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Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: The spotted wing drosophila, Drosophila suzukii Matsumura, is a South-East Asian vinegar fly that is a serious worldwide economic threat to the small fruit industry. Typical control consists of weekly pesticide applications, which can have nontarget effects, increase residual pesticides and lead to the development of resistance within pest populations. One potential alternate method of control is the planting of aromatic intercrops to attract the natural enemies of D. suzukii and/or repel the flies directly. We intercropped strawberry rows with flowering sweet alyssum or ryegrass-clover (control) to evaluate their efficacy at mitigating D. suzukii infestation through the attraction of two specialized larval parasitoids, Leptopilina japonica (Novkovic and Kimura) and Ganaspis brasiliensis (Ihering)., Results: Our study did not demonstrate any significant effect of sweet alyssum intercropping on the infestation rate of D. suzukii in strawberries or parasitism level. However, we found that advanced sampling date and recorded numbers of D. suzukii larvae and parasitoids were positively correlated, indicating higher populations at the end of the strawberry-growing season., Conclusions: Sweet alyssum intercrops did not reduce D. suzukii infestation rates or increase parasitism levels, likely due to low population numbers in early season berry varieties. Aromatic intercrops may be more effective for increasing pest control in later season crops. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Exosomes are 40-100 nm nano-sized extracellular vesicles and are receiving increasing attention as novel structures that participate in intracellular communication. We previously found that miRNA-1 (miR-1) functions as a tumor suppressor in renal cell carcinoma (RCC). In this study, we investigated the function of exosomal miR-1 and the possibility that the exosome constitutes a tumor maker in RCC. First, we established the method to collect exosomes from cell lysates and human serum by a spin column-based method. Next, we assessed exosomes using Nanosight nanoparticle tracking analysis and Western blot analysis with exosome marker CD63. We confirmed that exosomes labeled with PKH26 fused with recipient cells. Moreover, miR-1 expression was elevated in RCC cells treated with exosomes derived from miR-1-transfected cells. Functional analyses showed that exosomal miR-1 significantly inhibited cell proliferation, migration and invasion compared to control treatment. Our analyses with TCGA database of RCCs showed that miR-1 expression was significantly downregulated in clinical RCC samples compared to that in normal kidney samples, and patients with low miR-1 expression had poorer overall survival in comparison to patients with high expression. Furthermore, RNA sequence analyses showed that expression levels of several genes were altered by exposure to exosomal miR-1. The analyses with TCGA database indicated that high expression of MYO15A was associated with a poorer outcome in RCC. In addition, RT-qPCR analysis of exosomes from clinical patients' sera showed that MYO15A was significantly upregulated in RCC patients compared to that in healthy controls. This study showed that treatment with exosomal miR-1 might be an effective approach to treating RCCs. In addition, exosomal MYO15A could be a diagnostic tumor marker in RCCs., Competing Interests: Declaration of competing interest None of the authors has any direct or indirect commercial financial incentives associated with the publication of this article entitled “Exosomal microRNA-1 and MY6O15A as a target for therapy and diagnosis in renal cell carcinoma”., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Two species of larval parasitoids of the globally invasive fruit pest, Drosophila suzukii (Diptera: Drosophilidae), Leptopilina japonica, and Ganaspis brasiliensis (both Hymenoptera: Figitidae), were detected in British Columbia, Canada in 2016 and 2019, respectively. Both are presumed to have been unintentionally introduced from Asia; however, the extent of their establishment across different habitats with diverse host plants used by D. suzukii was unclear. In addition, there was no knowledge of the temporal dynamics of parasitism of D. suzukii by these two parasitoids. To address these gaps, we repeatedly sampled the fruits of known host plants of D. suzukii over the entire 2020 growing season in British Columbia. We documented the presence of L. japonica and G. brasiliensis and estimated the apparent percentage of D. suzukii parasitized among host plant species. Across a large region of southwestern British Columbia, both L. japonica and G. brasiliensis were found to be very common across a variety of mostly unmanaged habitats over the entire course of the season (May-October) in the fruits of most host plants known to host D. suzukii larvae. Parasitism of D. suzukii was variable (0-66% percent parasitism) and appeared to be time-structured. Our study demonstrates that the close association between the two larval parasitoids and D. suzukii that exists in Asia has evidently been reconstructed in North America, resulting in the highest parasitism levels of D. suzukii yet recorded outside of its area of origin., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Natural Resources Canada, 2022.)

