Dihydrofolate reductase (DHFR) plays a key role in folate metabolism and is a target molecule of methotrexate. An increase in the cellular expression level of DHFR is one of the mechanisms of tumor ...resistance to methotrexate. The present study investigated the possibility that adenosine-to-inosine RNA editing, which causes nucleotide conversion by adenosine deaminase acting on RNA (ADAR) enzymes, might modulate DHFR expression. In human breast adenocarcinoma-derived MCF-7 cells, 26 RNA editing sites were identified in the 3′-UTR of DHFR. Knockdown of ADAR1 decreased the RNA editing levels of DHFR and resulted in a decrease in the DHFR mRNA and protein levels, indicating that ADAR1 up-regulates DHFR expression. Using a computational analysis, miR-25-3p and miR-125a-3p were predicted to bind to the non-edited 3′-UTR of DHFR but not to the edited sequence. The decrease in DHFR expression by the knockdown of ADAR1 was restored by transfection of antisense oligonucleotides for these miRNAs, suggesting that RNA editing mediated up-regulation of DHFR requires the function of these miRNAs. Interestingly, we observed that the knockdown of ADAR1 decreased cell viability and increased the sensitivity of MCF-7 cells to methotrexate. ADAR1 expression levels and the RNA editing levels in the 3′-UTR of DHFR in breast cancer tissues were higher than those in adjacent normal tissues. Collectively, the present study demonstrated that ADAR1 positively regulates the expression of DHFR by editing the miR-25-3p and miR-125a-3p binding sites in the 3′-UTR of DHFR, enhancing cellular proliferation and resistance to methotrexate.
Bisphosphonate-related osteonecrosis of the jaw (BRONJ) worsens oral health-related quality of life. Most BRONJ occurs in multiple myeloma or metastatic breast cancer patients treated with ...bisphosphonate/chemotherapeutic combination therapies. Cyclophosphamide (CY), an alkylating chemotherapeutic drug, is used to treat multiple myeloma, although its use has been recently reduced. The aim of this study was to clarify the effects of CY dose on tooth extraction socket healing when CY is used with or without bisphosphonate in mice. Low-dose CY (50 mg/kg; CY-L), moderate-dose CY (100 mg/kg; CY-M), high-dose CY (150 mg/kg; CY-H), and bisphosphonate Zometa (ZA): 0.05 mg/kg were administered for 7 weeks. Each dose of CY and ZA in combination was also administered for 7 weeks. Both maxillary first molars were extracted at 3 weeks after the initiation of drug administration. Euthanasia was performed at 4 weeks post-extraction. Gross wound healing, microcomputed tomography analysis, histomorphometry, and immunohistochemistry were used to quantitatively evaluate osseous and soft tissue wound healing of tooth extraction sockets. ZA monotherapy induced no BRONJ-like lesions in mice. CY monotherapy rarely induced open wounds, though delayed osseous wound healing occurred in a CY dose-dependent manner. In contrast, CY/ZA combination therapy prevalently induced BRONJ-like lesions with compromised osseous and soft tissue healing in a CY dose-dependent manner. Interestingly, anti-angiogenesis was noted regardless of CY dose and ZA administration, even though only CY-M/ZA and CY-H/ZA combination therapies induced BRONJ-like lesions. Our findings suggest that high-dose CY may be associated with the development of BRONJ following tooth extraction only when CY is used together with ZA. In addition to anti-angiogenesis, other factors may contribute to the pathoetiology of BRONJ.
•Impaired tooth socket healing rarely occurred with cyclophosphamide (CY) monotherapy.•Bisphosphonate (ZA) monotherapy did not induce BRONJ-like lesions in mice.•High-dose CY in combination with ZA and tooth extraction induced BRONJ-like lesions in mice.•BRONJ-like lesions prevalently increased in a CY dose-dependent manner.
The current state of portable/wearable electromyographic (EMG) devices for assessment of bruxism was reviewed.
A search of full-text articles relevant to portable/wearable EMG devices capable of ...being used at home was performed. The data source used was MEDLINE via PubMed from January 1970 to July 2019.
There were nine kinds of wearable EMG devices capable of being used under unrestrained conditions. Ultra-miniaturized wearable EMG devices with a level of performance equivalent to that of conventional stationary EMG devices have been developed and are being used during sleep and in the daytime. The devices have a high level of diagnostic accuracy for sleep bruxism. A definite cut-off value for awake bruxism has not been established.
Assessment of sleep bruxism with a high level of accuracy can be performed using a portable/wearable EMG device. However, a definite cut-off value is required for assessment of awake bruxism.
