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Skeletal muscle atrophy impairs quality of life and increases the risk of disease, but current methods for assessment of muscle mass have several limitations. We here investigated the urinary concentration of a fragment of the muscle protein titin as a potential biomarker for the early detection of skeletal muscle atrophy. Four mouse models with different atrophy pathways were studied: those of cardiotoxin-induced acute muscle injury, cast-induced muscle immobilization, lipopolysaccharide-induced sepsis, and streptozotocin-induced diabetes. In all four models, urinary titin levels increased early, concurrent with or preceding upregulation of the atrophy-related genes for atrogin-1 and MuRF-1. The increase in the urinary titin concentration was thus associated with initial muscle damage and the onset of proteolysis, rather than with late-stage muscle wasting. Our findings suggest that urinary titin is a promising biomarker for detection of the onset of skeletal muscle catabolism and prediction of the subsequent development of atrophy in different catabolic states. Noninvasive measurement of urinary titin may therefore allow the earlier detection of skeletal muscle proteolysis compared with conventional techniques., 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 Author(s). Published by Elsevier Inc. All rights reserved.)

The tumor suppressor p53 prevents cancer development by regulating dozens of target genes with diverse biological functions. Although numerous p53 target genes have been identified to date, the dynamics and function of the regulatory network centered on p53 have not yet been fully elucidated. We herein identified inhibitor of DNA-binding/differentiation-3 (ID3) as a direct p53 target gene. p53 bound the distal promoter of ID3 and positively regulated its transcription. ID3 expression was significantly decreased in clinical lung cancer tissues, and was closely associated with overall survival outcomes in these patients. Functionally, ID3 deficiency promoted the metastatic ability of lung cancer cells through its effects on the transcriptional regulation of CDH1. Furthermore, the ectopic expression of ID3 in p53-knockdown cells restored E-cadherin expression. Collectively, the present results demonstrate that ID3 plays a tumor-suppressive role as a downstream effector of p53 and impedes lung cancer cell metastasis by regulating E-cadherin expression., 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 Elsevier Inc. All rights reserved.)

The skin is a protective interface between the internal organs and environment and functions not only as a physical barrier but also as an immune organ. However, the immune system in the skin is not fully understood. A member of the thermo-sensitive transient receptor potential (TRP) channel family, TRPM4, which acts as a regulatory receptor in immune cells, was recently reported to be expressed in human skin and keratinocytes. However, the function of TRPM4 in immune responses in keratinocytes has not been investigated. In this study, we found that treatment with BTP2, a known TRPM4 agonist, reduced cytokine production induced by tumor necrosis factor (TNF) α in normal human epidermal keratinocytes and in immortalized human epidermal keratinocytes (HaCaT cells). This cytokine-reducing effect was not observed in TRPM4-deficient HaCaT cells, indicating that TRPM4 contributed to the control of cytokine production in keratinocytes. Furthermore, we identified aluminum potassium sulfate, as a new TRPM4 activating agent. Aluminum potassium sulfate reduced Ca 2+ influx by store-operated Ca 2+ entry in human TRPM4-expressing HEK293T cells. We further confirmed that aluminum potassium sulfate evoked TRPM4-mediated currents, showing direct evidence for TRPM4 activation. Moreover, treatment with aluminum potassium sulfate reduced cytokine expression induced by TNFα in HaCaT cells. Taken together, our data suggested that TRPM4 may serve as a new target for the treatment of skin inflammatory reactions by suppressing the cytokine production in keratinocytes, and aluminum potassium sulfate is a useful ingredient to prevent undesirable skin inflammation through TRPM4 activation., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Mitochondria play a substantial role in cytosolic Ca 2+ buffering and energy metabolism. We recently demonstrated that mitofusin 2 (Mfn2) regulated Ca 2+ signaling by tethering mitochondria and sarcoplasmic reticulum (SR), and thus, facilitated mitochondrial function and the proliferation of vascular smooth muscle cells (VSMCs). However, the physiological role of mitofusin 1 (Mfn1) on Ca 2+ signaling and mitochondrial function remains unclear. Herein, the roles of Mfn1 and Mfn2 in mitochondrial function underlying Ca 2+ signaling, ATP production, and cell proliferation were examined in rat aortic smooth muscle A10 cells. Following an arginine vasopressin-induced increase in cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ), Mfn2 siRNA (siMfn2) reduced cytosolic Ca 2+ removal and mitochondrial Ca 2+ uptake. However, Mfn1 siRNA (siMfn1) attenuated mitochondrial Ca 2+ uptake, facilitated Ca 2+ removal from mitochondria, and resulted in increased [Ca 2+ ] cyt , which was mediated by the downregulation of mitochondrial Ca 2+ uniporter (MCU) expression and the upregulation of mitochondrial Na + /Ca 2+ exchanger (NCLX) expression. Furthermore, siMfn1 increased the mitochondrial membrane potential, ATP production by adenine nucleotide translocase (ANT), and cell proliferation, whereas siMfn2 exhibited the opposite responses. In conclusion, Mfn1 modulates the expressions of MCU, NCLX, and ANT, and Mfn2 tethers mitochondria to SR, which demonstrates their different mitochondrial functions for Ca 2+ signaling, ATP production, and the proliferation of VSMCs., 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.)

