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The success rate of flap tissue reconstruction has increased in recent years owing to advancements in microsurgical techniques. However, complications, such as necrosis, are still more prevalent in diabetic patients compared to non-diabetic individuals, presenting an ongoing challenge. To address this issue, many previous studies have examined vascular anastomoses dilation and stability, primarily concerning surgical techniques or drugs. In contrast, in the present study, we focused on microvascular damage of the peripheral microvessels in patients with diabetes mellitus and the preventative impact of nafamostat mesylate. Herein, we aimed to investigate the effects of hyperglycemia on glycocalyx (GCX) levels in mice with type 2 diabetes. We examined the endothelial GCX (eGCX) in skin flap tissue of 9-12-week-old type 2 diabetic mice (db/db mice) using a perforator skin flap and explored treatment with nafamostat mesylate. The growth rates were compared after 1 week. Heterotype (db/+) mice were used as the control group. Morphological examination of postoperative tissues was performed at 1, 3, 5, and 7 days post-surgery. In addition, db/db mice were treated with 30 mg/kg/day of nafamostat mesylate daily and were evaluated on postoperative day 7. Seven days after surgery, all db/db mice showed significant partial flap necrosis. Temporal observation of the skin flaps revealed a stasis-like discoloration and necrosis starting from the contralateral side of the remaining perforating branch. The control group did not exhibit flap necrosis, and the flap remained intact. In the quantitative assessment of endothelial glycans using lectins, intensity scoring showed that the eGCX in the db/db group was significantly thinner than that in the db/+ group. These results were consistent with the scanning electron microscopy findings. In contrast, treatment with nafamostat mesylate significantly improved the flap engraftment rate and suppressed eGCX injury. In conclusion, treatment with nafamostat mesylate improves the disrupted eGCX structure of skin flap tissue in db/db mice, potentially ameliorating the impaired capillary-to-venous return in the skin flap tissue., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Takeshi Murata reports financial support was provided by Collaboration of Structure Analysis for ADMETox Related Proteins. Takeshi Murata reports financial support was provided by Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) from AMED. Takeshi Murata reports financial support was provided by Japan Society for the Promotion of Science (JSPS). 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 Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Ovarian cancer is the leading cause of death among women with gynecological cancer, and novel treatment options are urgently needed. Extracellular vesicles (EVs), including exosomes, may be one of the most promising therapeutic tools for various diseases. In this study, we aimed to investigate the therapeutic effects of adipose-derived stem cell-derived EVs (ADSC-EVs) on ovarian cancer cell lines., Materials and Methods: ADSCs and the ovarian cancer cell lines SKOV3 and OV90 were used for analysis. ADSC-EVs were isolated through ultracentrifugation and validated using a cryotransmission electron microscope, nanoparticle tracking analysis, and western blotting. Then, the effect of ADSC-EVs on ovarian cancer cells was investigated using IncuCyte and microRNA sequencing. Moreover, the potential functions of miRNAs were evaluated by gain-of function analysis and in silico analysis., Results: ADSC-EVs suppressed SKOV3 and OV90 cell proliferation. In particular, small EVs (sEVs) from ADSCs exhibited a stronger antitumor effect than ADSC-medium/large EVs (m/lEVs). Comparison of the miRNA profiles between ADSC-sEVs and ADSC-m/lEVs, along with downstream pathway analysis, suggested the involvement of the let-7 family. Overexpression of hsa-let-7b-5p and hsa-let-7e-5p significantly suppressed the proliferation of SKOV3 cells. In silico analysis revealed that four potential target genes of hsa-let-7b-5p and hsa-let-7e-5p were significantly associated with the prognoses of the patients., Conclusion: ADSC-sEVs had a stronger antitumor effect than ADSC-m/lEVs. Hsa-let-7b-5p and hsa-let-7e-5p, which are highly abundant in ADSC-sEVs, suppressed cell proliferation. These findings may open up new possibilities for therapeutic approaches using ADSC-sEVs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Akira Yokoi reports financial support was provided by Japan Science and Technology Agency. Akira Yokoi reports financial support was provided by Princess Takamatsu Cancer Research Fund. Akira Yokoi reports financial support was provided by Aichi Cancer Research Foundation., (Copyright © 2023 Elsevier Inc. All rights reserved.)

