MCL is characterized by overexpression of cyclin D1 and high tumor proliferation. In this study, high expression of CD99 on MCL cell lines was confirmed. Our generated anti-CD99 monoclonal antibody (mAb), termed MT99/3, exerted potent antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities against mantle B-cell lymphoma without direct cytotoxic effects. The anti-tumor activities of mAb MT99/3 were more effective in MCL than in other B-cell lymphomas. Moreover, in a mouse xenograft model using Z138 MCL cell line, treatment of mAb MT99/3 reduced tumor development and growth. Our study indicated that mAb MT99/3 is a promising immunotherapeutic candidate for mantle cell lymphoma therapy.
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http://dx.doi.org/10.1007/s00262-020-02789-0DOI Listing
November 2020

High level of interleukin-33 in cancer cells and cancer-associated fibroblasts correlates with good prognosis and suppressed migration in cholangiocarcinoma.

J Cancer 2020 23;11(22):6571-6581. Epub 2020 Sep 23.

Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

Interleukin 33 (IL-33) promotes cholangiocarcinoma (CCA) genesis in a mouse model, however, its function in human CCA has not been clearly understood. This study was aimed to investigate IL-33 level in CCA tissues and its clinicopathological correlations. The results revealed that IL-33 was found in both cancer cells and stromal cancer-associated fibroblast (CAFs) staining patterns which were divided into high (CH) and low level (CL) in cancer cells; and presence (FP) and absence (FA) in CAFs. Kaplan-Meier analysis showed that patients in the CL group were significantly correlated with a short 2-year survival time ( = 0.027). The CL/FP group had a shorter survival time compared to the other groups with statistical significance for 2-year ( = 0.030) and 5-year ( = 0.023) survivals. In contrast, CH/FP patients had significantly greater 2-year ( = 0.003) and 5-year ( = 0.003) survivals. Univariate and multivariate analysis confirmed that CL/FP was a significantly independent risk factor whereas CH/FP was a significant protective factor in CCA patients. High IL-33 expressing CCA cells had low migration, but they showed increased migration when IL-33 expression was knocked down. The low level of recombinant human IL-33 (rhIL-33) (0.002 - 2 ng/ml) could promote CCA cell migration, in contrast to the suppressive effect at a high dose (20 - 200 ng/ml). In conclusion, the combination of high IL-33 level in cancer cells and CAFs is a potentially good prognosis marker in CCA patients. The migration suppressive effect of IL-33 may be the potential mechanism supporting its role as a good prognostic marker in CCA patients. The obtained results strengthen IL-33 as a promising predictor and therapeutic target for CCA.
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http://dx.doi.org/10.7150/jca.48327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545672PMC
September 2020

Upregulation of S100A10 in metastasized breast cancer stem cells.

Cancer Sci 2020 Dec 11;111(12):4359-4370. Epub 2020 Oct 11.

Department of Biochemistry, Fujita Health University School of Medicine, Toyoake, Japan.

Metastatic progression remains the major cause of death in human breast cancer. Cancer cells with cancer stem cell (CSC) properties drive initiation and growth of metastases at distant sites. We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44 cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, the expression levels of S100A10 and its family proteins were much higher in the CD44 cancer cells metastasized to the liver than those at the primary site. Knockdown of S100A10 in breast cancer cells suppressed and overexpression of S100A10 in breast cancer PDX cells enhanced their invasion abilities and 3D organoid formation capacities in vitro. Mechanistically, S100A10 regulated the matrix metalloproteinase activity and the expression levels of stem cell-related genes. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers.
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http://dx.doi.org/10.1111/cas.14659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734155PMC
December 2020

Matrin-3 plays an important role in cell cycle and apoptosis for survival in malignant melanoma.

J Dermatol Sci 2020 Nov 7;100(2):110-119. Epub 2020 Sep 7.

Department of Comprehensive Molecular Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. Electronic address:

Background: A previous study revealed that matrin-3 is an essential component in maintaining fibroblast growth factor 2 (FGF2)-mediated undifferentiation of neural stem cells (NSCs) using a proteomics approach. Malignant melanoma (MM) arises from melanocytes that originate from neural crest stem cells during development. Additionally, it has been reported that the expression of FGF2 is positively correlated with the progression of MM.

Objective: We expected that matrin-3, as a downstream component of FGF2, might be associated with the aggressiveness or differentiation of MM.

Methods: Matrin-3 expression was measured in human melanoma patient tissues and human MM cell lines. We analyzed the effect of matrin-3 siRNA on the proliferation of human MM cell lines and focused on cell cycle progression and apoptosis. We carried out in vivo xenograft tumor experiments by implanting A375 cells transfected with matrin-3 shRNA.