The Chikungunya virus (CHIKV), an enveloped RNA virus that has been identified in over 40 countries and is considered a growing threat to public health worldwide. However, there is no preventive vaccine or specific therapeutic drug for CHIKV infection. To identify a new inhibitor against CHIKV infection, this study constructed a subgenomic RNA replicon expressing the secretory Gaussia luciferase (Gluc) based on the CHIKV SL11131 strain. Transfection of in vitro-transcribed replicon RNA to BHK-21 cells revealed that Gluc activity in culture supernatants was correlated with the intracellular replication of the replicon genome. Through a chemical compound library screen using the Gluc reporter CHIKV replicon, we identified several compounds that suppressed CHIKV infection in Vero cells. Among the hits identified, CP-154,526, a non-peptide antagonist of the corticotropin-releasing factor receptor type-1 (CRF-R1), showed the strongest anti-CHIKV activity and inhibited CHIKV infection in Huh-7 cells. Interestingly, other CRF-R1 antagonists, R121919 and NGD 98-2, also exhibited inhibitory effects on CHIKV infection. Time-of-drug addition and virus entry assays indicated that CP-154,526 suppressed a post-entry step of infection, suggesting that CRF-R1 antagonists acted on a target in the intracellular replication process of CHIKV. Therefore, the Gluc reporter replicon system established in this study would greatly facilitate the development of antiviral drugs against CHIKV infection., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Youichi Suzuki reports financial support was provided by Japan Agency for Medical Research and Development., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca 2+ concentration following the activation of nicotinic acetylcholine receptors in parasympathetic systems. We previously reported that pineal Ca 2+ oscillations were regulated by voltage-dependent Ca 2+ channels and large-conductance Ca 2+ -activated K + (BK Ca ) channels, which inhibited melatonin production. In the present study, the contribution of small- and intermediate-conductance Ca 2+ -activated K + (SK Ca and IK Ca ) channels to the regulation of spontaneous Ca 2+ oscillations was examined in rat pinealocytes. The amplitude and frequency of spontaneous Ca 2+ oscillations were increased by a SK Ca channel blocker (100 nM apamin), but not by an IK Ca channel blocker (1 μM TRAM-34). On the other hand, they were decreased by a SK Ca channel opener (100 μM DCEBIO), but not by an IK Ca channel opener (1 μM DCEBIO). Expression analyses using quantitative real-time PCR, immunocytochemical staining, and Western blotting revealed that the SK Ca 2 channel subtype was abundantly expressed in rat pinealocytes. Moreover, the enhanced amplitude of Ca 2+ oscillations in the presence of apamin was further increased by a BK Ca channel blocker (1 μM paxilline). These results suggest that the activity of SK Ca 2 channels regulates cytosolic Ca 2+ signaling and melatonin production during parasympathetic activation in pineal glands., 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.)

Simple, highly sensitive detection technologies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for the effective implementation of public health policies. We used the systematic evolution of ligands by exponential enrichment with a modified DNA library, including a base-appended base (uracil with a guanine base at its fifth position), to create an aptamer with a high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. The aptamer had a dissociation constant of 1.2 and < 1 nM for the RBD and spike trimer, respectively. Furthermore, enzyme-linked aptamer assays confirmed that the aptamer binds to isolated authentic SARS-CoV-2 wild-type and B.1.617.2 (delta variant). The binding signal was larger that of commercially available anti-SARS-CoV-2 RBD antibody. Thus, this aptamer as a sensing element will enable the highly sensitive detection of SARS-CoV-2., Competing Interests: Declaration of competing interest This work was partly supported by grants for the Japan Program for Infectious Diseases Research and Infrastructure (JP21wm0225003 and JP21fk0108104j) from Japan Agency for Medical Research and Development (AMED) and fund from NEC Solution Innovators, Ltd., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In pulmonary arterial smooth muscle cells (PASMCs), an increase in the cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) is involved in many physiological processes such as cell contraction and proliferation. However, chronic [Ca 2+ ] cyt increases cause pulmonary vasoconstriction and vascular remodeling, resulting in pulmonary arterial hypertension (PAH). Therefore, [Ca 2+ ] cyt signaling plays a substantial role in the regulation of physiological and pathological functions in PASMCs. In the present study, the effects of SKF96365 on [Ca 2+ ] cyt were examined in PASMCs from normal subjects and idiopathic pulmonary arterial hypertension (IPAH) patients. SKF96365 is widely used as a blocker of non-selective cation channels. SKF96365 did not affect the resting [Ca 2+ ] cyt in normal-PASMCs. However, SKF96365 increased [Ca 2+ ] cyt in IPAH-PASMCs in a concentration-dependent manner (EC 50  = 18 μM). The expression of Ca 2+ -sensing receptors (CaSRs) was higher in IPAH-PASMCs than in normal-PASMCs. The SKF96365-induced [Ca 2+ ] cyt increase was inhibited by CaSR antagonists, NPS2143 and Calhex 231. The CaSR-mediated [Ca 2+ ] cyt increase was facilitated by SKF96365 and the activation was blocked by NPS2143 or Calhex 231. In addition, the SKF96365-induced [Ca 2+ ] cyt increase was reduced by siRNA knockdown of CaSRs. Taken together, SKF96365 activates CaSRs in IPAH-PASMCs and promotes [Ca 2+ ] cyt signaling., 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.)

