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Impella is a mechanical circulatory support device of a catheter-based intravascular microaxial pump for left ventricular support and unloading. However, nonclinical studies assessing the effects of the extended duration of left ventricular unloading on cardiac recovery are lacking. An animal model using Impella implanted with a less invasive procedure to enable long-term support is required. This study aimed to evaluate the feasibility of an animal model for long-term support with Impella 5.5 implanted through carotid artery access in sheep.Impella 5.5 was implanted in four sheep through the proximal region of the left carotid artery without a thoracotomy, and myocardial injuries were induced by coronary microembolization. Support by Impella 5.5 was maintained for 4 weeks, and the animals were observed. The position of Impella 5.5 and cardiac function was evaluated using cardiac computer tomography at 2 and 4 weeks after implantation.All four animals completed the 4-week study without major complications. The discrepancy in the Impella 5.5 flow rate between the conscious and anesthetized states was observed depending on the device's position. Animals in whom the inflow was above the left ventricular papillary muscle had a relatively high flow rate under the maximum performance level without a suction alarm during the conscious state. Pathological changes in the aortic valve were observed. Cardiac function under the minimum performance level was observed with no remarkable deterioration.The animal model with myocardial injuries supported for 4 weeks by Impella 5.5 implanted through carotid artery access in sheep was feasible., Competing Interests: Declarations. Conflict of interest: T Nishinaka and S. Miyagawa have a sponsored research agreement with Abiomed Japan K.K.. The other authors have no conflicts of interest to declare., (© 2024. The Japanese Society for Artificial Organs.)

This study aimed to investigate whether belt electrode skeletal muscle electrical stimulation (B-SES) would improve postoperative lower limb function and walking ability in patients with diabetes who have undergone minor amputations. Diabetic patients who had undergone minor amputations were assigned randomly to a B-SES or control group. The B-SES group underwent conventional physical therapy for 20 min and B-SES for 20 min. The control group underwent only the 20-min conventional physical therapy. In both groups, rehabilitation was introduced by the physical therapists for 14 days from postoperative day 1. The outcome measures were range of motion in the ankle joint, knee extension muscle strength, ambulation status, and quality of life score. All these were evaluated before the intervention and 2 and 4 weeks after the intervention. From the 84 patients initially assessed, 32 were assigned to either the B-SES ( N  = 16) or control ( N  = 16) group. Preoperatively, there were no significant differences in all endpoints. The B-SES group showed significant improvement in the ankle dorsiflexion angle at 2 weeks postoperatively and knee joint extension strength at 4 weeks postoperatively. Postoperative B-SES with standard physical therapy might improve the range of motion of dorsiflexion of the ankle joint and extensor strength of the knee joint in patients with diabetes who have undergone minor amputations. B-SES is a useful tool to improve postoperative physical function in diabetic patients who have undergone minor amputations. A multicenter study is needed to determine the effective B-SES combined with regular physiotherapy for minor amputation., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Substances released outside of the cells during cell necrosis are collectively called danger-associated molecular patterns (DAMPS) or alarmins. A pro-inflammatory cytokine, interleukin-1α (IL-1α) is known as a typical alarmin. IL-1α transmits signals by binding to IL-1 receptor 1 (IL-1R1), type I protein, expressed on the cell membrane of target cells, but detection of IL-1R1 at the protein and mRNA levels is difficult. Although the reasons are not elucidated, we attempted to add the HiBiT-tag to the N-terminus (N'-R1) or C-terminus (C'-R1) of IL-1R1 to examine whether the detection sensitivity can be augmented. Increase in detection sensitivity will allow the investigation of its function and subcellular localization much further. Using uterine cervical cancer-derived HeLa cells and its derivative CR-R1-4 cells lacking IL-1R1, C'-R1 was demonstrated to significantly increase the detection sensitivity of IL-1R1. Furthermore, the signal transduction function of neither N'-R1 nor C'-R1 was affected. Immunofluorescence cell staining revealed that wild-type IL-1R1 is mainly localized in the nucleus, whereas C'-R1 is localized both in the nucleus and the cytoplasm. The above results showed that adding a tag to the C-terminus of IL-1R1 increases detection sensitivity while maintaining its function. In the future, we would like to further investigate the relationship between changes in the intracellular localization of C'-R1 and increases in detection sensitivity.

