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The adenovirus (Ad) serotype 5 genome encodes two non-coding small RNAs (virus-associated RNAs: VA-RNAI and II), which are approximately 160nt-long RNAs transcribed by RNA polymerase III. It is well-known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (mivaRNAI, II); however, we and another group recently demonstrated that mivaRNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and VA-RNAII-derived miRNA (mivaRNAII) in Ad replication have remained to be clarified. In this study, we demonstrate mivaRNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3'-mivaRNAII-138, one of the most abundant mivaRNAII, significantly enhanced Ad replication, while the other species of mivaRNAII did not. We identified 8 putative target genes of 3'-mivaRNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3'-mivaRNAII-138 via post-transcriptional gene silencing, indicating that CUL4A is a target gene of 3'-mivaRNAII-138 and mivaRNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that mivaRNAII-mediated down-regulation of CUL4A enhanced JNK signaling, and thereby promoted Ad infection. [ABSTRACT FROM AUTHOR]

Hemophilia B is an inherited hemorrhagic disorder characterized by a deficiency of blood coagulation factor IX (FIX) that results in abnormal blood coagulation. The blood coagulation is already evident in hemophiliacs at the fetal stage, and thus intracranial hemorrhage and other bleeding complications can occur at birth, leading to sequelae. Therefore, it is important to develop effective treatments for hemophiliacs in utero. In this study, in order to transplacentally deliver FIX from pregnant mice to their fetuses, an improved adenovirus (Ad) vector expressing human FIX fused with the IgG Fc domain (FIX Fc fusion protein), which plays a crucial role in neonatal Fc receptor (FcRn)-mediated transcytosis across the placenta, was intravenously administered to E13.5 pregnant mice. Significant levels of FIX Fc fusion protein were detected in 0-day-old newborn mice whose mothers were administered an Ad vector expressing FIX Fc fusion protein. Wild-type FIX overexpressed in the pregnant mice was not delivered to the fetuses. Plasma FIX levels in the newborn mice were relatively well correlated with those in their mothers, although transplacental delivery efficiencies of FIX Fc fusion protein were slightly reduced when the FIX Fc fusion protein was highly expressed in the mother mice. Plasma FIX levels in the newborn mice were about 3.6-6.4% of those in their mothers, Transplacental delivery of FIX Fc fusion protein to their fetuses successfully improved the blood clotting ability in the newborn mice., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Small extracellular vesicles (SEV) have attracted much attention both as mediators of intercellular communication and as drug delivery systems. In addition, recent studies have shown that SEV containing virus components and virus particles are released from virus-infected cells. Oncolytic viruses, which efficiently kill tumor cells by tumor cell-specific replication, have been actively studied as novel anticancer agents in clinical and preclinical studies. However, it remains to be fully elucidated whether SEV released from oncolytic virus-infected cells are involved in the antitumor effects of oncolytic viruses. In this study, we examined the tumor cell killing efficiencies and innate immune responses following treatment with SEV released from oncolytic reovirus-infected tumor cells in vitro and in vivo. Reovirus-infected B16 cells secreted SEV associated with or containing reovirus particles (Reo-SEV) with a diameter of approximately 130 nm and a zeta potential of -17 mV, although death of reovirus-infected B16 cells was not observed. The secreted Reo-SEV also contained interferon (IFN)-β, tumor antigens, and damage-associated molecular patterns (DAMPs), including heat shock proteins (HSPs). Reo-SEV were secreted from the tumor tissues of reovirus-injected mice. Inhibition of the SEV secretion pathway using GW4869, which is a neutral sphingomyelinase inhibitor, resulted in significant reduction in the infectious titers of reovirus in the culture supernatants, suggesting that the cells released progeny virus via the SEV secretion pathway. Reo-SEV more efficiently killed mouse tumor cells and induced innate immune responses in mouse bone marrow-derived dendritic cells than reovirus. Reovirus and Reo-SEV mediated efficient and comparable levels of growth suppression of B16 subcutaneous tumors and induction of tumor infiltration of CD8+ T cells following intravenous administration. These results indicate that Reo-SEV are a promising oncolytic agent and that SEV are an effective delivery vehicle for oncolytic virus., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Adenovirus (Ad) vectors based on human adenovirus serotype 5 (Ad5) have attracted significant attention as vaccine vectors for infectious diseases. However, the effectiveness of Ad5 vectors as vaccines is often inhibited by the anti-Ad5 neutralizing antibodies retained by many adults. To overcome this drawback, we focused on human adenovirus serotype 35 (Ad35) vectors with low seroprevalence in adults. Although Ad35 vectors can circumvent anti-Ad5 neutralizing antibodies, vector yields of Ad35 vectors are often inferior to those of Ad5 vectors. In this study, we developed novel Ad35 vectors containing the Ad5 E4 orf 4, 6, and 6/7 or the Ad5 E4 orf 6 and 6/7 for efficient vector production, and compared their properties. These E4-modified Ad35 vectors efficiently propagated to a similar extent at virus titers comparable to those of Ad5 vectors. An Ad35 vector containing the Ad5 E4 orf 4, 6, and 6/7 mediated more efficient transduction than that containing the Ad5 E4 orf 6 and 6/7 in human cultured cells. Furthermore, insertion of an arginine-glycine-aspartate (RGD) peptide in the fiber region of an Ad35 vector containing the Ad5 E4 orf 4, 6, and 6/7 significantly improved the transgene product-specific antibody production following intramuscular administration in mice. The Ad35 vector containing the RGD peptide mediated efficient vaccine effects even in the mice pre-immunized with an Ad5., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hiroyuki Mizuguchi reports financial support was provided by Ministry of Education, Culture, Sports, Sciences, and Technology of Japan. Hiroyuki Mizuguchi reports financial support was provided by Japanese Agency for Medical Research and Development. Hiroyuki Mizuguchi reports financial support was provided by BIKEN Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Hypoxia inducible factor-1α (HIF-1α) is a crucial therapeutic target in various diseases, including cancer and fibrosis. We previously demonstrated that transfection with double-stranded RNA (dsRNA), including polyI:C and the dsRNA genome of mammalian orthoreovirus, resulted in significant reduction in HIF-1α protein levels in cultured cells; however, it remained to be elucidated how dsRNA induced down-regulation of HIF-1α protein levels. In this study, we examined the mechanism of dsRNA-mediated down-regulation of HIF-1α protein levels. We found that among the various cellular factors involved in dsRNA-mediated innate immunity, knockdown and knockout of protein kinase R (PKR) significantly restored HIF-1α protein levels in dsRNA-transfected cells, indicating that PKR was involved in dsRNA-mediated down-regulation of HIF-1α. Proteasome inhibitors significantly restored the HIF-1α protein levels in dsRNA-transfected cells. Ubiquitination levels of HIF-1α were increased by transfection with dsRNA. These findings indicated that degradation of HIF-1α in a ubiquitin-proteasome pathway was promoted in a PKR-dependent manner following dsRNA transfection. Expression of not only HIF-1α but also several proteins, including CDK4 and HER2, was down-regulated following dsRNA transfection. These data provide important clues for elucidation of the mechanism of dsRNA-mediated cellular toxicity, as well as for therapeutic application of dsRNA., (© 2023. The Author(s).)