Results: Matrin-3 was highly expressed in MM, and matrin-3 knockdown inhibited the proliferation of melanoma cellsin vivo and in vitro. Furthermore, we found that matrin-3 knockdown led to an accumulation of cells in the G1 phase and an increase in apoptotic cell number.

Conclusion: Our results suggest that matrin-3 could be a new therapeutic target for the treatment of MM.
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http://dx.doi.org/10.1016/j.jdermsci.2020.08.013DOI Listing
November 2020

The critical role of germinal center-associated nuclear protein in cell biology, immunohematology, and hematolymphoid oncogenesis.

Exp Hematol 2020 10 20;90:30-38. Epub 2020 Aug 20.

Department of Diagnostic Pathology, Fujita Health University School of Medicine, Toyoake, Japan. Electronic address:

Germinal center-associated nuclear protein (GANP) is a unique and multifunctional protein that plays a critical role in cell biology, neurodegenerative disorders, immunohematology, and oncogenesis. GANP is an orthologue of Saccharomyces Sac3, one of the components of the transcription export 2 (TREX-2) complex and a messenger RNA (mRNA) nuclear export factor. GANP is widely conserved in all mammals, including humans. Although GANP was originally discovered as a molecule upregulated in the germinal centers of secondary lymphoid follicles in peripheral lymphoid organs, it is expressed ubiquitously in many tissues. It serves numerous functions, including making up part of the mammalian TREX-2 complex; mRNA nuclear export via nuclear pores; prevention of R-loop formation, genomic instability, and hyper-recombination; and B-cell affinity maturation. In this review, we first overview the extensive analyses that have revealed the basic functions of GANP and its ancestor molecule Sac3, including mRNA nuclear export and regulation of R-loop formation. We then describe how aberrant expression of GANP is significantly associated with cancer development. Moreover, we discuss a crucial role for GANP in B-cell development, especially affinity maturation in germinal centers. Finally, we illustrate that overexpression of GANP in B cells leads to lymphomagenesis resembling Hodgkin lymphoma derived from germinal center B cells, and that GANP may be involved in transdifferentiation of B cells to macrophages, which strongly affects Hodgkin lymphomagenesis.
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http://dx.doi.org/10.1016/j.exphem.2020.08.007DOI Listing
October 2020

Establishment of bone marrow-derived M-CSF receptor-dependent self-renewing macrophages.

Cell Death Discov 2020 23;6:63. Epub 2020 Jul 23.

Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, 860-0811 Japan.

Recent studies have revealed that tissue macrophages are derived from yolk sac precursors or fetal liver monocytes, in addition to bone marrow monocytes. The relative contribution of these cells to the tissue macrophage pool is not fully understood, but embryo-derived cells are supposed to be more important because of their capacity to self-renew. Here, we show the presence of adult bone marrow-derived macrophages that retain self-renewing capacity. The self-renewing macrophages were readily obtained by long-term culture of mouse bone marrow cells with macrophage colony-stimulating factor (M-CSF), a key cytokine for macrophage development. They were non-tumorigenic and proliferated in the presence of M-CSF in unlimited numbers. Despite several differences from non-proliferating macrophages, they retained many features of cells of the monocytic lineage, including the differentiation into dendritic cells or osteoclasts. Among the transcription factors involved in the self-renewal of embryonic stem cells, Krüppel-like factor 2 (KLF2) was strongly upregulated upon M-CSF stimulation in the self-renewing macrophages, which was accompanied by the downregulation of MafB, a transcription factor that suppresses KLF2 expression. Indeed, knockdown of KLF2 led to cell cycle arrest and diminished cell proliferation in the self-renewing macrophages. Our new cell model would be useful to unravel differences in phenotype, function, and molecular mechanism of proliferation among self-renewing macrophages with different origins.
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http://dx.doi.org/10.1038/s41420-020-00300-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378060PMC
July 2020

Kiss1R Identification and Biodistribution Analysis Employing a Western Ligand Blot and Ligand-Derivative Stain with a FITC-Kisspeptin Derivative.

ChemMedChem 2020 Sep 20;15(18):1699-1705. Epub 2020 Aug 20.

Department of Pathology and Experimental Medicine Graduate School of Medical Sciences, Kumamoto University, Honjyo 1-1-1, Chyuo-ku, Kumamoto, 860-855, Japan.