The efficacy of n-3 polyunsaturated fatty acids (PUFAs) in improving outcomes in a renal ischemia-reperfusion injury (IRI) model has previously been reported. However, the underlying mechanisms remain poorly understood and few reports demonstrate how dietary n-3 PUFAs influence the composition of membrane phospholipids in the kidney. Additionally, it has not been elucidated whether perilla oil (PO), which is mainly composed of the n-3 alpha-linolenic acid, mitigates renal IRI. In this study, we investigated the effect of dietary n-3 PUFAs (PO), compared with an n-6 PUFA-rich soybean oil (SO) diet, on IRI-induced renal insufficiency in a rat model. Levels of membrane phospholipids containing n-3 PUFAs were higher in the kidney of PO-rich diet-fed rats than the SO-rich diet-fed rats. Levels of blood urea nitrogen and serum creatinine were significantly higher in the ischemia-reperfusion group than the sham group under both dietary conditions. However, no significant differences were observed in blood urea nitrogen, serum creatinine, or histological damage between PO-rich diet-fed rats and SO-rich diet-fed rats. In the kidney of PO-rich diet-fed rats, levels of arachidonic acid and arachidonic acid-derived pro-inflammatory lipid mediators were lower than SO-rich diet-fed rats. Eicosapentaenoic acid and eicosapentaenoic acid-derived lipid mediators were significantly higher in the kidney of PO-rich than SO-rich diet-fed rats. These results suggest that dietary n-3 PUFAs alter the fatty acid composition of membrane phospholipids and lipid mediators in the kidney; however, this does not attenuate renal insufficiency or histological damage in a renal IRI model., Competing Interests: Declaration of competing interest The authors declare no conflicts of interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The controlled release of medications using nanoparticle-based drug delivery carriers is a promising method to increase the efficacy of pharmacotherapy and gene therapy. One critical issue that needs to be overcome with these drug delivery carriers is their target specificity. We focused on the cell tropism of a virus to solve this issue, i.e., we attempted to apply hepatitis B virus-like particle (HBV-VLP) as a novel hepatic cell-selective carrier for medication and DNA. To prepare HBV-VLP, 293T cells were transfected with expression plasmids carrying HBV envelope surface proteins, large envelope protein (L), and small envelope protein (S). After 72 h post-transfection, VLP-containing culture supernatants were harvested, and HBV-VLP was labeled with red fluorescent dye (DiI) and was purified by sucrose gradient ultracentrifugation. An anticancer drugs (geldanamycin or doxorubicin) and GFP-expressing plasmid DNA were incorporated into HBV-VLP, and medication- and plasmid DNA-loaded VLPs were prepared. We evaluated their delivery capabilities into hepatocytes, other organ-derived cells, and hepatocytes expressing sodium taurocholate cotransporting polypeptide (NTCP), which functions as the cellular receptor for HBV by binding to HBV L protein. HBV-VLP selectively delivered both anticancer drugs and plasmid DNA not into HepG2, Huh7, and other organ cells but into HepG2 cells expressing NTCP. In summary, we developed a novel delivery nanocarrier using HBV-VLP that could be used as a hepatitis selective drug- and DNA-carrier for cancer treatment and gene therapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Air spaces and material surfaces in a pathogen-contaminated environment can often be a source of infection to humans, and disinfection has become a common intervention focused on reducing the contamination levels. In this study, we examined the efficacy of SAIW, a unique electrolyzed water with chlorine-free, high pH, high concentration of dissolved hydrogen, and low oxygen reduction potential, for the inactivation of several viruses and bacteria. Infectivity assays revealed that initial viral titers of enveloped and non-enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, herpes simplex virus type 1, human coronavirus, feline calicivirus, and canine parvovirus, were reduced by 2.9- to 5.5-log10 within 30 s of SAIW exposure. Similarly, the culturability of three Gram-negative bacteria (Escherichia coli, Salmonella, and Legionella) dropped down by 1.9- to 4.9-log10 within 30 s of SAIW treatment. Mechanistically, treatment with SAIW was found to significantly decrease the binding and subsequent entry efficiencies of SARS-CoV-2 on Vero cells. Finally, we showed that this chlorine-free electrolytic ion water had no acute inhalation toxicity in mice, demonstrating that SAIW holds promise for a safer antiviral and antibacterial disinfectant., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Articular chondrocytes are exposed to dynamic osmotic environments during normal joint loading, and thus, require effective volume regulatory mechanisms. A regulatory volume decrease (RVD) is one of the mechanisms for protecting chondrocytes from swelling and damage. Swelling-activated Cl - currents (I Cl,swell ) are responsible for the RVD, but the molecular identity in chondrocytes is largely unknown. In this study, we reveal that in human OUMS-27 chondrocytes, I Cl,swell can be elicited by hypoosmotic stimulation (180 mOsm) and be inhibited by classical Cl - channel