T follicular regulatory (Tfr) cells, a subset of CD4 + Foxp3 + regulatory T (Treg) cells, locate to the lymphoid follicle and germinal center (GC) and regulate antibody responses. Tfr cells express the functional molecules of follicular helper T (Tfh) cells, including CXCR5 and Bcl6. CD25 - mature Tfr cells differentiate from CD25 + Treg cells through CD25 + immature Tfr cells. Others and we have shown that Achaete-scute complex homolog 2 (Ascl2) plays a role in Tfh cell development; however, the role of Ascl2 in the development of Tfr cells remains unclear. Here, we found that Ascl2 was highly and preferentially expressed in CD25 + Tfr cells and CD25 - Tfr cells, and that the differentiation from CD25 + Tfr cells to CD25 - Tfr cells was impaired by the absence of Ascl2. Furthermore, the forced Ascl2 expression in Treg cells downregulated CD25 expression and suppressed IL-2-induced phosphorylation of STAT5, which is known to suppress CD25 - Tfr cell development. Finally, we found that the downregulation of CD25 by Ascl2 in Treg cells is independent of Bach2, which also regulates CD25 downregulation in CD25 + Tfr cells. These results suggest that Ascl2 plays a vital role in developing Tfr cells, possibly by downregulating CD25 expression in a Bach2-independent mechanism., Competing Interests: Declaration of compoeting interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kotaro Suzuki reports financial support was provided by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. Hiroshi Nakajima reports financial support was provided by Moonshot R&D. KAZUMA IIDA reports financial support was provided by LGS (Leading Graduate School) Program., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their spontaneous neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs containing excitatory and inhibitory neurons showed mature neural activity with an unsynchronized pattern. The CAs represent a powerful in vitro model for studying excitatory and inhibitory neuron interactions, cortical dynamics, and their dysfunction. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery., Competing Interests: Declaration of competing interest 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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In this study, we measured conjugated and unconjugated free bile acids (BAs) in plasma from patients with schizophrenia and healthy subjects to examine the possibility of BA as a biomarker for schizophrenia. Although the levels of each BA conjugate showed no significant differences, significant differences for three unconjugated bile acids were observed in the plasma between patients with schizophrenia and healthy subjects. Additionally, a more than three times difference between patients and healthy subjects was observed in the mean value of the total concentrations of primary BAs. These results indicate that cholic acid and chenodeoxycholic acid levels in plasma may be novel diagnostic markers for a sub-population of patients with schizophrenia. Thus, future studies should elucidate the relationship between this increase in BA levels and the pathology of schizophrenia and verify the potential of unconjugated BA in plasma as biomarkers for schizophrenia., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The vertebrate body is organized along the dorsal-ventral (DV) and anterior-posterior (AP) axes by the BMP and Wnt pathways, respectively. We previously reported that Xenopus Zbtb14 promotes dorsalization (neural induction) of ectoderm by inhibiting BMP signaling and also posteriorizes neural tissue by activating Wnt signaling, thereby coordinating the patterning of the DV and AP axes during early development. Although it has been reported that human ZBTB21 binds to ZBTB14 and is involved in gene expression in cultured mammalian cells, the function of Zbtb21 in early embryonic development remains unknown. Here, we show that Xenopus Zbtb21 plays an essential role in AP axis formation in the early Xenopus embryo. zbtb21 and zbtb14 are co-expressed in the dorsal region of embryos during gastrulation. Simultaneous overexpression of Zbtb21 and Zbtb14 in ectodermal explants enhances the neural-inducing activity of Zbtb14. Moreover, knockdown experiments showed that Zbtb21 is required for the formation of posterior neural tissue and the suppression of anterior neural development. Collectively, these results suggest that in cooperation with Zbtb14, Zbtb21 has a crucial function in AP patterning during early Xenopus embryogenesis., Competing Interests: Declaration of competing interest To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Accumulated studies have suggested that bone morphogenetic proteins (BMPs) are critical for skin development. However, it remains elusive how BMP signaling via ALK2 (aka ACVR1), one of the