Circulating tumor cells (CTCs) are present in the blood of cancer patients from the early stage of cancer development, and their presence has been correlated with patient prognosis and treatment responses. Accordingly, CTCs have been attracting attention as a novel biomarker for early detection of cancer and monitoring of treatment responses. However, since patients typically have only a few CTCs per milliliter of blood, development of an accurate and highly sensitive CTC detection method is crucial. We previously developed a CTC detection method using a novel conditionally replicating adenovirus (Ad) that expresses green fluorescence protein (GFP) in a tumor cell-specific manner by expressing the E1 gene using a tumor-specific human telomerase reverse transcriptase (hTERT) promoter (rAdF35-142T-GFP). CTCs were efficiently detected using rAdF35-142T-GFP, but GFP expression levels in the CTCs and production efficiencies of rAdF35-142T-GFP were relatively low. In this study, in order to overcome these problems, we developed four types of novel GFP-expressing conditionally replicating Ads and examined their ability to visualize CTCs in the blood samples of lung cancer patients. Among the four types of novel recombinant Ads, the novel conditionally replicating Ad containing the 2A peptide and the GFP gene downstream of the E1A gene and the adenovirus death protein (ADP) gene in the E3 region (rAdF35-E1-2A-GFP-ADP) mediated the highest number of GFP-positive cells in the human cultured tumor cell lines. Titers of rAdF35-E1-2A-GFP-ADP were significantly higher (about 4-fold) than those of rAdF35-142T-GFP. rAdF35-E1-2A-GFP-ADP and rAdF35-142T-GFP efficiently detected CTCs in the blood of lung cancer patients at similar levels. GFP+/CD45- cells (CTCs) were found in 10 of 17 patients (58.8%) for both types of recombinant Ads., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ikemoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Non-alcoholic liver disease (NAFLD) is a condition caused by excessive fat accumulation in the liver and developed via multiple pathways. miR-27b has been suggested to play crucial roles in the development of NAFLD, assuming via targeting genes involved in lipid catabolism and anabolism. However, other pathways regulated by miR-27b are largely unknown. Here we show that lipid accumulation was induced in miR-27b-transfected human and mouse hepatic cells and that knockdowns of three miR-27b-target genes, β-1,4-galactosyltransferase 3 (B4GALT3), matrix AAA peptidase interacting protein 1 (MAIP1) and PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2), induced lipid accumulation. We also show that B4GALT3 and MAIP1 were direct targets of miR-27b and overexpression of MAIP1 ameliorated miR-27b-induced lipid accumulation. In addition, we show that hepatic Maip1 expression declined in mice fed a high-fat diet, suggesting the involvement of decreased Maip1 expression in the condition of fatty liver. Overall, we identified MAIP1/miR-27b axis as a mediator of hepatic lipid accumulation, a potential therapeutic target for NAFLD., (© 2023. The Author(s).)

Background/aim: Oncolytic adenoviruses (Ads) (OAds) are gaining attention as an effective remedy for pancreatic cancer. Most OAds are based on human Ad serotype 5 (Ad5) (OAd5); however, two major drawbacks of OAd5 have been reported. Expression of coxsackievirus-adenovirus receptor, a primary infection receptor of Ad5, is often decreased on malignant tumor cells, including pancreatic cancers. More than 60% of adults have neutralizing antibodies against Ad5. Previously, we developed an OAd composed of Ad serotype 35 (Ad35) (OAd35). Ad35 recognizes CD46, which is often up-regulated on pancreatic cancers. In addition, only 20% or fewer adults have anti-Ad35 neutralizing antibodies., Materials and Methods: We examined the tumor cell lysis activities of OAd35 in the four human pancreatic cancer cell lines in the presence and absence of human serum. The tumor growth suppression effects of OAd35 after local and systemic administration were evaluated in nude mice bearing human pancreatic tumors., Results: OAd35 showed higher levels of tumor cell lysis activities than OAd5 in the human pancreatic cancer cell lines AsPC-1 and BxPC-3. Although the in vitro tumor cell lysis activities of OAd5 against MIA PaCa-2 and PANC-1 cells were strongly attenuated in the presence of human serum, OAd35 mediated comparable levels of tumor cell lysis in the presence and absence of human serum. Systemic administration of OAd5 did not mediate significant growth inhibition against the subcutaneous BxPC-3 tumor. On the other hand, OAd35 significantly suppressed tumor growth., Conclusion: OAd35 would be suitable as an alternative anticancer agent for pancreatic cancer., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