blockers, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and be attenuated by siRNA knockdown of ClC-3. Our molecular analyses revealed that ClC-3A is expressed as a major splice variant in both human articular chondrocytes and OUMS-27 cells. The onset and early phase of RVD following hypoosmotic stress in OUMS-27 cells were affected by DIDS and ClC-3 knockdown. Hypoosmotic stimulation caused Ca 2+ influx and subsequent release of prostaglandin E 2 (PGE 2 ) in OUMS-27 cells, and both of these responses were reduced by DIDS and ClC-3 knockdown. These results strongly suggest that ClC-3 is responsible for I Cl,swell and RVD under the hypoosmotic environments. It is likely that ClC-3 is associated with the pathogenesis of cartilage degenerative diseases including osteoarthritis via PGE 2 release., 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 development of molecular targeted therapy has improved clinical outcomes in patients with life-threatening advanced lung cancers with driver oncogenes. However, selective treatment for KRAS-mutant lung cancer remains underdeveloped. We have successfully characterised specific molecular and pathological features of KRAS-mutant lung cancer utilising newly developed cell line models that can elucidate the differences in driver oncogenes among tissues with identical genetic backgrounds. Among these KRAS-mutation-associated specific features, we focused on the IGF2-IGF1R pathway, which has been implicated in the drug resistance mechanisms to AMG 510, a recently developed selective inhibitor of KRAS G12C lung cancer. Experimental data derived from our cell line model can be used as a tool for clinical treatment strategy development through understanding of the biology of lung cancer. The model developed in this paper may help understand the mechanism of anticancer drug resistance in KRAS-mutated lung cancer and help develop new targeted therapies to treat patients with this disease., 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.)

cDNA expression cloning has been shown to be a powerful approach in the search for cellular factors that control virus replication. In this study, cDNA library screening using a pool of cDNA derived from interferon-treated human cells was combined with the MinION sequencer to identify cellular genes inhibiting Chikungunya virus (CHIKV) replication. Challenge infection of CHIKV to Vero cells transduced with the cDNA library produced virus-resistant cells. Then, the MinION sequence of cDNAs extracted from the surviving cells revealed that the open reading frames of TOM7, S100A16, N-terminally truncated form of ECI1 (ECI1ΔN59), and RPL29 were inserted in many of the cells. Importantly, the transient expression of TOM7, S100A16, and ECI1ΔN59 was found to inhibit the replication of CHIKV in Huh7 cells, indicating that these cellular factors were potentially anti-CHIKV molecules. Thus, our study demonstrated that cDNA expression cloning combined with the MinION sequencer allowed a rapid and comprehensive detection of cellular inhibitors against CHIKV., Competing Interests: Declaration of competing interest The authors have no competing interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Multidrug-resistant bacteria are a growing issue worldwide. This study developed a convenient and effective method to downregulate the expression of a specific gene to produce a novel antimicrobial tool using a small (140 nucleotide) RNA with a 24-nucleotide antisense (as) region from an arabinose-inducible expression phagemid vector in Escherichia coli. Knockdown effects of rpoS encoding RNA polymerase sigma factor were observed using this inducible artificial asRNA approach. asRNAs targeting several essential E. coli genes produced significant growth defects, especially when targeted to acpP and ribosomal protein coding genes rplN, rplL, and rpsM. Growth inhibited phenotypes were facilitated in hfq - conditions. Phage lysates were prepared from cells harboring phagemids as a lethal-agent delivery tool. Targeting the rpsM gene by phagemid-derived M13 phage infection of E. coli containing a carbapenem-producing F-plasmid and multidrug-resistant Klebsiella pneumoniae containing an F-plasmid resulted in the death of over 99.99% of infected bacteria. This study provides a possible strategy for treating bacterial infection and can be applied to any F-pilus producing bacterial species., Competing Interests: Declaration of competing interest None declared., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pancreatic neuroendocrine tumors (pNETs) occur due to the abnormal growth of pancreatic islet cells and predominantly develop in the duodenal-pancreatic region. Somatostatinoma is one of the pNETs associated with tumors of pancreatic δ cells, which produce and secrete somatostatin. Limited information is currently available on the pathogenic mechanisms of somatostatinoma. The large-conductance Ca 2+ -activated K + (BK Ca ) channel is expressed in several types of cancer cells and regulates cell proliferation, migration, invasion, and metastasis. In the present study, the functional expression of the BK Ca channel was examined in a human somatostatinoma QGP-1 cell line. In QGP-1 cells, outward currents were elicited by membrane depolarization at pCa 6.5 (300 nM) in the pipette solution and inhibited by the specific