important BMP type I receptors, regulates keratinocyte differentiation. To address this question, we utilized a genetic system that enhances BMP signaling via ALK2 in an epidermis-specific manner in mice (hereafter ca-Alk2:K14-Cre). Ca-Alk2:K14-Cre mice displayed a sticky and hairless skin phenotype with a thinner epidermis incapable of differentiating. Although cellular proliferation and survival were comparable between wild-type and ca-Alk2:K14-Cre mice, skin differentiation was severely hampered in ca-Alk2:K14-Cre mice. To uncover the mechanism of altered keratinocyte differentiation, we performed a transcriptome analysis. As a result, we found that the expression levels of cell cycle inhibitor p21 were increased in ca-Alk2:K14-Cre mice. Our findings suggest that aberrant BMP signaling via ALK2 positively regulates p21 expression that attenuates keratinocyte differentiation, and further highlights the critical role of BMP signaling in skin development., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A20 (Tnfaip3), a ubiquitin-editing enzyme, inhibits NF-κB signaling pathways in response to pro-inflammatory cytokines. Previous studies have proved the anti-inflammatory roles of A20 in various cell types, including T cells, B cells, dendritic cells, and intestinal epithelial cells. Moreover, recent studies have shown that A20 expressed in lung epithelial cells is required for LPS-induced protection from asthma. In humans, a single-nucleotide polymorphism in TNFAIP3 is associated with asthma risk. However, the role of A20 expressed in T cells in asthmatic responses has not been elucidated. We addressed this point by generating mice lacking A20 expression in T cells (CD4-CreA20  fl/fl mice). We found that house dust mite (HDM)-induced allergic airway inflammation, mucus production, airway hyperresponsiveness, and Th2 cytokine production were significantly exacerbated in CD4-CreA20  fl/fl mice compared with those in control A20  fl/fl mice. In vitro differentiation of Th2 cells but not of Th1 cells or Th17 cells was enhanced in CD4 + T cells by the absence of A20. Consistently, enforced expression of A20 inhibited the differentiation of Th2 cells but not of Th1 cells or Th17 cells. Notably, the expression of GATA3 was significantly enhanced in A20-deficient CD4 + T cells, and the enhanced GATA3 expression was partly canceled by IL-2 neutralization. These results suggest that A20 functions as a stabilizing factor maintaining GATA3 levels during the induction of Th2 cells to prevent excessive Th2 cell differentiation., Competing Interests: Declaration of competing interest 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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Metastatic urothelial cancer is a lethal disease. Although recent advances in immunotherapies and targeted therapy against fibroblast growth factor receptor (FGFR)2/3 mutation (erdafitinib) have improved patient survival, there is still a critical need for novel therapeutic strategies for patients who do not benefit from these treatments. Evasion of apoptosis through amplifying anti-apoptotic Bcl-2 family proteins (Mcl-1, Bcl-xL, Bcl-2) is one mechanism responsible for treatment resistance in urothelial cancers, suggesting that targeting anti-apoptotic proteins may be a possible therapeutic strategy for urothelial cancers. Here, we showed that erdafitinib increased Mcl-1 degradation mainly through previously unknown mechanisms and synergized with a BH3 mimetic drug targeting Bcl-xL/Bcl-2 to induce apoptosis in FGFR wild-type urothelial cancer cells. Strikingly, clinical sequencing data showed amplification of MCL1 or BCL2L1 (encoding Bcl-xL) in subsets of FGFR mutation-negative bladder cancer tissues. In conclusion, these findings suggest that exploiting apoptosis pathways may be a promising treatment strategy for patients with FGFR wild-type metastatic urothelial cancer with Mcl-1 or Bcl-xL overexpression., Competing Interests: Declaration of competing interest 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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mycoplasma pneumoniae (Mp) is one of the most common causes of bacterial community-acquired pneumonia in humans. Because of the frequent epidemics and the emergence of antibiotic-resistant Mp, vaccines for Mp are urgently needed to ameliorate the pneumonia and secondary complications. The community-acquired respiratory distress syndrome (CARDS) toxin produced by Mp is a pathogenic factor that induces severe inflammatory responses in lung. Although blocking CARDS toxin is expected to mitigate the severity of Mp pneumonia, the potential of CARDS toxin as a vaccine antigen has not been assessed. Here, we examined the effectiveness of vaccine using recombinant CARDS toxin (rCARDS toxin) as an antigen in mice. Immunization