PEGylated liposomes (PEG-liposomes) are a promising drug delivery vehicle for tumor targeting because of their efficient tumor disposition profiles via the enhanced permeability and retention (EPR) effect. However, tumor targeting of PEG-liposomes, particularly their delivery inside the tumors, is often disturbed by physical barriers in the tumor, including tumor cells themselves, extracellular matrices, and interstitial pressures. In this study, B16 melanoma tumor-bearing mice were injected intravenously with oncolytic reovirus before administration of PEG-liposomes to enhance PEG-liposomes' tumor disposition. Three days after reovirus administration, significant expression of reovirus sigma 3 protein, elevation of apoptosis-related gene expression, and activation of caspase 3 in the tumors were found. Apoptotic cells were found inside the tumors. These data indicated that reovirus efficiently replicated in the tumors and induced apoptosis of tumor cells. The tumor disposition levels of PEG-liposomes were approximately doubled by reovirus pre-administration, compared with a PBS-pretreated group. PEG-liposomes were widely distributed in the tumors of reovirus-pretreated mice, whereas in the PBS-pretreated group, PEG-liposomes were found mainly around or inside the blood vessels in the tumors. Pre-treatment with reovirus also improved the tumor accumulation of PEG-liposomes in human pancreatic BxPC-3 tumors. 3D imaging analysis of whole BxPC-3 tumors demonstrated that pretreatment with reovirus led to the enhancement of PEG-liposome accumulation inside the tumors. Combination treatment with reovirus and paclitaxel-loaded PEG-liposomes (PTX-PEG-liposomes) significantly suppressed B16 tumor growth. These results provide important information for clinical use of combination therapy of reovirus and nanoparticle-based drug delivery system (DDS)., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Fremanezumab is a humanized monoclonal antibody against calcitonin gene-related peptide for subcutaneous use to suppress migraine attacks. A phase 3 study was conducted to investigate the safety of autoinjector (AI)-assisted self-injection of fremanezumab 225 mg., Research Design and Methods: The multicenter, open-label study involving 71 patients with migraine was conducted between June 2020 and November 2020 at ten institutions in Japan. The study consisted of a 4-week (28-day) screening period and an 8-week (57-day) treatment period. According to the investigator's instructions, all patients successfully performed self-injection for 4 weeks at the institutional site and at home and maintained eDiaries of their headaches. The primary endpoint was safety of the drug based on treatment-emergent adverse events (TEAEs)., Results: Treatment-emergent adverse events were more frequent after at-home injection than after at-site injection, but they were mainly injection site reactions and mostly mild. The safety profile was comparable, raising no concerns compared with what has been reported in previous studies. Both migraine days and headache days were decreased considerably., Conclusions: Overall, AI-assisted, at-home self-injection of fremanezumab was found to be generally safe and well-tolerated. This injection strategy is considered clinically meaningful in view of improved utility of and adherence to the drug.

Myeloid-derived suppressor cells (MDSCs), which accumulate in tumor bearers, are known to suppress anti-tumor immunity and thus promote tumor progression. MDSCs are considered a major cause of resistance against immune checkpoint inhibitors in patients with cancer. Therefore, MDSCs are potential targets in cancer immunotherapy. In this study, we modified an in vitro method of MDSC differentiation. Upon stimulating bone marrow (BM) cells with granulocyte-macrophage colony-stimulating factor in vitro , we obtained both lymphocyte antigen 6G positive (Ly-6G + ) and negative (Ly-6G - ) MDSCs (collectively, hereafter referred to as conventional MDSCs), which were non-immunosuppressive and immunosuppressive, respectively. We then found that MDSCs differentiated from Ly-6G - BM (hereafter called 6G - BM-MDSC) suppressed T-cell proliferation more strongly than conventional MDSCs, whereas the cells differentiated from Ly-6G + BM (hereafter called 6G + BM-MDSC) were non-immunosuppressive. In line with this, conventional MDSCs or 6G - BM-MDSC, but not 6G + BM-MDSC, promoted tumor progression in tumor-bearing mice. Moreover, we identified that activated glutathione metabolism was responsible for the enhanced immunosuppressive ability of 6G - BM-MDSC. Finally, we showed that Ly-6G + cells in 6G - BM-MDSC, which exhibited weak immunosuppression, expressed higher levels of Cybb mRNA, an immunosuppressive gene of MDSCs, than 6G + BM-MDSC. Together, these data suggest that the depletion of Ly-6G + cells from the BM cells leads to differentiation of immunosuppressive Ly-6G + MDSCs. In summary, we propose a better method for MDSC differentiation in vitro . Moreover, our findings contribute to the understanding of MDSC subpopulations and provide a basis for further research on MDSCs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Oncolytic adenoviruses (OAds), most of which are based on species C human adenovirus serotype 5 (Ad5) (OAd5), have recently received much attention as potential anticancer agents. High seroprevalence of anti-Ad5 neutralizing antibodies is a major hurdle for Ad5-based gene therapy. However, the impacts of anti-Ad5 neutralizing antibodies on OAd5-mediated transgene expression in the tumor and antitumor effects remain to be fully elucidated. In this study, we examined the impact of anti-Ad5 neutralizing antibodies on the OAd5-mediated antitumor effects and OAd5-mediated transgene expression. The luciferase expression of OAd-tAIB-Luc, which contains the cytomegalovirus promoter-driven luciferase gene, was inhibited in human cultured cells in the presence of human serum. Although the inhibitory effects of human serum possessing the low anti-Ad5 neutralizing antibody titers were overcome by long-term infection, the in vitro tumor cell lysis activities of OAd-tAIB-Luc were entirely attenuated by human serum containing the high titers of anti-Ad5 neutralizing antibodies. OAd-tAIB-Luc-mediated luciferase expression in the subcutaneous tumors 3 days after administration and tumor growth suppression levels following intratumoral administration were significantly lower in mice possessing the high titers of anti-Ad5 neutralizing antibodies, compared to those in control mice. These results suggested that pre-existing anti-Ad5 antibodies attenuated both transgene expression and potential antitumor effects of OAd5 following intratumoral administration., (© 2022. The Author(s).)

The liver is the main organ that regulates lipid and glucose metabolism. Ectopic lipid accumulation in the liver impairs insulin sensitivity and glucose metabolism. Lipoprotein lipase (LPL), mainly expressed in the adipose tissue and muscle, is a key enzyme that regulates lipid metabolism via the hydrolysis of triglyceride in chylomicrons and very-low-density lipoproteins. Here, we aimed to investigate whether the suppression level of hepatic lipid accumulation via overexpression of LPL in mouse liver leads to improved metabolism. To overexpress LPL in the liver, we generated an LPL-expressing adenovirus (Ad) vector using an improved Ad vector that exhibited considerably lower hepatotoxicity (Ad-LPL). C57BL/6 mice were treated with Ad vectors and simultaneously fed a high-fat diet (HFD). Lipid droplet formation in the liver decreased in Ad-LPL-treated mice relative to that in control Ad vector-treated mice. Glucose tolerance and insulin resistance were remarkably improved in Ad-LPL-treated mice compared to those in control Ad vector-treated mice. The expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1, and acyl-CoA oxidase 1, were 1.7-2.0-fold higher in Ad-LPL-treated mouse livers than that in control Ad-vector-treated mouse livers. Furthermore, hepatic LPL overexpression partly maintained mitochondrial content in HFD-fed mice. These results indicate that LPL overexpression in the livers of HFD-fed mice attenuates the accumulation of lipid droplets in the liver and improves glucose metabolism. These findings may enable the development of new drugs to treat metabolic syndromes such as type 2 diabetes mellitus and non-alcoholic fatty liver disease., Competing Interests: The authors have declared that no competing interests exist.