Hematopoietic stem cell (HSC)-independent hematopoiesis from hemogenic endothelial cells (HECs) in the mouse embryo has been recognized as a source of tissue-resident hematopoietic cells in adult mice. Connective tissue mast cells (MCs) have been reported to originate from VE-cadherin (VE-cad)-expressing HECs in the yolk sac and embryo proper (EP) by a VE-cad-Cre-mediated lineage-tracing analysis. However, it remains unclear whether MCs are generated via a conventional HSC-dependent hematopoietic differentiation pathway, or whether through a fast-track pathway bypassing the emergence of HSCs. Here, we investigated whether EP-derived VE-cad+ cells differentiate into MCs independently of HSCs. VE-cad+ cells isolated from the embryonic day (E) 9.5-10.5 EP robustly formed connective tissue-type MCs in a newly established co-culture system using PA6 stromal cells. In contrast, bone marrow (BM) reconstitution assays of cultured cells indicated that E9.5 VE-cad+ cells did not differentiate into transplantable HSCs in this culture condition. Lymphoid-biased HSCs with a limited self-renewal capacity were occasionally detected in some cultures of E10.5 VE-cad+ cells, while MC growth was constantly observed in all cultures examined. HSCs purified from adult BM required a more extended culture period to form MCs in the PA6 co-culture than the embryonic VE-cad+ cells. Furthermore, E9.5-E10.5 VE-cad+ cells contributed to tissue-resident MCs in postnatal mice when transplanted into the peritoneal cavity of newborn mice. These results suggest that EP-derived VE-cad+ cells generate MCs independently of HSC development in vitro and possess the potential of generating connective tissue MCs in vivo, although the exact differentiation program remains unsolved., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Definitive hematopoietic cells develop from fetal liver kinase 1 (Flk1) + mesodermal cells during the in vitro differentiation of mouse embryonic stem cells (ESCs). VE-cadherin + CD41 - CD45 - (V + 41 - 45 - ) hemogenic endothelial cells (HECs) and VE-cadherin + CD41 + CD45 - (V + 41 + 45 - ) cells mediate the definitive hematopoietic development from Flk1 + cells. Bone morphogenetic protein 4 (BMP4) is known to be essential for the formation of mesoderm. However, the role of BMP4 in differentiation of the VE-cadherin +  definitive hematopoietic precursors from the mesoderm has been elusive. We addressed this issue using a co-aggregation culture of ESC-derived Flk1 + cells with OP9 stromal cells. This culture method induced V + 41 - 45 - cells, V + 41 + 45 - cells, and CD45 + cells from Flk1 + cells. V + 41 + 45 - cells possessed potential for erythromyeloid and T-lymphoid differentiation. When Flk1 + cells were cultured in the presence of a high concentration of BMP4, the generation of V + 41 - 45 - cells was enhanced. The increase in V + 41 - 45 - cells led to the subsequent increase in V + 41 + 45 - and CD45 + cells. The addition of BMP4 also increased hematopoietic colony-forming cells of various lineages. Furthermore, BMP4 promoted the expansion of V + 41 + 45 - cells independently of the preceding V + 41 - 45 - cell stage. These results suggest that BMP4 has promotive effects on the differentiation of V + 41 - 45 - HECs from Flk1 + mesodermal cells and the subsequent proliferation of V + 41 + 45 - hematopoietic precursors. These findings may provide insights for establishing a culture system to induce definitive hematopoietic stem cells from ESCs., Competing Interests: Conflict of interest disclosure The authors declare that there are no potential conflicts of interest., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: Cisplatin-based chemotherapy is recommended as the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease. However, the benefits are limited due to the acquisition of drug resistance. The mechanisms of resistance remain unclear. Although there are some reports that some molecules are associated with cisplatin resistance in advanced BC, those reports have not been fully investigated. Therefore, we undertook a new search for cisplatin resistance-related genes targeted by tumor suppressive microRNAs as well as genes that were downregulated in cisplatin-resistant BC cells and clinical BC tissues., Methods: First, we established cisplatin-resistant BOY and T24 BC cell lines (CDDP-R-BOY, CDDP-R-T24). Then, Next Generation Sequence analysis was performed with parental and cisplatin-resistant cell lines to search for the microRNAs responsible for cisplatin resistance. We conducted gain-of-function analysis of microRNAs and their effects on cisplatin resistance, and we searched target genes comprehensively using Next Generation mRNA sequences., Results: A total of 28 microRNAs were significantly downregulated in both CDDP-R-BOY and CDDP-R-T24. Among them, miR-486-5p, a tumor suppressor miRNA, was negatively correlated with the TNM classification of clinical BC samples in The Cancer Genome Atlas (TCGA) database. Transfection of miRNA-486-5p significantly inhibited cancer cell proliferation, migration, and invasion, and also improved the cells' resistance to cisplatin. Among the genes targeted by miRNA-486-5p, we focused on enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase (EHHADH), which is involved in the degradation of fatty acids. EHHADH was directly regulated by miRNA-486-5p as determined by a dual-luciferase reporter assay. Loss-of-function study using EHHADH si-RNA showed significant inhibitions of cell proliferation, migration, invasion and the recovery of cisplatin sensitivity., Conclusion: Identification of EHHADH as a target of miRNA-486-5p provides novel insights into the potential mechanisms of cisplatin resistance in BC.