with rCARDS toxin induced both rCARDS toxin- and Mp-specific antibody responses, indicating that CARDS toxin is located on the surface of Mp. In addition, immunization with rCARDS toxin decreased not only lung injury, neutrophil infiltration, and the production of inflammatory cytokines but also the persistence of Mp in lung after Mp challenge. Furthermore, we elucidated that the CARDS toxin on the surface of Mp facilitates the adherence of Mp to epithelial cells. In conclusion, we have demonstrated the potential of rCARDS toxin as a vaccine antigen to ameliorate Mp pneumonia by suppressing the inflammatory responses induced by Mp and the persistence of Mp in lung. These data support the development of novel vaccines for Mp pneumonia., Competing Interests: Declaration of competing interest Y. Inoue, K. Suzuki, and Y. Yoshioka are employed by the Research Foundation for Microbial Diseases of Osaka University. The other authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

During vertebrate development, the formation of the central nervous system (CNS) is initiated by neural induction and patterning of the embryonic ectoderm. We previously reported that Cdc2-like kinase 2 (Clk2) promotes neural development in Xenopus embryos by regulating morphogen signaling. However, the functions of other Clk family members and their roles in early embryonic development remain unknown. Here, we show that in addition to Clk2, Clk1 and Clk3 play a role in the formation of neural tissue in Xenopus. clk1 and clk3 are co-expressed in the developing neural tissue during early Xenopus embryogenesis. We found that overexpression of clk1 and clk3 increases the expression of neural marker genes in ectodermal explants. Furthermore, knockdown experiments showed that clk3 is required for the formation of neural tissues. These results suggest that Xenopus Clk3 plays an essential role in promoting neural development during early embryogenesis., Competing Interests: Declaration of competing interest To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Amphibians such as Xenopus tropicalis exhibit a remarkable capacity for tissue regeneration after traumatic injury. Although transforming growth factor-β (TGF-β) receptor signaling is known to be essential for tissue regeneration in fish and amphibians, the role of TGF-β ligands in this process is not well understood. Here, we show that inhibition of TGF-β1 function prevents tail regeneration in Xenopus tropicalis tadpoles. We found that expression of tgfb1 is present before tail amputation and is sustained throughout the regeneration process. CRISPR-mediated knock-out (KO) of tgfb1 retards tail regeneration; the phenotype of tgfb1 KO tadpoles can be rescued by injection of tgfb1 mRNA. Cell proliferation, a critical event for the success of tissue regeneration, is downregulated in tgfb1 KO tadpoles. In addition, tgfb1 KO reduces the expression of phosphorylated Smad2/3 (pSmad2/3) which is important for TGF-β signal-mediated cell proliferation. Collectively, our results show that TGF-β1 regulates cell proliferation through the activation of Smad2/3. We therefore propose that TGF-β1 plays a critical role in TGF-β receptor-dependent tadpole tail regeneration in Xenopus., Competing Interests: Declaration of competing interest To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Inflammatory bowel disease (IBD) comprises two major subtypes, ulcerative colitis (UC) and Crohn's disease, which are multifactorial diseases that may develop due to genetic susceptibility, dysbiosis, or environmental factors. Environmental triggers of IBD include food-borne factors, and a previous nationwide survey in Japan identified pre-illness consumption of isoflavones as a risk factor for UC. However, the precise mechanisms involved in the detrimental effects of isoflavones on the intestinal mucosa remain unclear. The present study employed human colonic organoids (hCOs) to investigate the functional effect of two representative isoflavones, genistein and daidzein, on human colonic epithelial cells. The addition of genistein to organoid reformation assays significantly decreased the number and size of reformed hCOs compared with control and daidzein treatment, indicating an inhibitory effect of genistein on colonic cell/progenitor cell function. Evaluation of the phosphorylation status of 49 different receptor tyrosine kinases showed that genistein selectively inhibited phosphorylation of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR). We established a two-dimensional wound-repair model using hCOs and showed that genistein significantly delayed the overall wound-repair response. Our results collectively show that genistein may exert its detrimental effects on the intestinal mucosa via negative regulation of stem/progenitor cell function, possibly