This study aimed to clarify the real-world efficacy of sequential nivolumab for treating metastatic renal cancer after first-line molecular targeting therapy. Patients were divided into two groups (2014-2016 and 2017-2020) according to the year when they started primary treatment with molecular targeted drugs (MTDs). We compared the overall survival of patients and investigated a contributing factor for survival. The mean duration of overall survival was significantly longer in the 2017-2020 group (44.0 months) than in the 2014-2016 group (8.5 months). Univariate analysis showed that nivolumab treatment was a significant prognostic factor (P = .0021). Patients treated with nivolumab as second-line therapy had a significantly higher 5-year survival rate compared to that of other patients (70% vs 32%). In addition, the time from commencement of MTDs to switch to nivolumab was significantly shorter in the 2017-2020 group compared to the 2014-2016 group (8.94 vs 34.12 months, P = .03). In our study, cases with first-line MTDs had markedly prolonged outcomes after the 2017 guideline update, and sequential nivolumab with prompt switching to nivolumab was an important factor., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

Oncolytic adenoviruses (OAds) have attracted much attention as novel anticancer agents. Numerous studies have examined the antitumour effects of combinational use of an OAd and anticancer agents; however, few chemical compounds enhancing OAd infection have been reported. In this study, we screened a food and drug administration (FDA)-approved drug library containing 1134 small chemical compounds to identify chemical compounds that enhance OAd replication in human tumour cells. We found that domperidone, a dopamine D2 receptor antagonist, significantly enhanced the replication of an OAd in human tumour cells, including human pancreatic tumour cells, by two-fivefold, resulting in improvement of OAd-mediated tumour cell killing activities. The E1A mRNA levels were significantly increased in domperidone-pre-treated cells following OAd infection, which contributed to the promotion of OAd replication. However, mRNA levels of the dopamine D2 receptor (DRD2), which is known to be a target molecule of domperidone, were undetectable in most of the tumour cells by real-time reverse transcription (RT)-PCR analysis, indicating that domperidone promoted OAd replication by acting on a molecule other than DRD2. This study provides important clues for the improvement of OAd-mediated cancer therapy.

Conventional imaging techniques are available for clinical identification of tumor sites. However, detecting metastatic tumor cells that are spreading from primary tumor sites using conventional imaging techniques remains difficult. In contrast, fluorescence-based labeling systems are useful tools for detecting tumor cells at the single-cell level in cancer research. The ability to detect fluorescent-labeled tumor cells enables investigations of the biodistribution of tumor cells for the diagnosis and treatment of cancer. For example, the presence of fluorescent tumor cells in the peripheral blood of cancer patients is a predictive biomarker for early diagnosis of distant metastasis. The elimination of fluorescent tumor cells without damaging normal tissues is ideal for minimally invasive treatment of cancer. To capture fluorescent tumor cells within normal tissues, however, tumor-specific activated target molecules are needed. This review focuses on recent advances in tumor-targeted fluorescence labeling systems, in which indirect reporter labeling using tumor-specific promoters is applied to fluorescence labeling of tumor cells for the diagnosis and treatment of cancer. Telomerase promoter-dependent fluorescence labeling using replication-competent viral vectors produces fluorescent proteins that can be used to detect and eliminate telomerase-positive tumor cells. Tissue-specific promoter-dependent fluorescence labeling enables identification of specific tumor cells. Vimentin promoter-dependent fluorescence labeling is a useful tool for identifying tumor cells that undergo epithelial-mesenchymal transition (EMT). The evaluation of tumor cells undergoing EMT is important for accurately assessing metastatic potential. Thus, tumor-targeted fluorescence labeling systems represent novel platforms that enable the capture of tumor cells for the diagnosis and treatment of cancer., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Genome-wide association studies have identified more than 300 loci associated with type 2 diabetes mellitus; however, the mechanisms underlying their role in type 2 diabetes mellitus susceptibility remain largely unknown. Zinc finger AN1-type domain 3 (ZFAND3), known as testis-expressed sequence 27, is a type 2 diabetes mellitus-susceptibility gene. Limited information is available regarding the physiological role of ZFAND3 in vivo . This study aimed to investigate the association between ZFAND3 and type 2 diabetes mellitus. ZFAND3 was significantly upregulated in the liver of diabetic mice compared to wild-type mice. To overexpress ZFAND3, we generated a ZFAND3-expressing adenovirus (Ad) vector using an improved Ad vector exhibiting significantly lower hepatotoxicity (Ad-ZFAND3). Glucose tolerance was significantly improved in Ad-ZFAND3-treated mice compared to the control Ad-treated mice. ZFAND3 overexpression in the mouse liver also improved insulin resistance. Furthermore, gluconeogenic gene expression was significantly lower in primary mouse hepatocytes transduced with Ad-ZFAND3 than those transduced with the control Ad vector. The present results suggest that ZFAND3 improves glucose tolerance by improving insulin resistance and suppressing gluconeogenesis, serving as a potential novel therapeutic target for type 2 diabetes mellitus., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Background/aim: Efficient production of adenovirus vectors is crucial for their clinical use. Adenovirus death protein (ADP), which is encoded in the E3 region of the adenovirus genome, is involved in host-cell lysis and the subsequent release of progeny virus; however, the ADP gene is often removed from the adenovirus vector genome., Materials and Methods: We have developed adenovirus vectors that possess the ADP gene and maintain a relatively large insertion capacity for foreign genes by deleting the partial E3 region. Adenovirus vector-mediated transgene expression levels and virus titers were examined., Results: The adenovirus vectors maintaining the ADP gene showed cytopathic effect earlier than conventional adenovirus vector without the ADP gene following treatment of HEK293 cells, although there were no significant differences in total virus titers., Conclusion: The adenovirus vectors possessing the ADP gene showed efficient spread of progeny virus infection following transduction in HEK293 cells., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