BK Ca channel blocker, paxilline. Paxilline-sensitive currents were detected, even at pCa 8.0 (10 nM) in the pipette solution, in QGP-1 cells. In addition to the α and β2-4 subunits of the BK Ca channel, the novel regulatory γ1 subunit (BK Ca γ1) was co-localized with the α subunit in QGP-1 cells. Paxilline-sensitive currents at pCa 8.0 in the pipette solution were reduced by the siRNA knockdown of BK Ca γ1. Store-operated Ca 2+ entry was smaller in BK Ca γ1 siRNA-treated QGP-1 cells. The proliferation of QGP-1 cells was attenuated by paxilline or the siRNA knockdown of BK Ca γ1. These results strongly suggest that BK Ca γ1 facilitates the proliferation of human somatostatinoma cells. Therefore, BK Ca γ1 may be a novel therapeutic target for somatostatinoma., 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.)

Recent research has revealed that glioblastoma (GBM) avoids the immune system via strong expression of indoleamine 2,3-dioxygenase 1 (IDO1). IDO1, an enzyme involved in tryptophan metabolism, is now proposed as a new target in GBM treatment, since several reports have demonstrated that IDO1 expression is related to GBM malignancy. On the other hand, it is well known that glioma stem cells (GSCs) are strongly related to the malignancy of GBM. However, there is as yet no report evaluating the relationship between GSCs and IDO1. We therefore examined the expression levels of IDO1 in GSCs in order to identify a new therapeutic target for GBM based on the immune systems of GSCs. In the present study, we employed human GBM cell lines (U-138MG, U-251MG) and patient-derived GSC model cell lines (0125-GSC, 0222-GSC). GSC model cell lines Rev-U-138MG and Rev-U-251MG were established by culturing U-138MG and U-251MG in serum-free media, while differentiated GBM model cell lines 0125-DGC and 0222-DGC were established by culturing 0125-GSC and 0222-GSC in serum-containing media. The expression levels of stem cell markers (Nanog, Nestin, Oct4 and Sox2) and IDO1 protein and mRNA were determined. Rev-U-138MG and Rev-U-251MG formed spheres and their expression levels of stem cell markers were increased as compared to U-138MG and U-251MG. On the other hand, 0125-DGC and 0222-DGC suffered breakdown of sphere formation, despite the original 0125-GSC and 0222-GSC forming spheres, and their expression levels of the markers were decreased. IDO1 expressions were strongly recognized in Rev-U-138MG, Rev-U-251MG, 0125-GSC and 0222-GSC as compared to U-138MG, U-251MG, 0125-DGC and 0222-DGC. These findings demonstrate that GSCs exhibit treatment resistance with immunosuppression via high expression levels of IDO1, and could represent a novel target for GBM treatment., Competing Interests: Declaration of competing interest We have no potential conflicts of interest to disclosure., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) and play an essential role in the regulation of its functions. Oxidative stress accumulates excessive reactive oxygen species (ROS) and facilitates the death of BCECs, leading to a dysfunctional BBB. However, the mechanisms underlying the death of BCECs under oxidative stress remain unclear. In the present study, the effects of oxidative stress on cell viability, ROS production, intracellular Ca 2+ concentration, and protein expression were examined using a cell line derived from bovine BCECs, t-BBEC117. When t-BBEC117 cells were exposed to oxidative stress induced by hydrogen peroxide (H 2 O 2 , 10-100 μM), cell growth was inhibited in a dose-dependent manner. Oxidative stress by 30 μM H 2 O 2 increased the production of ROS and its effects were blocked by the ROS scavenger, 10 mM N-acetyl-l-cysteine (NAC). In addition, oxidative stress reduced store-operated Ca 2+ entry (SOCE) and this decrease was recovered by NAC or the Orai channel activator, 5 μM 2-aminoethyl diphenylborinate (2-APB). The siRNA knockdown of Orai1 revealed that Orai1 was mainly responsible for SOCE channels and its activity was decreased by oxidative stress. However, the protein expression of Orai1 and STIM1 was not affected by oxidative stress. Oxidative stress-induced cell death was rescued by 2-APB, NAC, or the STIM-Orai activating region. In conclusion, oxidative stress reduces Orai1-mediated SOCE and, thus, facilitates the death of BCECs., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) regulate membrane trafficking and actin cytoskeleton. The molecular mechanism of how Arf GAPs regulate actin cytoskeleton remains to be elucidated. We identified AGAP1, a subtype of Arf GAP, as a binding protein of FilGAP, a Rac-specific GAP, in mammalian cells. AGAP1 binds to C-terminus of FilGAP whereas FilGAP binds to N-terminus of AGAP1 containing GLD domain. FilGAP co-localized with AGAP1 at intracellular vesicles and targeting of FilGAP at the vesicles requires its interaction with AGAP1. Consistently, depletion of endogenous AGAP1 induced the accumulation of endogenous FilGAP into paxillin-positive focal adhesions and actin cytoskeletal structures. Knockdown of endogenous AGAP1 suppressed cell spreading on collagen and the suppression was released by depletion of endogenous FilGAP. Moreover, depletion of AGAP1 in MDA-MB-231 cells promoted cell invasion in extracellular matrices and depletion of FilGAP blocked the invasion. Taken together, the present study suggests that AGAP1 may