leading to sustained mucosal injury and the development of UC., Competing Interests: Declaration of competing interest 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Podoplanin (PDPN) is a highly O-glycosylated glycoprotein that is utilized as a specific lymphatic endothelial marker under pathophysiological conditions. We previously developed an anti-human PDPN (hPDPN) monoclonal antibody (mAb), clone LpMab-3, which recognizes the epitope, including both the peptides and the attached disialy-core-l (NeuAcα2-3Galβl-3 [NeuAcα2-6]GalNAcαl-O-Thr) structure at the Thr76 residue in hPDPN. However, it is unclear if the mAb binds directly to both the peptides and glycans. In this study, we synthesized the binding epitope region of LpMab-3 that includes the peptide (- 67 LVATSVNSV-T-GIRIEDLP 84 -) possessing a disialyl-core-1 O-glycan at Thr76, and we determined the crystal structure of the LpMab-3 Fab fragment that was bound to the synthesized glycopeptide at a 2.8 Å resolution. The six amino acid residues and two sialic acid residues are directly associated with four complementarity-determining regions (CDRs; H1, H2, H3, and L3) and four CDRs (H2, H3, L1, and L3), respectively. These results suggest that IgG is advantageous for generating binders against spacious epitopes such as glycoconjugates., Competing Interests: Declaration of competing interest 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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Alternative splicing of the pyruvate kinase M (PKM) pre-mRNA generates two isoforms, PKM1 and PKM2. PKM catalyzes the conversion of phosphoenol-pyruvate to pyruvate in glycolytic pathway. PKM1 exist as a stable tetramer that is at an active enzyme state, while PKM2 is in equilibrium among monomer, dimer and tetramer under the regulation of its allosteric activators. Many cancer cells show the feature of higher glucose uptake and lactate production in spite of oxygen availability, which is known as the Warburg effect. PKM2 is upregulated in most cancer types and the inactive PKM2 lead to the cancer metabolism. In addition, dimeric PKM2 induces its nuclear translocation through posttranslational modification and acts as a transcriptional co-activator for the expression of oncogenes. Therefore, it is important to elucidate mechanisms for modulation of an active or inactive state of PKM2, namely the tetramer-to-dimer-transition. The definitive difference between PKM1 and PKM2 is to constitutively form tetramer or not in the cytoplasm, which is ascribed to 22 amino acids derived from exon 9 (PKM1) or exon 10 (PKM2). In this study, we generated 22 different PKM1-mimetic point mutants of PKM2, and demonstrated that replacement of cysteine424 residue of PKM2 with leucine424 conserved in PKM1 (C424L) promote its tetramerization. PKM2(C424L) formed a tetramer without allosteric activator, and escaped the inhibitory effects by oxidative stress, like PKM1. Our findings intensely suggest that C424 or L424 determines the different catalytic and modulatory properties between PKM splicing isoforms., Competing Interests: Declaration of competing interest 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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-β. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1, Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-β. Surprisingly, addition of TGF-β induced a set of genes, including Slc28a2, Tubb2a, and Cpe, that were expressed preferentially in fetal IECs. Moreover, TGF-β significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs., Competing Interests: Declaration of competing interest Okamoto reports grants from Cooperation Program between TMDU and Sony IP&S, Inc., during the study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

FLCN is a tumor suppressor gene which controls energy homeostasis through regulation of a variety of metabolic pathways including mitochondrial oxidative metabolism and autophagy. Birt-Hogg-Dubé (BHD) syndrome which is driven by germline alteration of the FLCN gene, predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas, pulmonary cysts and less frequently, salivary gland tumors. Here, we report metabolic roles for FLCN in the salivary gland as well as their clinical relevance. Screening of salivary glands of BHD patients using ultrasonography demonstrated increased cyst formation in the salivary gland. Salivary gland tumors that developed in BHD patients exhibited an upregulated mTOR-S6R pathway as well as increased GPNMB expression, which are characteristics of FLCN-deficient cells. Salivary gland-targeted Flcn knockout mice developed cytoplasmic clear cell formation in ductal cells with increased mitochondrial biogenesis, upregulated mTOR-S6K pathway, upregulated TFE3-GPNMB axis and upregulated lipid metabolism. Proteomic