N-Methyl-d-aspartate receptors (NMDARs) are members of the ionotropic glutamate receptor family and play a crucial role in learning and memory by regulating synaptic plasticity. Activation of NMDARs containing GluN2A, one of the NMDAR subunits, has recently attracted attention as a promising therapeutic approach for neuropsychiatric diseases such as schizophrenia, depression, and epilepsy. In the present study, we developed potent and brain-penetrable GluN2A-selective positive allosteric modulators. Lead compound 2b was generated by scaffold hopping of hit compound 1, identified from the internal alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-focused compound library through a high-throughput screening campaign. Subsequent optimization of the lead compound, including a structure-based drug design approach, resulted in the identification of a potent GluN2A PAM (R)-9, which possessed high selectivity against both subtypes of AMPAR and NMDAR. Furthermore, (R)-9 significantly enhanced long-term potentiation in the rat hippocampus 24 h after oral administration, indicating that this molecule is a potentially useful in vivo pharmacological tool for treating psychiatric diseases., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Replication-incompetent adenovirus (Ad) vectors have been widely used as gene delivery vehicles in both gene therapy studies and basic studies for gene function analysis due to their highly advantageous properties, which include high transduction efficiencies, relatively large capacities for transgenes, and high titer production. In addition, Ad vectors induce moderate levels of innate immunity and have relatively high thermostability, making them very attractive as potential vaccine vectors. Accordingly, it is anticipated that Ad vectors will be used in vaccines for the prevention of infectious diseases, including Ebola virus disease and acquired immune deficiency syndrome (AIDS). Much attention is currently focused on the potential use of an Ad vector vaccine for coronavirus disease 2019 (COVID-19). In this review, we describe the basic properties of an Ad vector, Ad vector-induced innate immunity and immune responses to Ad vector-produced transgene products. Development of novel Ad vectors which can overcome the drawbacks of conventional Ad vector vaccines and clinical application of Ad vector vaccines to several infectious diseases are also discussed., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2021 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Oncolytic viruses, which mediate tumor cell-specific infection, resulting in efficient tumor cell killing, have attracted much attention as a novel class of anti-cancer biopharmaceutical agents. Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment that strongly supports the growth, survival, and metastasis of tumor cells, suggesting that CAFs would have influence to the antitumor effects of oncolytic viruses; however, it remains to be fully evaluated whether oncolytic viruses affect the viabilities and properties of CAFs following treatment. Oncolytic reovirus, which is a non-enveloped virus that contains 10-segmented double-stranded RNA genome, shows efficient tumor cell lysis without apparent cytotoxicity to normal cells and has been tested worldwide in clinical trials against various types of tumors. In this study, we demonstrated that reovirus exhibited cytotoxicity against mouse primary CAFs isolated from subcutaneous tumors, but not against tail-tip fibroblasts. Infection with reovirus resulted in activation of caspase 3 and up-regulation of apoptosis-related gene expression, indicating that reovirus induced apoptosis of mouse primary CAFs. Intratumoral administration of reovirus induced apoptosis of mouse CAFs in the tumor. Taken together, these results indicate that reovirus has the potential to mediate antitumor effects by killing not only cancer cells but also CAFs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: To compare the surgical outcomes of laparoscopic sacrocolpopexy for pelvic organ prolapse between a group in which only sutures were used (standard method), and a group in which a combination of tackers and sutures were used (tacker combination method)., Methods: A total of 77 patients who underwent laparoscopic sacrocolpopexys from June 2016 to October 2019 were divided into a suture group (36 patients) and a suture + tacker group (41 patients). We retrospectively compared operation time, amount of blood loss, postoperative length of hospital stay, incidence of perioperative complications and anatomical cure rate 1 year after surgery. Lower urinary tract symptoms were evaluated using symptom questionnaires and objective parameters., Results: Operation time in the suture + tacker group was shorter (104.9 ± 27.0 vs 147.5 ± 33.7 min; P < 0.0001). The incidence of perioperative complications in the suture group and the suture + tacker group was 2.8% and 2.4%, respectively (P = 0.9409). Anatomical cure rates at 1 year after surgery were 94.4% and 100%, respectively (P = 0.2153). Both groups showed significant improvement after 1 year for International Prostate Symptom Score total and quality of life score, Overactive Bladder Symptom Score total score, voided volume, maximum urinary flow rate and post-void residual. [Corrections added on 7 September 2021 after first online publication: the first two P-values have been updated.] CONCLUSIONS: The combined use of sutures and tackers in laparoscopic sacrocolpopexy simplifies the procedure and translates into shorter operation time. Surgical outcomes at 1 year and improvement of lower urinary tract symptoms are similar regardless of the technique., (© 2021 The Japanese Urological Association.)

Human induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells., (© 2021. The Author(s).)

Background/aim: Oncolytic reovirus, which is a non-enveloped virus possessing a 10-segmented double-stranded RNA genome, has been anticipated as a novel class of antitumor agent. Hepatocellular carcinoma (HCC) is considered to be a target suitable for reovirus-mediated virotherapy. Transforming growth factor (TGF)-β plays an important role in the pathogenesis of HCC. TGF-β-signaling inhibitors have proceeded to clinical trials as potential antitumor agents for HCC. On the other hand, TGF-β is involved in induction of expression of cathepsins B and L, which are important for reovirus infection. It remains to be examined whether TGF-β signaling inhibitors affect reovirus-mediated lysis of HCC cells. The aim of this study was to evaluate the effects of TGF-β-signaling inhibitors on tumor cell lysis efficiency of reovirus in human HCC cells., Materials and Methods: Reovirus was added to four types of human HCC cell lines pretreated with one of three TGF-β type I receptor inhibitors: SB431542, A-83-01, or galunisertib (LY2157299). Cell viability, virus genome copy numbers, and virus protein expression were evaluated following reovirus infection., Results: SB431542 significantly inhibited reovirus-mediated killing of human HCC cell lines, while A-83-01 and galunisertib did not inhibit., Conclusion: These data indicate that SB431542 inhibited reovirus-mediated lysis of human HCC cells in a TGF-β signaling-independent manner., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The intestinal mucosa plays an important role as an immune barrier due to its continual exposure to invading pathogens, including viruses. It is thus highly important to evaluate virus infection profiles in the intestinal mucosa for prevention of virus infection and development of antivirus medicines; however, only a few enterocyte lines are available as in vitro intestinal models for the evaluation of virus infection. In this study, we evaluated profiles of infection and innate immune responses following infection with a mammalian orthoreovirus (hereafter reovirus), which has often been used as a tractable model for studies of viral pathogenesis, in human iPS cell-derived small intestinal epithelial-like cell (hiPS-SIEC) monolayers and cells of a human colon adenocarcinoma cell line, Caco-2. The levels of reovirus infection were similar between hiPS-SIEC and Caco-2 cell monolayers, which are often used as an intestinal model, after apical and basolateral infection. In hiPS-SIEC monolayers, more efficient replication of the virus genome was observed following basolateral infection than apical infection, while apical infection resulted in higher levels of virus protein expression and progeny virus production than basolateral infection. Reovirus significantly induced innate immune responses, including expression of type I and III interferons (IFNs), in hiPS-SIEC monolayers more efficiently than Caco-2 cells. Higher levels of type I and III interferon (IFN) expression were found in hiPS-SIEC monolayers following apical infection than basolateral infection. These results suggested that hiPS-SIECs are a promising in vitro model for the evaluation of virus infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Human adenoviruses (Ads), common pathogens that cause upper respiratory and gastrointestinal infections, are blocked by neutralizing antibodies (nAbs). However, Ads are not fully eliminated even in hosts with nAbs. In this study, we assessed the infectivity of progeny Ad serotype 5 (Ad5) in the presence of nAb. The infectivity of Ad5 was evaluated according to the expression of the Ad genome and reporter gene. Infection by wild-type Ad5 and Ad5 vector continued to increase until 3 days after infection even in the presence of nAb. We established an assay for determining the infection levels of progeny Ad5 using a sorting system with magnetic beads and observed little difference in progeny Ad5 counts in the presence and absence of nAb 1 day after infection. Moreover, progeny Ad5 in the presence of nAb more effectively infected coxsackievirus and adenovirus receptor (CAR)-positive cells than CAR-negative cells. We investigated the function of fiber proteins, which are the binding partners of CAR, during secondary infection, observing that fibre proteins spread from infected cells to adjacent cells in a CAR-dependent manner. In conclusion, this study revealed that progeny Ad5 could infect cells even in the presence of nAb, differing from the common features of the Ad5 infection cycle. Our findings may be useful for developing new therapeutic agents against Ad infection.