Plant germ cells, such as pollen grains, can be affected by exposure to metal nanoparticles (NPs) that have diffused into the environment. The germination and tube elongation of pollen grain (
Lilium ...longiflorum
) exposed to low-solubility NPs was observed. The germination rate of pollen grain exposed to 100 mg L
−1
ZnO NP dispersion decreased significantly from controls and exposure of the other NPs. On the other hand, when pollens were exposed to ionic solutions in which particles were removed from the 100 mg L
−1
ZnO NP dispersion, the germination rates were equivalent to those in the control pollens. On the contrary, decrease of the germination rate compared to controls was small in the exposure to ZnCl
2
solution, which contained a larger amount of water-soluble Zn
2+
than ZnO NP dispersion. From these results, it was concluded that fine ZnO NP may adhere to pollens, due to the cohesive property of nanoparticles, and Zn
2+
dissolved at the interface may be continuously absorbed by pollens. When pollen was exposed to ZnO NP, a spot with a high Ca
2+
local concentration was observed at the tip of the pollen tube. On the contrary, simultaneous exposure to antagonistic ZnO NP and CaCl
2
resulted in no decrease in the germination rate. From the above, it is considered that upon exposure to ZnO NP, cells absorb Zn
2+
depending on the specific solubility of ZnO NP.
Key Message
Despite the low solubility of zinc oxide nanoparticle, pollen cell-attached particles inhibited germination and elongation of pollen tube by continuous Zn
2+
dissolution from particles and Zn
2+
absorption by the cell.
Although it is now well documented that laboratory rats learn to avoid the flavored substance consumed immediately before running in activity wheels or swimming in water buckets, research on this ...activity-based flavor avoidance learning in other species is limited. Recently, running-based flavor avoidance learning has been demonstrated in laboratory mice by employing a method of resistance-to-habituation of neophobic reaction to novel food; mice that repeatedly experience running after encountering a novel food have a prolonged tendency to reject that food compared to control mice without paired running. The present article reports a series of attempts to obtain evidence of flavor avoidance learning based on swimming rather than running using this resistance-to-habituation method. Swimming-based flavor avoidance was clearly demonstrated in a differential conditioning paradigm; however, its demonstration in a simple conditioning paradigm requires a post-training choice test of the target food and another type of food. These results are likely due to the short swimming time (20 min) and the formation of weak flavor aversion.
•Mice learn to avoid a novel food repeatedly paired with 20-min swimming.•This is a kind of flavor avoidance learning based on swimming.•This learning is easily demonstrated in a differential conditioning paradigm.•However, its demonstration in a simple conditioning paradigm is difficult.•A post-training choice test reveals latent food-avoidance learning.
Denosumab-related osteonecrosis of the jaw (DRONJ), which mainly occurs in cancer patients receiving anti-receptor activator NF-kappaB ligand (RANKL) antibody, reduces oral health-related quality of ...life. However, the exact mechanisms of and definitive treatment strategies for DRONJ remain unknown. We hypothesized that cessation of denosumab heals and/or ameliorates DRONJ, since it is a protein-based antibody agent, although stopping denosumab should be avoided in clinical situations. Therefore, the aims of this study were: 1) to create a healing and/or amelioration murine model of DRONJ-like lesions induced by chemotherapy/anti-RANKL antibody (mAb) combination therapy and tooth extraction; and 2) to investigate histopathology and immunopathology in the extraction sockets by comparing the murine model of DRONJ-like lesions with the amelioration/healing model of DRONJ-like lesions. Eight-week-old, female C57B/6J mice received chemotherapeutic drug (cyclophosphamide: CY) and mAb combination therapy (CY/mAb) with tooth extraction. Open wounds were sustained in CY/mAb-treated mice at 2 and 4 weeks post-extraction. Impaired socket healing was diagnosed as CY/mAb-related ONJ-like lesions at 3 weeks post-extraction in this study. Next, mAb was discontinued for 2 and 4 weeks after diagnosis of CY/mAb-related ONJ-like lesions. mAb cessation for 2 weeks induced partial osseous wound healing and significantly improved soft tissue wound healing of the extraction sockets. Anti-angiogenesis and normal lymphangiogenesis with CY/mAb combination therapy was not changed by mAb discontinuation. However, mAb cessation for 2 weeks significantly increased the number of CD38+F4/80+ M1 and CD163+F4/80+ M2 macrophages, which significantly increased the M2/M1 ratio in the connective tissue of extraction sockets. No direct effects of mAb on macrophages were noted both in vivo and in vitro. Therefore, the developed healing and/or amelioration murine model of CY/mAb-related ONJ-like lesions is a useful tool to investigate the histopathology and immunopathology of DRONJ in humans. Dynamic polarization shifting from M1 to M2 macrophages induced by mAb cessation may play an important role in wound healing, rather than angiogenesis and lymphangiogenesis, in DRONJ.
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•Anti-RANKL antibody (mAb) cessation reduced cyclophosphamide (CY)/mAb-related osteonecrosis of the jaw-like lesions in mice.•mAb cessation did not affect anti-angiogenesis or lymphangiogenesis in the extraction sockets.•Dynamic polarization shifting from CD38+F4/80+ M1 to CD163+F4/80+ M2 macrophages was induced by mAb cessation.•mAb monotherapy did not directly affect macrophage polarization.