regulate subcellular localization of FilGAP and control cell migration and invasion through interaction with FilGAP., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Jiadifenolide has been reported to have neurotrophin-like activity in primary rat cortical neurons, and also possesses neurotrophic effects in neuronal precursor cells derived from human induced pluripotent stem cells (hiPSCs), as we have previously reported. However, the molecular mechanisms by which jiadifenolide exerts its neurotrophic effects in rat and human neurons are unknown. Thus, we aimed to investigate the molecular mechanisms and pathways by which jiadifenolide promotes neurotrophic effects. Here, we found that jiadifenolide activated cellular communication network factor (CCN) signaling pathways by up-regulating mRNA level expression of CCN genes in human neuronal cells. We also found that CCN2 (also known as connective tissue growth factor, CTGF) protein promotes neurotrophic effects through activation of the p44/42 mitogen-activated protein kinase signaling pathway. This is the first discovery which links neurotrophic activity with CCN signaling., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The mechanisms underlying neuronal cell death induced by the rise of intracellular Na + concentration ([Na + ] i ) following abnormal hyperexcitation are not fully understood. Previously, we have established a recombinant cell line derived from HEK293 cells, in which the occurrence of a sustained action potential (AP) induces cell death. Mutated voltage-gated Nav1.5 channel (IFM/QQQ) lacking inactivation, and inward rectifying K + channel (Kir2.1) were co-expressed in HEK293 cells (IFM/QQQ + Kir2.1 cells). In this cell line, the rise of [Na + ] i due to a sustained AP reached maximum within 15 min without concomitant [Ca 2+ ] i rise, and then elicited significant externalization of phosphatidylserine and enhancement of caspase activity. Marked decreases in mitochondrial transmembrane potential and ATP concentration were also detected. The significant cell death occurred at 3 h from the AP onset and reached a steady state at around 12 h. The significant release of lactate dehydrogenase was not detected even after 12 h. These results provide novel findings that Na + accumulation or/and possibly concomitant K + loss elicits apoptosis presumably due to the mitochondrial dysfunction, which is attributable to neither the membrane depolarization nor [Ca 2+ ] i change. This apoptotic mechanism may be involved, at least in part, in neuronal cell death under pathophysiological settings with abnormal hyperexcitability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The interaction of high mobility group box 1 (HMGB1), which is secreted from immune and dying cells during cellular infection and injury, and receptor for advanced glycation end-products (RAGE) appears to be critical for acute and chronic inflammatory disorders. Here we designed a unique cyclic β-hairpin peptide (Pepb2), which mimics the predicted RAGE-binding domain of HMGB1. Pepb2 competitively inhibited HMGB1/RAGE interaction. We then identified papaverine as a Pepb2 mimetic by in silico 3D-structural similarity screening from the DrugBank library. Papaverine was found to directly inhibit HMGB1/RAGE interaction. It also suppressed the HMGB1-mediated production of pro-inflammatory cytokines, IL-6 and TNF-α, in mouse macrophage-like RAW264.7 cells and bone marrow-derived macrophages. In addition, papaverine attenuated mortality in cecal ligation puncture-induced sepsis model mice. Taken together, these findings indicate that papaverine could become a useful therapeutic against HMGB1/RAGE-mediated sepsis and other inflammatory diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Conversion of intracellular Ca 2+ signals to electrical activity results in multiple and differing physiological impacts depending on cell types. In some organs such as gastrointestinal and urinary systems, spontaneous Ca 2+ oscillation in pacermaker cells can function essentially as a Ca 2+ clock mechanism, which has been originally found in pacemaking in sinoatrial node cell of the heart. The conversion of discrete Ca 2+ clock events to spontaneous electrical activity is an essential step for the initiation and propagation of pacemaker activity through the multicellular organs resulting in synchronized physiological functions. Here, a model of intracellular signal transduction from a Ca 2+ oscillation to initiation of electrical slow waves and their propagation were reconstituted in HEK293 cells. This was accomplished based on ryanodine receptor (RyR) type 3, Ca 2+ -activated ion channels, i.e. small conductance Ca 2+ -activated K + channel (SK2) or Ca 2+ -activated Cl - channel (TMEM16A), and connexin43 being heterologously co-expressed. The propagation of electrical waves was abolished or substantially reduced by treatment with selective blockers of the expressed channels and 18β-glycyrrhetinic acid, a gap junction inhibitor, respectively. Thus, we demonstrated that the conversion of Ca 2+ oscillation to electrical signals with cell to cell propagation can be reconstituted as a model of Ca 2+ clock pacemaker activity by combinational expression of critical elements in heterologous expression system., (Copyright © 2019 Elsevier Inc. All rights reserved.)