Background: Surgical treatments for renal cell carcinoma reduces kidney volume to some degree and may derive postsurgical chronic kidney disease. We made a new marker for postoperative renal function using CT volumetry. To determine the impact of various parameters including this marker, we observed pre- and postsurgical renal function of experienced cases., Methods: From 2004 to 2014, we underwent total or partial nephrectomy for 181 patients with renal carcinoma in a single institution. Of the total, 138 cases with presurgical CT volumetry were included in this study. We evaluated parameters for assessments of peri- and postoperative renal function including age, gender, serum creatinine, eGFR, performed surgery, pathology, estimated residual kidney volume and associated disease. Presence or absence of acute kidney injury (AKI) and chronic kidney disease (CKD) were also evaluated before, immediately after and 5 years after surgery., Results: Multiple logistic regression analysis identified AKI, preoperative eGFR and estimated residual kidney volume as significant prognostic factors for the postoperative CKD. Moreover, cases with triple positive of these factors suffer postoperative CKD more significantly than those with one or two positives., Conclusion: Using these predictive factors, we may determine patients with high risk for CKD who require an early intervention of renal protective treatment.

Background/aim: Oncolytic adenoviruses (OAds) have attracted much attention as novel anticancer therapeutics. The proper design of an expression cassette containing the E1A gene, which is indispensable for self-replication of the Ad genome, is crucial for efficient tumor cell-specific infection of an OAd. Various types of oncolytic adenoviruses (OAds) possessing different types of the E1A gene expression cassettes have been developed, but their oncolytic activities and safety profiles have not been systematically evaluated. Herein we examined the oncolytic activities and safety profiles of five types of OAds possessing different types of the E1A gene expression cassette in order to optimize the E1A gene expression cassette for development of an efficient and safe OAd., Materials and Methods: We prepared five types of OAds containing different types of E1 gene expression cassettes, and examined the oncolytic activities and safety profiles of the OAds., Results: Among the OAds examined, OAd-Δ24, which had a 24-bp deletion in the E1A gene, mediated the most efficient oncolytic activities against the human tumor cell lines, although OAd-Δ24 showed slightly higher cytotoxicity to normal human cells than the other OAds., Conclusion: These results provide important clues for the development of safe and efficient OAds., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Oncolytic adenoviruses (OAds) are among the most promising oncolytic viruses. Almost all oncolytic adenoviruses are composed of human adenovirus serotype 5 (Ad5) (OAd5). However, expression of the primary infection receptor for Ad5, coxsackievirus-adenovirus receptor (CAR), often declines on malignant tumor cells, resulting in inefficient infection in CAR-negative tumor cells. In addition, at least 80% of adults have neutralizing antibodies against Ad5. In this study, we developed a novel OAd fully composed of OAd35. OAd35 recognizes CD46, which is ubiquitously expressed on almost all human cells and is often upregulated on malignant tumor cells, as an infection receptor. Moreover, 20% or fewer adults have neutralizing antibodies against Ad35. OAd35 mediated efficient cell lysis activities at levels similar to OAd5 in CAR-positive tumor cells, while OAd35 showed higher levels of cell lysis activities than OAd5 in CAR-negative tumor cells. Anti-Ad5 serum significantly inhibited in vitro tumor cell lysis activities of OAd5, whereas OAd35 exhibited comparable levels of in vitro tumor cell lysis activities in the presence of anti-Ad5 and naive serum. OAd35 significantly suppressed growth of the subcutaneous CAR-positive and CAR-negative tumors following intratumoral administration. These results indicated that OAd35 is a promising alternative oncolytic virus for OAd5., Competing Interests: F.S. and H.M. have the potential to receive patent royalities from Oncolys BioPharm, Inc. The other authors declare no conflicts of interest., (© 2021 The Author(s).)