It is not always easy to establish specific antibodies against receptors. Most receptors are hydrophobic and have complicated three-dimensional structures, making them difficult to use as immunogens. Thus, we developed receptor detection methods with a fluorescein-labeled ligand as an antibody alternative, which we referred to as a western ligand blot (WLB) and ligand derivative stain (LDS). Kisspeptin receptor (Kiss1R) was detected by its ligand. Kiss1R expression was confirmed in eight human cell lines by the WLB and in four pathological tissues by the LDS. Next, Kiss1R was stained by LDS in organs, revealing Kiss1R expression by [ Ga]Ga-DOTA-kisspeptin 10 accumulation. As a result, Kiss1R-expressing cells in each organ could be stained with fluorescein-labeled kisspeptin 14 instead of an antibody and observed by light microscopy. The combination of the WLB and LDS allows identification of receptors in tissues, which can be readily applied to target receptor detection by a synthetic ligand derivative.
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http://dx.doi.org/10.1002/cmdc.202000356DOI Listing
September 2020

Insulin2 (Kuma) mutant mice develop diabetes with dominant inheritance.

Sci Rep 2020 07 22;10(1):12187. Epub 2020 Jul 22.

School of Life Science and Technology, Tokyo Institute of Technology, 4259-B-25 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa, 226-8501, Japan.

Insulin gene mutations have been identified to cause monogenic diabetes, and most of which developed permanent neonatal diabetes at young ages before 6 months of age in humans. To establish an animal model of permanent diabetes, we performed genome editing using the CRISPR/Cas9 system. We generated a novel Kuma mutant mice with p.Q104del in the Insulin2 (Ins2) gene in a BRJ background that exhibits a severe immune deficiency. Kuma mutant mice are non-obese and developed hyperglycemia from 3 weeks after birth in both males and females, which are inherited in a dominant mode. Kuma mutant mice displayed reduced insulin protein levels from 3-weeks-old, which seem to be caused by the low stability of the mutant insulin protein. Kuma mutant showed a reduction in islet size and islet mass. Electron microscopic analysis revealed a marked decrease in the number and size of insulin granules in the beta-cells of the mutant mice. Hyperglycemia of the mutant can be rescued by insulin administration. Our results present a novel insulin mutation that causes permanent early-onset diabetes, which provides a model useful for islet transplantation studies.
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http://dx.doi.org/10.1038/s41598-020-68987-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376009PMC
July 2020

Targeting aberrant DNA hypermethylation as a driver of ATL leukemogenesis by using the new oral demethylating agent OR-2100.

Blood 2020 08;136(7):871-884

Department of Clinical Laboratory Medicine, Faculty of Medicine, and.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Most HTLV-1-infected individuals are asymptomatic, and only 3% to 5% of carriers develop ATL. Here, we describe the contribution of aberrant DNA methylation to ATL leukemogenesis. HTLV-1-infected T-cells and their uninfected counterparts were separately isolated based on CADM1 and CD7 expression status, and differentially methylated positions (DMPs) specific to HTLV-infected T cells were identified through genome-wide DNA methylation profiling. Accumulation of DNA methylation at hypermethylated DMPs correlated strongly with ATL development and progression. In addition, we identified 22 genes downregulated because of promoter hypermethylation in HTLV-1-infected T cells, including THEMIS, LAIR1, and RNF130, which negatively regulate T-cell receptor (TCR) signaling. Phosphorylation of ZAP-70, a transducer of TCR signaling, was dysregulated in HTLV-1-infected cell lines but was normalized by reexpression of THEMIS. Therefore, we hypothesized that DNA hypermethylation contributes to growth advantages in HTLV-1-infected cells during ATL leukemogenesis. To test this idea, we investigated the anti-ATL activities of OR-1200 and OR-2100 (OR21), novel decitabine (DAC) prodrugs with enhanced oral bioavailability. Both DAC and OR21 inhibited cell growth, accompanied by global DNA hypomethylation, in xenograft tumors established by implantation of HTLV-1-infected cells. OR21 was less hematotoxic than DAC, whereas tumor growth inhibition was almost identical between the 2 compounds, making it suitable for long-term treatment of ATL patient-derived xenograft mice. Our results demonstrate that regional DNA hypermethylation is functionally important for ATL leukemogenesis and an effective therapeutic target.
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http://dx.doi.org/10.1182/blood.2019003084DOI Listing
August 2020

Biofilm formation and transfer of a streptomycin resistance gene in enterococci from fermented pork.

J Glob Antimicrob Resist 2020 09 24;22:434-440. Epub 2020 Apr 24.