White adipose tissue (WAT) is not only the main tissue for energy storage but also an endocrine organ that secretes adipokines. Obesity is the most common metabolic disorder and is related to alterations in WAT characteristics, such as chronic inflammation and increasing oxidative stress. WW domain containing E3 ubiquitin protein ligase 1 (WWP1) is a HECT-type ubiquitin E3 ligase that has been implicated in various pathologies. In the present study, we found that WWP1 was upregulated in obese WAT in a p53-dependent manner. To investigate the functions of WWP1 in adipocytes, a proteome analysis of WWP1 overexpression (OE) and knockdown (KD) 3T3-L1 cells was performed. This analysis showed a positive correlation between WWP1 expression and the abundance of several antioxidative proteins. Thus, we measured reactive oxygen species (ROS) in WWP1 OE and KD cells. Consistent with the proteome results, WWP1 OE reduced ROS levels, whereas KD increased them. These findings indicate that WWP1 is an obesity-inducible E3 ubiquitin ligase that can protect against obesity-associated oxidative stress in WAT., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic obstructive pulmonary disease (COPD) is predominantly a cigarette smoke (CS)-triggered disease with features of chronic systemic inflammation. Oxidants derived from CS can induce DNA damage and stress-induced premature cellular senescence in the respiratory system, which play significant roles in COPD. Therefore, antioxidants should provide benefits for the treatment of COPD; however, their therapeutic potential remains limited owing to the complexity of this disease. Recently, molecular hydrogen (H 2 ) has been reported as a preventive and therapeutic antioxidant. Molecular H 2 can selectively reduce hydroxyl radical accumulation with no known side effects, showing potential applications in managing oxidative stress, inflammation, apoptosis, and lipid metabolism. However, there have been no reports on the efficacy of molecular H 2 in COPD patients. In the present study, we used a mouse model of COPD to investigate whether CS-induced histological damage in the lungs could be attenuated by administration of molecular H 2 . We administered H 2 -rich pure water to senescence marker protein 30 knockout (SMP30-KO) mice exposed to CS for 8 weeks. Administration of H 2 -rich water attenuated the CS-induced lung damage in the SMP30-KO mice and reduced the mean linear intercept and destructive index of the lungs. Moreover, H 2 -rich water significantly restored the static lung compliance in the CS-exposed mice compared with that in the CS-exposed H 2 -untreated mice. Moreover, treatment with H 2 -rich water decreased the levels of oxidative DNA damage markers such as phosphorylated histone H2AX and 8-hydroxy-2'-deoxyguanosine, and senescence markers such as cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase inhibitor 1, and β-galactosidase in the CS-exposed mice. These results demonstrated that H 2 -rich pure water attenuated CS-induced emphysema in SMP30-KO mice by reducing CS-induced oxidative DNA damage and premature cell senescence in the lungs. Our study suggests that administration of molecular H 2 may be a novel preventive and therapeutic strategy for COPD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An ideal antibiofilm strategy is to control both in the quality and quantity of biofilm while maintaining the benefits derived from resident microflora. Vizantin, a recently developed immunostimulating compound, has also been found to have antibiofilm property. This study evaluated the influence on biofilm formation of Streptococcus mutans in the presence of sulfated vizantin and biofilm development following bacterial adhesion on a hydroxyapatite disc coated with sulfated vizantin. Supplementation with sulfated vizantin up to 50 μM did not affect either bacterial growth or biofilm formation, whereas 50 μM sulfated vizantin caused the biofilm to readily detach from the surface. Sulfated vizantin at the concentration of 50 μM upregulated the expression of the gtfB and gtfC genes, but downregulated the expression of the gtfD gene, suggesting altered architecture in the biofilm. Biofilm development on the surface coated with sulfated vizantin was inhibited depending on the concentration, suggesting prevention from bacterial adhesion. Among eight genes related to bacterial adherence in S. mutans, expression of gtfB and gtfC was significantly upregulated, whereas the expression of gtfD, GbpA and GbpC was downregulated according to the concentration of vizantin, especially with 50 μM vizantin by 0.8-, 0.4-, and 0.4-fold, respectively. These findings suggest that sulfated vizantin may cause structural degradation as a result of changing gene regulation related to