Adenovirus (Ad) vector-mediated transduction can cause hepatotoxicity during two phases, at ∼2 and 10 days after administration. Early hepatotoxicity is considered to involve inflammatory cytokines; however, the precise mechanism remains to be clarified. We examined the mechanism of early Ad vector-induced hepatotoxicity by using a conventional Ad vector, Ad-CAL2, and a modified Ad vector, Ad-E4-122aT-CAL2. Ad-E4-122aT-CAL2 harbors sequences complementary to the liver-specific miR-122a in the 3' untranslated region of E4 , leading to significant suppression of leaky Ad gene expression in the liver via posttranscriptional gene silencing and a significant reduction in late-phase hepatotoxicity. We found that Ad-E4-122aT-CAL2 transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT-CAL2 and Ad-CAL2 induced comparable cytokine expression levels in the liver and spleen. IL-6, a major inflammatory cytokine induced by Ad vectors, significantly enhanced leaky Ad gene expression and cytotoxicity in primary mouse hepatocytes following Ad-CAL2 but not Ad-E4-122aT-CAL2 transduction. Furthermore, leaky Ad gene expression and cytotoxicity in Ad-CAL2-treated hepatocytes in the presence of IL-6 were significantly suppressed upon inhibition of JAK and STAT3. Ad vector-mediated acute hepatotoxicities and leaky Ad expression were significantly reduced in IL-6 knockout mice compared with those in wild-type mice. Thus, Ad vector-induced IL-6 promotes leaky Ad gene expression, leading to acute hepatotoxicity., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Replication-incompetent adenovirus (Ad) vectors are promising gene delivery vehicles, especially for hepatocytes, due to their superior hepatic tropism; however, in vivo application of an Ad vector often results in hepatotoxicity, mainly due to the leaky expression of Ad genes from the Ad vector genome. In order to reduce the Ad vector-induced hepatotoxicity, we previously developed an Ad vector containing the sequences perfectly complementary to a liver-specific microRNA (miRNA), miR-122a, in the 3'-untranslated region (UTR) of the E4 gene. This improved Ad vector showed a significant reduction in the leaky expression of Ad genes and hepatotoxicity in the mouse liver and primary mouse hepatocytes; however, the safety profiles and transduction properties of this improved Ad vector in human hepatocytes remained to be elucidated. In this study, we examined the transgene expression and safety profiles of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in human hepatocytes from chimeric mice with humanized liver. The transgene expression levels of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene were significantly higher than those of the conventional Ad vectors. The leaky expression levels of Ad genes of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in the primary human hepatocytes were largely reduced, compared with the conventional Ad vectors, resulting in an improvement in Ad vector-induced cytotoxicity. These data indicated that this improved Ad vector was a superior gene delivery vehicle without severe cytotoxicity for not only mouse hepatocytes but also human hepatocytes.

IL-10 is an immune regulatory cytokine and its genetic defect leads to gastrointestinal inflammation in humans and mice. Moreover, the IL-23/Th17 axis is known to be involved in these inflammatory disorders. IL-17A, a representative cytokine produced by Th17 cells, has an important role for the pathological process of inflammatory diseases. However, the precise function of IL-17A in inflammatory bowel disease (IBD) remains controversial. In this study, we evaluated the effect of IL-17A on colitis in IL-10-deficient (Il10-/-) mice. Mice lacking both IL-10 and IL-17A (Il10-/-Il17a-/-) suffered from fatal wasting and manifested more severe colitis compared with Il10-/-Il17a+/- mice. Moreover, we found that CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) accumulated in the bone marrow, spleen and peripheral blood of Il10-/-Il17a-/- mice. These MDSCs highly expressed inducible nitric oxide synthase (iNOS) (Nos2) and suppressed the T-cell response in vitro in a NOS-dependent manner. In correlation with these effects, the concentration of nitric oxide was elevated in the serum of Il10-/-Il17a-/- mice. Surprisingly, the severe colitis observed in Il10-/-Il17a-/- mice was ameliorated in Il10-/-Il17a-/-Nos2-/- mice. Our findings suggest that IL-17A plays suppressive roles against spontaneous colitis in Il10-/- mice in an iNOS-dependent manner and inhibits MDSC differentiation and/or proliferation., (© The Japanese Society for Immunology. 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Arming of an oncolytic adenovirus (OAd) by inserting expression cassettes of therapeutic transgenes into the OAd genome is a promising approach to enhance the therapeutic effects of an OAd. Ideally, this approach would simultaneously promote the replication of an OAd in tumor cells and transgene product-mediated antitumor effects by expressing therapeutic transgenes. We previously demonstrated that knockdown of cullin 4A (CUL4A), which is an E3 ubiquitin ligase, significantly promoted adenovirus replication by increasing the c-JUN protein level. In addition, previous studies reported that CUL4A was highly expressed in various types of tumor, and was involved in tumor growth and metastasis., Materials and Methods: In this study, we developed a novel OAd expressing a short-hairpin RNA (shRNA) against CUL4A (OAd-shCUL4A)., Results: OAd-shCUL4 mediated higher levels of cytotoxic effects on various types of human tumor cell than a conventional OAd. Higher levels of OAd genome copy numbers were found in the tumor cells for OAd-shCUL4A, compared with a conventional OAd., Conclusion: OAd-shCUL4A showed efficient antitumor effects by both enhancing OAd replication and inhibiting tumor cell growth., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The direct monofluorination of N -protected pyridone derivatives has been developed using a stable electrophilic fluorinating reagent, N -fluorobenzenesulfonimide (NFSI). Interestingly, the fluorine atom is regioselectively introduced at the position opposite the carbonyl group in the pyridone substrate during the reaction. This method is applicable to a wide range of substrates and allows the regioselective late-stage monofluorination of pyridone scaffolds.

Background: MicroRNAs (miRNAs) have gained much attention as cellular factors regulating hepatitis C virus (HCV) infection. miR-27b has been shown to regulate HCV infection in the hepatocytes via various mechanisms that have not been fully elucidated. In this study, therefore, we examined the mechanisms of miR-27b-mediated regulation of HCV infection., Methods: In silico screening analysis, transfection with miR-27b mimic, and a cell-based reporter assay were performed to identify miR-27b target genes. Cell cultured-derived HCV (HCVcc) was added to Huh7.5.1 cells knocked down for aquaporin (AQP) 11 (AQP11) and overexpressing AQP11. HCV replication levels were evaluated by real-time RT-PCR analysis of HCVcc genome., Results: Infection of Huh7.5.1 cells with HCVcc resulted in significant elevation in miR-27b expression levels. In silico analysis revealed that AQP11, which is an AQP family member and is mainly localized in the endoplasmic reticulum (ER), was a candidate for a target gene of miR-27b. Transfection of a miR-27b mimic significantly reduced AQP11 expression, but a cell-based reporter assay demonstrated that miR-27b did not suppress the expression of a reporter gene containing the 3'-untranslated region of the AQP11 gene, suggesting that miR-27b indirectly suppressed AQP11 expression. AQP11 expression levels were significantly reduced by infection with HCVcc in Huh7.5.1 cells. Knockdown and over-expression of AQP11 significantly reduced and increased HCVcc genome levels in the cells following infection, respectively, however, AQP11 knockdown did not show significant effects on the HCVcc titers in the culture supernatants., Conclusions: These results indicated that HCV infection induced a miR-27b-mediated reduction in AQP11 expression, leading to a modest reduction in HCV genome levels in the cells, not HCV titers in the culture supernatants.