d-3-Phosphoglycerate dehydrogenase (PHGDH) conducts an important step in the synthesis of serine. Importantly, the PHGDH gene is often amplified in certain cancers. Our previous studies revealed that PHGDH gene amplification was associated with poor overall survival in clear cell renal cell carcinoma (ccRCC) and that metabolic reprogramming of serine synthesis through PHGDH recruitment allowed ccRCC cells to survive in unfavorable environments. There have been no investigations of the role of PHGDH expression in bladder cancer (BC). In this investigation, we examined the clinical importance of PHDGH in BC. Furthermore, we asked whether PHGDH expression could be exploited for BC therapy. Finally, we investigated the regulatory mechanisms that modulated the expression of PHGDH. Using data from The Cancer Genome Atlas, we found that patients with high-grade BC had significantly higher PHGDH expression levels than did those with low-grade BC. In addition, patients with high PHGDH expression did not survive as long as those with low expression. PHGDH downregulation by si-RNAs or an inhibitor in BC cell lines significantly inhibited proliferative ability and induced apoptosis. Furthermore, combined treatment using a PHGDH inhibitor and gemcitabine/cisplatin achieved synergistic tumor suppression compared to use of a single agent both in vitro as well as in vivo. Mechanistic analyses of PHGDH regulation showed that PHGDH expression might be associated with DNA copy number and hypomethylation in BC. These findings suggest novel therapeutic strategies could be used in BC. Finally, our data enhance our understanding of the role of PHGDH in BC., (© 2020 Kagoshima University. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The alcohol-abuse drug disulfiram has antitumor effects against diverse cancer types via inhibition of the ubiquitin-proteasome protein nuclear protein localization protein 4 (NPL4). However, the antitumor effects of NPL4 and disulfiram in clear cell renal cell carcinoma (ccRCC) are unclear. Here, we evaluated the therapeutic potential of targeting the ubiquitin-proteasome pathway using disulfiram and RNA interference and investigated the mechanisms underlying disulfiram in ccRCC. According to data from The Cancer Genome Atlas, NPL4 mRNA expression was significantly upregulated in clinical ccRCC samples compared with that in normal kidney samples, and patients with high NPL4 expression had poor overall survival compared with patients with low NPL4 expression. Disulfiram and NPL4 siRNA inhibited ccRCC cell proliferation in vitro, and disulfiram inhibited ccRCC tumor growth in a xenograft model. Synergistic antiproliferative effects were observed for combination treatment with disulfiram and sunitinib in vitro and in vivo. In RCC cells from mice treated with disulfiram and/or sunitinib, several genes associated with serine biosynthesis and aldose reductase were downregulated in cells treated with disulfiram or sunitinib alone and further downregulated in cells treated with both disulfiram and sunitinib. These findings provided insights into the mechanisms of disulfiram and suggested novel therapeutic strategies for RCC treatment., Competing Interests: The authors have declared that no competing interests exist.

Exosomes are 40-100 nm nano-sized extracellular vesicles. They are released from many cell types and move into the extracellular space, thereby transferring their components to recipient cells. Exosomes are receiving increasing attention as novel structures participating in intracellular communication. RAB27B is one of the leading proteins involved in exosome secretion, and oncogenic effects have been reported in several cancers. In recent years, molecularly targeted agents typified by sunitinib are widely used for the treatment of metastatic or recurrent renal cell carcinoma (RCC). However, intrinsic or acquired resistance to sunitinib has become a major issue. The present study aimed to elucidate the role of RAB27B in RCC including sunitinib-resistant and its role in exosomes. Bioinformatic analyses revealed that high expression of RAB27B correlates with progression of RCC. The expression of RAB27B protein in RCC cell lines was significantly enhanced compared with that in normal kidney cell lines. Furthermore, RAB27B protein expression was enhanced in all of the tested sunitinib-resistant RCC cell lines compared to parental cells. Although no specific effect of RAB27B on exosomes was identified in RCC cells, loss-of-function studies demonstrated that knockdown of RAB27B suppressed cell proliferation, migration and invasive activities. Moreover, anti-tumor effects of RAB27B downregulation were also observed in sunitinib-resistant RCC cells. RNA sequence and pathway analysis suggested that the oncogenic effects of RAB27B might be associated with MAPK and VEGF signaling pathways. These results showed that RAB27B is a prognostic marker and a novel therapeutic target in sunitinib-sensitive and -resistant RCCs. Further analyses should improve our understanding of sunitinib resistance in RCC., Competing Interests: The authors have declared that no competing interests exist.