Bleeding complications have emerged as major causes of morbidity and mortality in patients with implantable left ventricular assist devices (LVAD). We hypothesized that the hemodynamics after LVAD implantation may influence the occurrence of bleeding complications after LVAD implantation. We retrospectively evaluated 78 patients who underwent continuous-flow LVAD implantation and hemodynamic ramp test after LVAD implantation between July 2017 and July 2023 at Osaka University. The bleeding complication occurred in 13 patients. The rates of freedom from bleeding complications at 1, 3, and 5 years were 94%, 85%, and 74%. Gastrointestinal bleeding, nose bleeding, and intraperitoneal hemorrhage occurred in six, three, and two patients, respectively. Preoperative average brachial-ankle pulse wave velocity (baPWV) was positively associated with bleeding complication (1,276 ± 280 vs. 1,098 ± 190 cm/s p = 0.04). In the hemodynamic ramp test, systemic vascular resistance (SVR) in patients with bleeding complications was higher than that in patients without bleeding complications (SVR: 1,359 ± 341 vs. 1,150 ± 217 dyne sec/cm5, p = 0.01). High preoperative baPWV and high SVR in the hemodynamic ramp test were significantly associated with bleeding complications after LVAD implantation. Arteriosclerosis is a risk factor for bleeding complications after LVAD implantation., Competing Interests: Disclosure: The authors have no conflicts of interest to report., (Copyright © ASAIO 2024.)

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has epoxide hydrolase activity and phosphatase activity. Our earlier study revealed that lysophosphatidic acids are a substrate of the phosphatase activity of sEH in vitro, but its physiological function remained unknown. Herein, we used the CRISPR/Cas9 system and i-GONAD method to generate mice that are deficient in sEH phosphatase activity. In the mouse brain, sEH was highly expressed in the olfactory bulb. Deletion of the sEH phosphatase activity resulted in decreased levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), which is a dephosphorylated form of 2-arachidonoyl-lysophosphatidic acid in the olfactory bulb. The sEH-deficient mice showed depressive-like behavior. These results indicate that sEH can regulate the production of 2-AG and brain function in vivo., (© 2024 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

This study investigated the impact of diabetes on the physical function of patients undergoing dialysis. This study included 22 patients undergoing outpatient dialysis with continued exercise therapy during dialysis at our hospital between January 2021 and August 2021. The participants were divided into two groups based on the presence or absence of diabetes, and various parameters were compared between the groups. To compare each physical function assessment and measurement of anterior thigh muscle thickness, repeated-measures analysis of variance was conducted to test for the presence of interactions and main effects. Significant differences were observed in the absence of dyslipidemia (p < 0.01), high-density lipoprotein cholesterol level (p < 0.01), and foot sole skin perfusion pressure (p < 0.02). In terms of physical function, a main effect between the groups was observed in the five-time sit-to-stand test, and anterior thigh muscle thickness showed a main effect over time. Significant differences in the anterior thigh muscle thickness were observed between three and six months after the intervention (p < 0.05). In patients undergoing dialysis with diabetes complications, a decrease in physical activity and lack of exercise can lead to a reduction in overall physical activity levels. Additionally, impairments such as peripheral neuropathy may contribute to an accelerated decrease in skeletal muscle mass., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Minata et al.)