Division of Hematopoiesis, Joint Research Center for Human Retrovirus Infection & Graduate School of Medical Sciences, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, Japan. Electronic address:

Objectives: Multidrug-resistant (MDR) enterococci are found extensively in food samples. This study characterized the phenotypic virulence factors and the ability of horizontal gene transfer of a streptomycin resistance gene among enterococci isolated from fermented pork.

Methods: Thirty-six MDR enterococci were subjected to screening of gelatinase, biofilm formation at various temperatures (4 °C, 25 °C and 37 °C), clumping ability and conjugation.

Results: All gelatinase-positive and clumping-positive strains were Enterococcus faecalis (41.7% and 38.9%, respectively). None of Enterococcus faecium and Enterococcus hirae demonstrated both phenotypes. Moderate and strong biofilm formations were found mostly at optimal temperatures in all the three species tested. However, moderate and weak biofilm formations could be found in 52.8% at 4 °C. No association was observed between biofilm formation and asa1, efaA, gelE and esp genes. Surprisingly, our data revealed evidence of the streptomycin resistance gene (aadE) being transferred among meat E. faecalis isolates as characterized by the pheromone-clumping response.

Conclusions: Here we report the co-existence of some virulence factors and MDR enterococci from fermented pork. Our data demonstrated for the first time that the aadE gene could be transferred via conjugation among enterococci isolated from meat, contributing to streptomycin resistance. This study highlights the importance of horizontal gene transfer within the food chain reservoir and that transfer to humans might be possible, causing harm or untreatable diseases.
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http://dx.doi.org/10.1016/j.jgar.2020.04.016DOI Listing
September 2020

Protein Arginine -methyltransferases 5 and 7 Promote HIV-1 Production.

Viruses 2020 03 23;12(3). Epub 2020 Mar 23.

Viral Infectious Disease Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

Current therapies for human immunodeficiency virus type 1 (HIV-1) do not completely eliminate viral reservoirs in cells, such as macrophages. The HIV-1 accessory protein viral protein R (Vpr) promotes virus production in macrophages, and the maintenance of Vpr is essential for HIV-1 replication in these reservoir cells. We identified two novel Vpr-binding proteins, i.e., protein arginine -methyltransferases (PRMTs) 5 and 7, using human monocyte-derived macrophages (MDMs). Both proteins found to be important for prevention of Vpr degradation by the proteasome; in the context of PRMT5 and PRMT7 knockdowns, degradation of Vpr could be prevented using a proteasome inhibitor. In MDMs infected with a wild-type strain, knockdown of PRMT5/PRMT7 and low expression of PRMT5 resulted in inefficient virus production like Vpr-deficient strain infections. Thus, our findings suggest that PRMT5 and PRMT7 support HIV-1 replication via maintenance of Vpr protein stability.
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http://dx.doi.org/10.3390/v12030355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150949PMC
March 2020

Functional and genetic characterization of three cell lines derived from a single tumor of an Opisthorchis viverrini-associated cholangiocarcinoma patient.

Hum Cell 2020 Jul 23;33(3):695-708. Epub 2020 Mar 23.

Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand.

Three cholangiocarcinoma (CCA) cell line-formerly named, M156, M213 and M214 have been intensively used with discrepancy of their tumor origins. They were assumed to be originated from three different donors without authentication. To verify the origins of these cell lines, the short tandem repeat (STR) analysis of the currently used cell lines, the cell stocks from the establisher and the primary tumor of a CCA patient were performed. Their phenotypic and genotypic originality were compared. The currently used 3 CCA cell lines exhibited similar STR as CCA patient ID-M213 indicating the same origin of these cells. The cell stocks from the establisher, however, revealed the same STR of M213 and M214 cells, but not M156. The misidentification of M214 and M156 is probably due to the mislabeling and cross-contamination of M213 cells during culture. These currently used cell lines were renamed as KKU-213A, -213B and -213C, for the formerly M213, M214 and M156 cells, respectively. These cell lines were established from a male with an intrahepatic mass-forming CCA stage-4B. The tumor was an adenosquamous carcinoma with the liver fluke ova granuloma in evidence. All cell lines had positive CK19 with differential CA19-9 expression. They exhibited aneuploidy karyotypes, distinct cell morphology, cell growth, cytogenetic characteristic and progressive phenotypes. KKU-213C formed a adenosquamous carcinoma, whereas KKU-213A and KKU-213B formed poorly- and well-differentiated squamous cell carcinomas in xenografted mice. mRNA microarray revealed different expression profiles among these three cell lines. The three cell lines have unique characteristics and may resemble the heterogeneity of tumor origin.
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http://dx.doi.org/10.1007/s13577-020-00334-wDOI Listing
July 2020