Sunitinib, a multitargeted receptor tyrosine kinase inhibitor including vascular endothelial growth factor, has been widely used as a first-line treatment against metastatic renal cell carcinoma (mRCC). However, mRCC often acquires resistance to sunitinib, rendering it difficult to treat with this agent. Recently, Rapalink-1, a drug that links rapamycin and the mTOR kinase inhibitor MLN0128, has been developed with excellent therapeutic effects against breast cancer cells carrying mTOR resistance mutations. The aim of the present study was to evaluate the in vitro and in vivo therapeutic efficacy of Rapalink-1 against renal cell carcinoma (RCC) compared to temsirolimus, which is commonly used as a small molecule inhibitor of mTOR and is a derivative of rapamycin. In comparison with temsirolimus, Rapalink-1 showed significantly greater effects against proliferation, migration, invasion and cFolony formation in sunitinib-naïve RCC cells. Inhibition was achieved through suppression of the phosphorylation of substrates in the mTOR signal pathway, such as p70S6K, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and AKT. In addition, Rapalink-1 had greater tumor suppressive effects than temsirolimus against the sunitinib-resistant 786-o cell line (SU-R 786-o), which we had previously established, as well as 3 additional SU-R cell lines established here. RNA sequencing showed that Rapalink-1 suppressed not only the mTOR signaling pathway but also a part of the MAPK signaling pathway, the ErbB signaling pathway and ABC transporters that were associated with resistance to several drugs. Our study suggests the possibility of a new treatment option for patients with RCC that is either sunitinib-sensitive or sunitinib-resistant., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

We report here five pediatric patients with human T-lymphotropic virus type 1 (HTLV-I) uveitis. The patients were one boy and four girls aged between 3 and 14 years. The transmission route was considered to be breast feeding from their mothers. All patients had unilateral uveitis and the ocular symptoms were similar to those in HTLV-I uveitis in adults. The ocular inflammation responded to therapy with topical or systemic corticosteroids, but recurred in three patients. HTLV-I provirus DNA was detected by polymerase chain reaction (PCR) from infiltrating cells in the anterior chamber in one patient. The percentage of HTLV-I-infected cells in the peripheral blood mononuclear cells was measured by quantitative PCR, and the values were high (2.9 approximately 7.3%) in three cases tested as compared with an asymptomatic carrier. These five cases show that HTLV-I uveitis can be induced in a relatively short period (3 approximately 10 years) after the viral infection, and that HTLV-I uveitis should be considered as one possible etiology of uveitis in children, particularly in a viral endemic area.

The optical resolution of racemic 4-[alpha-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoic acid (dl-I), a new potent and long lasting thromboxane synthetase inhibitor, was investigated. (S)-3-(3,5-Dinitrobenzoylthio)-2-methylpropionyl group was introduced to alpha-hydroxy moiety of methyl ester of dl-I by three steps of reaction to give the corresponding ester compound (VIa). Then, two diastereomers of VIa, alpha-VIa and beta-VIa, were separated by column chromatography using on silica gel. Alkaline hydrolysis of alpha-VIa and beta-VIa, followed by reactions with sodium ethylate gave optically pure sodium salts of (-)-I and (+)-I, respectively. The separative determination methods for the diastereomers of VIa, and for the enantiomers of the sodium salts of dl-I by high performance liquid chromatography were also established.

The pro-drugs of alpha,2-dimethyl-5H-[1]benzopyrano[2,3-b]pyridine-7-acetic acid(I) with a potent anti-inflammatory activity were synthesized in order to reduce its gastrointestinal side effects. Various esters synthesized were evaluated for their anti-inflammatory activity and ulcerogenicity. Among the compounds maintaining a potent activity of I, N,N-dimethylcarbamoylmethyl alpha,2-dimethyl-5H-[1]benzopyrano[2,3-b]pyridine-7-acetate (II-18) showed excellent biopharmaceutical characteristics. The ulcerogenic effect of II-18 on the rat gastric mucosa was about 3 times less than that of I. It was suggested that II-18 may be an useful biolabile pro-drug for I among the compounds tested.

The urinary metabolites of sodium 4-[alpha-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoat e (Y-20811) in dog were investigated. The main metabolite was isolated by high performance liquid chromatography and subsequent preparative thin layer chromatography. The structure of this metabolite was established as 4-[alpha-hydroxy-2-hydroxymethyl-5-(1-imidazolyl)benzyl]-3,5- dimethylbenzoic acid on the basis of spectral analyses and confirmed by its total synthesis.

Several imidazolylpyridinemethanols and imidazolylbenzenemethanols were prepared and evaluated for an inhibitory activity against arachidonic acid-induced platelet aggregation. The result shows that the arylmethanol moiety is essential for the activity and may correspond to the 15-OH group of prostagrandin H2 (PGH2). Among the compounds tested, 4-[alpha-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoic acid (XV) was found to have a potent inhibitory activity and a long duration of action. Structure-activity relationships are also discussed briefly.