and metabolite analysis using LC/MS and GC/MS revealed that Flcn inactivation in salivary gland triggers metabolic reprogramming towards the pentose phosphate pathway which consequently upregulates nucleotide synthesis and redox regulation, further supporting that Flcn controls metabolic homeostasis in salivary gland. These data uncover important roles for FLCN in salivary gland; metabolic reprogramming under FLCN deficiency might increase nucleotide production which may feed FLCN-deficient salivary gland cells to trigger tumor initiation and progression, providing mechanistic insight into salivary gland tumorigenesis as well as a foundation for development of novel therapeutics for salivary gland tumors., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Xenopus tropicalis tadpoles can regenerate an amputated tail, including spinal cord, muscle and notochord, through cell proliferation and differentiation. However, the molecular mechanisms that regulate cell proliferation during tail regeneration are largely unknown. Here we show that JunB plays an important role in tail regeneration by regulating cell proliferation. The expression of junb is rapidly activated and sustained during tail regeneration. Knockout (KO) of junb causes a delay in tail regeneration and tissue differentiation. In junb KO tadpoles, cell proliferation is prevented before tissue differentiation. Furthermore, TGF-β signaling, which is activated just after tail amputation, regulates the induction and maintenance of junb expression. These findings demonstrate that JunB, a downstream component of TGF-β signaling, works as a positive regulator of cell proliferation during Xenopus tail regeneration., Competing Interests: Declaration of competing interest To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise., (Copyright © 2019 Elsevier Inc. All rights reserved.)

JTE-607 is a small molecule that was developed as an inflammatory cytokine inhibitor and also as an anti-leukemia reagent for monocytic leukemia. However, the mode of action of JTE-607 remains unknown. In this study, we identified JTE-607 to be a prodrug compound that is converted to an active form by ester hydrolysis. Furthermore, we determined that the active form of JTE-607 bound cleavage and polyadenylation specificity factor subunit 3 (CPSF3), using compound-immobilized affinity chromatography. CPSF3 is a 73-kDa subunit of the cleavage and polyadenylation specificity factor complex, which functions as an RNA endonuclease. The protein is involved in the 3'-end processing of messenger RNA precursors (pre-mRNAs) at the cleavage site located downstream of the poly(A) addition signal. We found that treatment with JTE-607 caused accumulation of pre-mRNAs. Furthermore, knockdown experiments showed that CPSF3 deficiency also caused accumulation of pre-mRNAs and suppressed the expression of inflammatory cytokines, like JTE-607. These findings indicated that CPSF3 is a direct target of JTE-607 and a new potential target for the treatment of disease-related abnormal cytokine production., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purification of undifferentiated cells by removing differentiated parts is an essential step in pluripotent stem cell culture. This process has been traditionally performed manually using a fine glass capillary or plastic tip under a microscope, or by culturing in a selective medium supplemented with anti-differentiation inhibitors. However, there are several inevitable problems associated with these methods, such as contamination or biological side-effects. Here, we developed a laser-assisted cell removing (LACR) technology that enables precise, fast, and contact-less cell removal. Using LACR combined with computational image recognition/identification-discriminating technology, we achieved automatic cell purification (A-LACR). Practicability of A-LACR was evaluated by two demonstrations: selective removal of trophoblast stem (TS) cells from human iPS and TS cell co-cultures, and purification of undifferentiated iPS cells by targeting differentiated cells that spontaneously developed. Our results suggested that LACR technology is a novel approach for stem cell processing in regenerative medicine., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor necrosis factor (TNF)-α is responsible for expressions of several clock genes and affects joint symptoms of rheumatoid arthritis (RA) with diurnal fluctuation. We tried to determine the mechanism involved in over-expression of Bmal1, induced by TNF-α, in primary cultured rheumatoid synovial cells. Cells were incubated with intra-cellular Ca 2+ chelator BAPTA-AM, calcineurin inhibitor FK506 and p300/CBP (CREB binding protein) inhibitor C646, respectively, or transfected with p300 and CBP small interfering RNA (siRNA) before stimulation