bacterial adhesion and glucan production of S. mutans., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ultraviolet (UV) radiation in sunlight can result in DNA damage and an inflammatory reaction of the skin commonly known as sunburn, which in turn can lead to cutaneous tissue disorders. However, little has been known about how UV-induced DNA damage mediates the release of inflammatory mediators from keratinocytes. Here, we show that UVB radiation intensity-dependently increases NLRP3 gene expression and IL-1β production in human keratinocytes. Knockdown of NLRP3 with siRNA suppresses UVB-induced production of not only IL-1β, but also other inflammatory mediators, including IL-1α, IL-6, TNF-α, and PGE2. In addition, inhibition of DNA damage repair by knockdown of XPA, which is a major component of the nucleotide excision repair system, causes accumulation of cyclobutane pyrimidine dimer (CPD) and activation of NLRP3 inflammasome. In vivo immunofluorescence analysis confirmed that NLRP3 expression is also elevated in UV-irradiated human epidermis. Overall, our findings indicate that UVB-induced DNA damage initiates NLRP3 inflammasome activation, leading to release of various inflammatory mediators from human keratinocytes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4-5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba(2+)-sensitive inward rectifier K(+) current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca(2+) imaging study revealed that the hypoxic stress enhanced store-operated Ca(2+) (SOC) entry, which was significantly reduced in the presence of 100 μM Ba(2+). On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba(2+). We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca(2+) entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The crotonylation of histones is an important post-translational modification, and epigenetically functions in the regulation of genomic DNA activity. The histone modifications in the structured "histone-fold" domains are considered to have an especially important impact on the nucleosome structure and dynamics. In the present study, we reconstituted the human nucleosome containing histone H3.2 crotonylated at the Lys122 residue, and determined its crystal structure at 2.56 Å resolution. We found that the crotonylation of the H3 Lys122 residue does not affect the overall nucleosome structure, but locally impedes the formation of the water-mediated hydrogen bond with the DNA backbone. Consistently, thermal stability assays revealed that the H3 Lys122 crotonylation, as well as the H3 Lys122 acetylation, clearly reduced the histone-DNA association., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Chemerin is a chemoattractant cytokine (chemokine) produced by adipocytes and hepatocytes; it regulates insulin sensitivity and adipocyte differentiation. The objective of this study was to investigate the effect of chemerin on the expression of genes related to lactogenesis and the regulators of chemerin signaling in a bovine mammary epithelial cell line (MAC-T). Two types of chemerin receptors, chemokine like-receptor 1 (CMKLR1) and chemokine (C-C motif) receptor-like 2 (CCRL2), were detected in cultured MAC-T cells, whereas chemerin was not detected. G protein-coupled receptor 1 (GPR1), another receptor of chemerin, was undetectable in MAC-T cells. Chemerin upregulated transcript expression of CMKLR1, CCRL2, and genes associated with fatty acid synthesis, glucose uptake, insulin signaling, and casein synthesis in MAC-T cells. Lactogenic hormones (insulin, growth hormone, and prolactin) downregulated the expression of CMKLR1 in MAC-T cells. Adiponectin suppressed CMKLR1 expression. TNF-α suppressed CMKLR1, but induced CCRL2 expression. These data suggest chemerin is a novel regulator of lactogenesis via its own receptor in bovine mammary epithelial cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Store-operated Ca(2+) entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca(2+) influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this study, we investigated cellular uptake and metabolism of phosphatidylcholine hydroperoxide (PCOOH) in human hepatoma HepG2 cells by high performance liquid chromatography-tandem mass spectrometry, and then evaluated whether PCOOH or its metabolites cause pathophysiological effects such as cytotoxicity and apoptosis. Although we found that most PCOOH was reduced to PC hydroxide in HepG2 cells, the remaining PCOOH caused cytotoxic effects that may be mediated through an unusual apoptosis pathway. These results will enhance our fundamental understanding of how PCOOH, which is present in oxidized low density lipoproteins, is involved in the development of atherosclerosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)