This study aimed to identify factors associated with the Cumulated Ambulation Score (CAS) in the first week post-surgery (Week 1 CAS) in patients with proximal femoral fractures. Proximal femoral fractures are prevalent in the elderly, with rising incidence rates and significant social and functional implications. The ability to walk postoperatively is a critical determinant of patient prognosis. The study included 53 patients out of 79 who underwent surgery for proximal femoral fractures at the orthopedics department of Oita Oka Hospital from January 2021 to December 2021. Exclusion criteria were pre-existing walking difficulties, inability to be evaluated in the first postoperative week, non-weight bearing post-surgery, and complications during hospitalization. The physical therapy program followed Oita Oka Hospital's clinical path, starting ambulation with a walker within the first week post-surgery. Data collected included patient background, surgical techniques, pre-injury ambulatory status, and pre-admission residential environment. Physical function assessments one week postoperatively included range of motion (ROM), manual muscle testing (MMT), pain evaluation (NRS), and CAS. Statistical analyses involved the Shapiro-Wilk test, independent t-test, Mann-Whitney U test, chi-square test, and decision tree analysis using classification and regression trees (CART). Patients were categorized into 'favorable' and 'poor' groups based on Week 1 CAS. Significant differences in dementia presence and pre-admission living environment were noted between groups. Knee extension MMT at Week 1 postoperatively showed a significant difference. The decision tree analysis identified knee extension MMT as the primary variable, with a threshold of 3.5. In patients with MMT below 3.5, dementia presence was a secondary factor, with 81% in the poor CAS group. In patients with MMT above 3.5, knee extension strength continued to be significant. The model's accuracy was 64%, with precision at 71%, recall at 63%, and an F1-score of 0.67. The study highlights knee extension MMT and dementia presence as significant factors influencing Week 1 CAS in patients with proximal femoral fractures. The poor CAS group had a higher proportion of dementia and weaker knee extension MMT. Dementia hinders rehabilitation effectiveness, impacting recovery in knee extension strength and CAS. The decision tree analysis provided an intuitive understanding of variable interrelationships, emphasizing knee extension strength as the primary factor, followed by dementia in cases with lower MMT scores. This study elucidated factors related to Week 1 CAS in postoperative patients with proximal femoral fractures. Knee extension MMT emerged as the initial factor, followed by the presence of dementia, influencing Week 1 CAS. These findings are crucial for rehabilitation planning and long-term prognostic predictions in this patient population. However, the study's limitations include potential selection bias and a small sample size, necessitating further research for enhanced predictive accuracy., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Hiramatsu et al.)

Asynchronous rotational-speed modulation of a continuous-flow left ventricular assist device (LVAD) can increase pulsatility; however, the feasibility of hemodynamic modification by asynchronous modulation of an LVAD has not been sufficiently verified. We evaluated the acute effect of an asynchronous-modulation mode under LVAD support and the accumulated effect of 6 consecutive hours of driving by the asynchronous-modulation mode on hemodynamics, including both ventricles, in a coronary microembolization-induced acute-myocardial injury sheep model. We evaluated 5-min LVAD-support hemodynamics, including biventricular parameters, by switching modes from constant-speed to asynchronous-modulation in the same animals ("acute-effect evaluation under LVAD support"). To determine the accumulated effect of a certain driving period, we evaluated hemodynamics including biventricular parameters after weaning from 6-hour (6 h) LVAD support by constant-speed or asynchronous-modulation mode ("6h-effect evaluation"). The acute-effect evaluation under LVAD support revealed that, compared to the constant-speed mode, the asynchronous-modulation mode increased vascular pulsatility but did not have significantly different effects on hemodynamics, including both ventricles. The 6 h-effect evaluation revealed that the hemodynamics did not differ significantly between the two groups except for some biventricular parameters which did not indicate negative effects of the asynchronous-modulation mode on both ventricles. The asynchronous-modulation mode could be feasible to increase vascular pulsatility without causing negative effects on hemodynamics including both ventricles. Compared to the constant-speed mode, the asynchronous-modulation mode increased pulsatility during LVAD support without negative effects on hemodynamics including both ventricles in the acute phase. Six hours of LVAD support with the asynchronous-modulation mode exerted no negative effects on hemodynamics, including both ventricles, after weaning from the LVAD., (© 2023. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Interleukin-1α (IL-1α), a cytokine released by necrotic cells, causes sterile inflammation. On the other hand, IL-1α is present in the nucleus and also regulates the expression of many proteins. A protein substrate containing a classical nuclear localization signal (cNLS) typically forms a substrate/importin α/β complex, which is subsequently transported to the nucleus. To the best of our knowledge, no study has directly investigated whether IL-1α-which includes cNLS-is imported into the nucleus in an importin α/β-dependent manner. In this study, we noted that all detected importin α subtypes interacted with IL-1α. In HeLa cells, importin α1-mediated nuclear translocation of IL-1α occurred at steady state and was independent of importin β1. Importin α1 not only was engaged in IL-1α nuclear transport but also concurrently functioned as a molecule that regulated IL-1α protein level in the cell. Furthermore, we discussed the underlying mechanism of IL-1α nuclear translocation by importin α1 based on our findings., (© 2024. The Author(s).)