with TNF-α. Oscillation phase and amplitude of Bmal1, transcriptional activator Rorα, transcriptional repressor Rev-erbα, and histone acetyltransferases (p300 and Cbp) were evaluated by quantitative real-time PCR. As results, TNF-α did not influence the oscillation phase of Rev-erbα, while enhanced those of Rorα, resulting in over-expression of Bmal1. When Ca 2+ influx was inhibited by BAPTA-AM, TNF-α-mediated up-regulation of Rorα was cancelled, however, that of Bmal1 was still apparent. When we further explored another pathway between TNF-α and Bmal1, TNF-α suppressed the expression of Rev-erbα in the absence of Ca 2+ influx, as well as those of p300 and Cbp genes. Finally, actions of TNF-α, in increasing Bmal1/Rorα and decreasing Rev-erbα, were cancelled by C646 treatment or silencing of both p300 and Cbp. In conclusion, we determined a novel role of TNF-α in inducing Bmal1 via dual calcium dependent pathways; Rorα was up-regulated in the presence of Ca 2+ influx and Rev-erbα was down-regulated in the absence of that. Results proposed that inhibition of p300/CBP could be new therapeutic targets for RA., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Euglena gracilis Z is a micro-algae that is used as a food or nutritional supplement. Paramylon, the carbohydrate storage substance of Euglena gracilis Z has β-1, 3-glucan structure. Euglena gracilis Z and paramylon are reported to affect the immune system. In this study, we investigated the protective effects of Euglena gracilis Z and paramylon against influenza virus infection in mice. Euglena gracilis Z and paramylon were administered to mice as a 2% dietary mixture ad libitum. At 2 weeks after initiation of dietary administration, mice were infected intranasally with influenza virus A/PR/8/34 (H1N1). Survival rate was monitored 10 days after infection. In addition, we performed virus titer and cytokine profiles in the lung. High survival rates were observed for Euglena gracilis Z and paramylon-treated groups compared to the control group. Significantly lower virus titer in the lung was observed in the Euglena gracilis Z and paramylon-treated groups compared to the control group from day 1 after infection. Higher amount of IL-1β, IL-6, IL-12 (p70), IFN-γ, and IL-10 was observed in the paramylon groups compared to the control group. Our data therefore reveals a novel immunoregulatory role of the Euglena gracilis Z and paramylon which provides protection against influenza virus infection., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Although several antibodies developed to target programmed cell death-1 (PD-1) and its ligand (PD-L1) have demonstrated great promise for the treatment of non-small cell lung cancer (NSCLC), and other malignancies, these therapeutic antibodies can cause pneumonitis. Furthermore, epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-induced pneumonitis was reported after treatment with anti PD-1 antibodies. We previously demonstrated that mice with naphthalene-induced airway epithelial injury developed severe gefitinib-induced pneumonitis through a neutrophil-dependent mechanism. The present study aimed to investigate whether treatment with afatinib, an EGFR-TKI that effectively targets EGFR mutation-positive NSCLC, and anti PD-1 antibodies induces pneumonitis in mice. C57BL/6J mice were treated intraperitoneally with naphthalene (200 mg/kg) on day 0. Afatinib (20 mg/kg) was administered orally on days -1 to 13. An anti-PD-1 antibody (0.2 mg/mice) was also administered intraperitoneally every 3 days from day 1 until day 13. The bronchoalveolar lavage fluid (BALF) and lung tissues were sampled on day 14. As observed previously with gefitinib, afatinib significantly increased the severity of histopathologic findings and the level of protein in BALF on day 14, compared to treatment with naphthalene alone. A combined anti-PD-1 antibody and afatinib treatment after naphthalene administration had yielded the same histopathological grade of lung inflammation as did afatinib treatment alone. Our results suggest that anti-PD-1 antibody treatment has little effect on afatinib-induced lung injury., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Gefitinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), is an effective therapeutic agent for non-small cell lung cancer with EGFR mutations. It can cause severe acute pneumonitis in some patients. We previously demonstrated that mice with naphthalene-induced airway epithelial injury developed severe gefitinib-induced pneumonitis and that neutrophils played important roles in the development of the disease. This study aimed to investigate the effects of the neutrophil elastase inhibitor sivelestat on gefitinib-induced pneumonitis in mice. C57BL/6J mice received naphthalene (200 mg/kg) intraperitoneally on day 