Human induced pluripotent stem cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical research and regenerative medicine. In general, human induced pluripotent stem (iPS) cells are differentiated into hepatocyte-like cells through definitive endoderm cells and hepatoblast-like cells using various growth factors that are essential for liver development. Although recombinant bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are widely used in the hepatoblast differentiation, hepatoblast differentiation process has not been fully modified. In this study, we examined the roles of BMPs and FGFs in the hepatoblast differentiation from human iPS cells. Surprisingly, the gene expression levels of hepatoblast markers were upregulated by the removal of FGFs. In addition, the percentages of hepatoblast markers-positive cells were increased by the removal of FGFs. Furthermore, the hepatocyte differentiation potency was also significantly increased by the removal of FGFs. To examine whether FGF signals are completely unnecessary for the hepatoblast differentiation, the expression levels of endogenous FGF ligands and receptors were examined. The definitive endoderm cells highly expressed the FGF ligand, FGF2, and the FGF receptor, FGFR1. To examine the role of endogenous FGF signals, an FGFR inhibitor was treated during the hepatoblast differentiation. The hepatoblast differentiation was promoted by using FGFR inhibitor, suggesting that endogenous FGF signals are also unnecessary for the hepatoblast differentiation. In conclusion, we found that FGF signals are not essential for hepatoblast differentiation. We believe that our finding will be useful for generating functional hepatocyte-like cells for medical applications.

Reovirus, which possesses a 10-segmented double-stranded RNA genome, mediates superior antitumor effects via not only virus replication in a tumor cell-specific manner but also other mechanisms distinct from virus replication. Several groups, including ours, reported the reovirus-mediated downregulation of hypoxia inducible factor-1α (HIF-1α) following infection in cultured tumor cells; however, it remained to be clarified whether reovirus downregulates the expression of HIF-1α and its target genes in tumor-bearing hosts. We found that reovirus induced significant downregulation of protein levels of HIF-1α and its target genes in the subcutaneous tumors at 120 h post-systemic administration. Expression of reovirus capsid protein σ3 was found in the pimonidazole-positive hypoxic area in the tumor. Significant levels of tumor cell apoptosis were not found in the tumors of reovirus-treated mice at this time point, suggesting that reovirus-mediated tumor cell killing did not largely contribute to the downregulation of HIF-1α protein levels in the tumors. UV-inactivated reovirus did not induce downregulation of HIF-1α expression in the tumors, indicating that virus replication was indispensable for downregulation of HIF-1α expression in the subcutaneous tumors. This study provides important information for the development of reovirus-mediated virotherapy against various types of tumors.

The small intestine plays an important role in the absorption and metabolism of oral drugs. In the current evaluation system, it is difficult to predict the precise absorption and metabolism of oral drugs. In this study, we generated small intestinal epithelial-like cells from human induced pluripotent stem cells (hiPS-SIECs), which could be applied to drug absorption and metabolism studies. The small intestinal epithelial-like cells were efficiently generated from human induced pluripotent stem cell by treatment with WNT3A, R-spondin 3, Noggin, EGF, IGF-1, SB202190, and dexamethasone. The gene expression levels of small intestinal epithelial cell (SIEC) markers were similar between the hiPS-SIECs and human adult small intestine. Importantly, the gene expression levels of colonic epithelial cell markers in the hiPS-SIECs were much lower than those in human adult colon. The hiPS-SIECs generated by our protocol exerted various SIEC functions. In conclusion, the hiPS-SIECs can be utilized for evaluation of drug absorption and metabolism., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatocyte-like cells differentiated from human iPS cells are expected to be utilized in pharmaceutical research and regenerative medicine. Recently, various culture methods for human iPS cell maintenance have been developed. However, it is not well known whether human iPS cell maintenance method affects hepatic differentiation potency. In this study, we cultured human iPS cells using four maintenance methods: ReproStem medium with feeder cells (mouse embryonic fibroblasts), AK02N medium with iMatrix-511 (E8 fragments of laminin511), Essential 8 medium with Vitronectin N (N-terminal domain of vitronectin), TeSR-E8 medium with Vitronectin XF (xeno-free vitronectin). Then, these human iPS cells were differentiated into the hepatocyte-like cells. Interestingly, the gene expression levels of definitive endoderm markers in the definitive endoderm cells generated from human iPS cells cultured with ReproStem or TeSR-E8 medium were higher than those in other groups. The gene expression level of foregut marker, HHEX, in the definitive endoderm cells generated from human iPS cells cultured with ReproStem medium was higher than that in other groups. Consistently, the expression levels of hepatocyte markers, albumin and urea secretion capacity, and CYP3A4 activity in the hepatocyte-like cells generated from human iPS cells cultured with ReproStem medium were higher than those in the other groups. Our data indicated that the most suitable human iPS cell maintenance method for efficient hepatic differentiation was the on-feeder method which uses mouse embryonic fibroblasts, but not feeder-free methods. In conclusion, human iPS cell maintenance method largely affects hepatic differentiation potency., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Pre-existing anti-adenovirus (Ad) neutralizing antibodies (AdNAbs) are a major barrier in clinical gene therapy using Ad vectors and oncolytic Ads; however, it has not been fully elucidated which Ad capsid protein-specific antibodies are involved in AdNAb-mediated inhibition of Ad infection in vivo. In this study, mice possessing antibodies specific for each Ad capsid protein were prepared by intramuscular electroporation of each Ad capsid protein-expressing plasmid. Ad vector-mediated hepatic transduction was efficiently inhibited by more than 100-fold in mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid. An Ad vector pre-coated with FX before administration mediated more than 100-fold lower transduction efficiencies in the liver of warfarinized mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid, compared with those in the liver of warfarinized non-immunized mice. These data suggest that anti-fiber protein and anti-penton base antibodies bind to an Ad vector even though FX has already bound to the hexon, and inhibit Ad vector-mediated transduction. This study provides important clues for the development of a novel Ad vector that can circumvent inhibition with AdNAbs.

The isolation and structure elucidation of collimonins A-D (1-4) from the fungus-feeding bacterium Collimonas fungivorans Ter331 are reported. Collimonins are new derivatives of polyoxygenated hexadecanoic acid, including an ene-triyne moiety. Their absolute configurations were fully determined by combining spectroscopic, chemical, and crystalline sponge methods. Collimonins showed antifungal or pigmentation activities against the fungus Aspergillus niger ATCC 9029.