Carboxylesterase (CES) and arylacetamide deacetylase (AADAC) are the major enzymes responsible for the hydrolysis of various clinical drugs. Our recent study demonstrated that the identity of the ...responsible hydrolase can be roughly surmised based on the chemical structures of compounds in humans. Dogs are used for preclinical studies in drug development, but the substrate specificities of dog CES and AADAC remain to be clarified. The purpose of this study is to characterize their substrate specificities. We prepared recombinant dog CES1, CES2, and AADAC. p-Nitrophenyl acetate, a general substrate for esterases, was hydrolyzed by dog CES1 and AADAC, while it was not hydrolyzed by CES2. CES2 protein was not substantially detected in the recombinant system or in the dog liver and intestinal microsomes by Western blot using anti-human CES2 antibodies. In silico analyses demonstrated slight differences in the three-dimensional structures of dog CES2 and human CES2, indicating that dog CES2 might be unstable or inactive. By evaluating the hydrolase activities of 22 compounds, which are known to be substrates of human CES and/or AADAC, we found that the activities of dog recombinant CES1 and AADAC as well as dog tissue preparations for nearly all compounds were lower than those of human enzymes. The dog enzymes that were responsible for the hydrolysis of most compounds corresponded to the human enzymes, but the following differences were observed: oseltamivir, irinotecan, and rifampicin were not hydrolyzed in the dog liver or by any of the recombinant esterases and procaine, a human CES2 substrate, was hydrolyzed by dog CES1. In conclusion, the present study could provide new finding to facilitate our understanding of species differences in drug hydrolysis, which can facilitate drug development and drug safety evaluation.
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Adenosine to inosine (A-to-I) RNA editing is the most frequent type of post-transcriptional nucleotide conversion in humans, and it is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. ...In this study we investigated the effect of RNA editing on human aryl hydrocarbon receptor (AhR) expression because the AhR transcript potentially forms double-stranded structures, which are targets of ADAR enzymes. In human hepatocellular carcinoma-derived Huh-7 cells, the ADAR1 knockdown reduced the RNA editing levels in the 3′-untranslated region (3′-UTR) of the AhR transcript and increased the AhR protein levels. The ADAR1 knockdown enhanced the ligand-mediated induction of CYP1A1, a gene downstream of AhR. We investigated the possibility that A-to-I RNA editing creates miRNA targeting sites in the AhR mRNA and found that the miR-378-dependent down-regulation of AhR was abolished by ADAR1 knockdown. These results indicated that the ADAR1-mediated down-regulation of AhR could be attributed to the creation of a miR-378 recognition site in the AhR 3′-UTR. The interindividual differences in the RNA editing levels within the AhR 3′-UTR in a panel of 32 human liver samples were relatively small, whereas the differences in ADAR1 expression were large (220-fold). In the human liver samples a significant inverse association was observed between the miR-378 and AhR protein levels, suggesting that the RNA-editing-dependent down-regulation of AhR by miR-378 contributes to the variability in the constitutive hepatic expression of AhR. In conclusion, this study uncovered for the first time that A-to-I RNA editing modulates the potency of xenobiotic metabolism in the human liver.
Background IL-22 functions as both a proinflammatory cytokine and an anti-inflammatory cytokine in various inflammations, depending on the cellular and cytokine milieu. However, the roles of IL-22 in ...the regulation of allergic airway inflammation are still largely unknown. Objective We sought to determine whether IL-22 is involved in the regulation of allergic airway inflammation. Methods We examined IL-22 production and its cellular source at the site of antigen-induced airway inflammation in mice. We also examined the effect of IL-22 neutralization, as well as IL-22 administration, on antigen-induced airway inflammation. We finally examined the effect of IL-22 on IL-25 production from a lung epithelial cell line (MLE-15 cells). Results Antigen inhalation induced IL-22 production in the airways of sensitized mice. CD4+ T cells, but not other lymphocytes or innate cells, infiltrating in the airways produced IL-22, and one third of IL-22–producing CD4+ T cells also produced IL-17A. The neutralization of IL-22 by anti–IL-22 antibody enhanced antigen-induced IL-13 production, eosinophil recruitment, and goblet cell hyperplasia in the airways. On the other hand, intranasal administration of recombinant IL-22 attenuated antigen-induced eosinophil recruitment into the airways. Moreover, anti–IL-22 antibody enhanced antigen-induced IL-25 production in the airways, and anti–IL-25 antibody reversed the enhancing effect of anti–IL-22 antibody on antigen-induced eosinophil recruitment into the airways. Finally, IL-22 inhibited IL-13–mediated enhancement of IL-25 expression in IL-1β– or LPS-stimulated MLE-15 cells. Conclusion IL-22 attenuates antigen-induced airway inflammation, possibly by inhibiting IL-25 production by lung epithelial cells.
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