0. Gefitinib (250 or 300 mg/kg) was orally administered to mice from day -1 until day 13. Sivelestat (150 mg/kg) was administered intraperitoneally from day 1 until day 13. Bronchoalveolar lavage fluid (BALF) and lung tissues were sampled on day 14. Sivelestat treatment significantly reduced the protein level, neutrophil count, neutrophil elastase activity in BALF, and severity of histopathologic findings on day 14 for mice administered with 250 mg/kg of gefitinib. Moreover, sivelestat treatment significantly improved the survival of mice administered with 300 mg/kg of gefitinib. These results indicate that sivelestat is a promising therapeutic agent for severe acute pneumonitis caused by gefitinib., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Objective: As a member of the epidermal growth factor family, amphiregulin contributes to the regulation of cell proliferation. Amphiregulin was reported to be upregulated in damaged lung tissues in patients with chronic obstructive pulmonary disease and asthma and in lung epithelial cells in a ventilator-associated lung injury model. In this study, we investigated the effect of amphiregulin on lipopolysaccharide (LPS)-induced acute lung injury in mice., Methods: Acute lung injury was induced by intranasal instillation of LPS in female C57BL/6 mice, and the mice were given intraperitoneal injections of recombinant amphiregulin or phosphate-buffered saline 6 and 0.5 h before and 3 h after LPS instillation. The effect of amphiregulin on apoptosis and apoptotic pathways in a murine lung alveolar type II epithelial cell line (LA-4 cells) were examined using flow cytometry and western blotting, respectively., Results: Recombinant amphiregulin suppressed epithelial cell apoptosis in LPS-induced lung injury in mice. Western blotting revealed that amphiregulin suppressed epithelial cell apoptosis by inhibiting caspase-8 activity., Conclusion: Amphiregulin signaling may be a therapeutic target for LPS-induced lung injury treatment through its prevention of epithelial cell apoptosis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Thyroglobulin (Tg) stored in thyroid follicles exerts a potent negative-feedback effect on each step of pre-hormone biosynthesis, including Tg gene transcription and iodine uptake and organification, by suppressing the expression of specific transcription factors that regulate these steps. Pre-hormones are stored in the follicular colloid before being reabsorbed. Following lysosomal proteolysis of its precursor, thyroid hormone (TH) is released from thyroid follicles. Although the suppressive effects of follicular Tg on each step of pre-hormone biosynthesis have been extensively characterized, whether follicular Tg accumulation also affects hormone reabsorption, proteolysis, and secretion is unclear. In this study we explored whether follicular Tg can regulate the expression and function of the lysosomal endopeptidases cathepsins. We found that in the rat thyroid cell line FRTL-5 follicular Tg induced cathepsin H mRNA and protein expression, as well as cathepsin H enzyme activity. Double immunofluorescence staining showed that Tg endocytosis promoted cathepsin H translocalization into lysosomes where it co-localized with internalized Tg. These results suggest that cathepsin H is an active participant in lysosome-mediated pre-hormone degradation, and that follicular Tg stimulates mobilization of pre-hormones by activating cathepsin H-associated proteolysis pathways., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ligand-dependent activation of Notch signaling is required to maintain the stem-cell niche of normal intestinal epithelium. However, the precise role of Notch signaling in the maintenance of the intestinal tumor stem cell niche and the importance of the RBPJ-independent non-canonical pathway in intestinal tumors remains unknown. Here we show that Notch signaling was activated in LGR5 +ve cells of APC-deficient mice intestinal tumors. Accordingly, Notch ligands, including Jag1, Dll1, and Dll4, were expressed in these tumors. In vitro studies using tumor-derived organoids confirmed the intrinsic Notch activity-dependent growth of tumor cells. Surprisingly, the targeted deletion of Jag1 but not RBPJ in LGR5 +ve tumor-initiating cells resulted in the silencing of Hes1 expression, disruption of the tumor stem cell niche, and dramatic reduction in the proliferation activity of APC-deficient intestinal tumors in vivo. Thus, our results highlight the importance of ligand-dependent non-canonical Notch signaling in the proliferation and maintenance of the tumor stem cell niche in APC-deficient intestinal adenomas., (Copyright © 2016 Elsevier Inc. All rights reserved.)

LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status., (Copyright © 2015 Elsevier Inc. All rights reserved.)