Aims: The aim of the present study is to elucidate the mechanism of CYP2E1 induction as a causative factor of Alcoholic Hepatitis (AH) and its relationship with inflammation., Background: Chronic alcohol consumption induces CYP2E1, which is involved in the development of Alcoholic Hepatitis (AH). However, the mechanisms underlying the induction of CYP2E1 by alcohol remain unclear. Therefore, we herein investigated the induction of drug-metabolizing enzymes, particularly CYP2E1, by hydrogen peroxide (H 2 O 2 ), the concentration of which is elevated under inflammatory conditions., Objective: The mechanisms underlying the induction of CYP2E1 by H 2 O 2 were examined with a focus on Keap1, a target factor of H 2 O 2 ., Methods: We assessed changes in the expression of drug-metabolizing enzymes in the human hepatoma cell line, Hep3B, following treatment with H 2 O 2 , and evaluated changes in the expression of the NF-kB-related factor RelA(p65) after the knockdown of Keap1, a regulator of Nrf2 expression by reactive oxygen species. We also performed a promoter analysis using the upstream region of the CYP2E1 gene. We herein used the GSE89632 series for non-alcoholic hepatitis (NASH) and the GSE28619 series for AH., Results: The induction of CYP2E1 by H 2 O 2 was significantly stronger than that of other drugmetabolizing enzymes. On the other hand, the knockdown of Keap1, a target of H 2 O 2 , markedly increased RelA(p65), an NFkB factor. Furthermore, the overexpression of RelA(p65) strongly induced the expression of CYP2E1. Four candidate p65-binding sequences were identified upstream of the CYP2E1 gene, and promoter activity assays showed that the third sequence was responsive to the overexpression of RelA(p65). We used the GSE89632 series for NASH and the GSE28619 series for AH in the present study. The expression of CYP2E1 mRNA in the liver was significantly lower in AH patients than in HC patients, but was similar in HC patients and NASH patients., Conclusion: We herein demonstrated that the expression of CYP2E1 was induced by H 2 O 2 . The overexpression of RelA(p65) also induced CYP2E1 mRNA expression, whereas H 2 O 2 did not after the knockdown of RelA. These results suggest that H 2 O 2 acts on Keap1 to upregulate RelA (p65) in the NFkB system. One of the mechanisms underlying the induction of CYP2E1 was dependent on the H 2 O 2 -Keap1-RelA axis. The results of the database analysis revealed that the expression of CYP2E1 in the liver was significantly lower in AH patients than in NASH patients, suggesting that CYP2E1 is not the main cause of AH; however, CYP2E1 may exacerbate the pathogenesis of AH., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The first enantioselective total synthesis of kopsiyunnanine B, which has a unique folded and complex pentacyclic structure containing six contiguous chiral centers, has been achieved along our originally proposed biosynthetic pathway. The key reaction of this synthesis includes a bioinspired cascade that builds three ring structures and three chiral centers in one step and features the stereoselective reduction of a β-acrylate and oxidation to an oxindole.

Few studies have focused on the clinical outcomes and risk factors of left ventricular assist device (LVAD) pump infection, and no standard treatment for LVAD pump infection has been established. Therefore, we used a therapeutic flowchart to manage LVAD pump infections. We retrospectively evaluated 220 patients who underwent continuous-flow LVAD implantation between January 2005 and March 2021 at Osaka University, Japan. First, we performed wound debridement, negative-pressure wound therapy, antibiotic treatment, and omental flap transposition. Subsequently, we administered conservative treatment, scheduled implantable LVAD exchange, or emergent removal of the implantable LVAD and exchange for extracorporeal LVAD or percutaneous LVAD (IMPELLA). Pump infections occurred in 32 patients. The survival rates of patients with pump infections during LVAD support were 93%, 74%, and 61% at 180 days, 1 year, and 2 years after LVAD pump infection, respectively. Fifteen patients underwent successful heart transplantation. Bridge-to-bridge surgery, preoperative use of venoarterial extracorporeal membrane oxygenation or percutaneous LVAD, high lactate dehydrogenase levels, and driveline infection were significantly associated with pump infection. Our study reveals that poor preoperative condition and driveline infection were significant risk factors for LVAD pump infection., Competing Interests: Disclosure: The authors have no conflicts of interest to report., (Copyright © ASAIO 2023.)

We developed a new artificial placenta (AP) system consisting of a loop circuit configuration extracorporeal membrane oxygenation (ECMO) with a bridge circuit designed to be applied to the fetus in the form of an umbilical arterial-venous connection. We aimed to evaluate the feasibility of the AP system by performing a hydrodynamic simulation using a mechanical mock circulation system and fetal animal experiment. The effect of the working condition of the AP system on the fetal hemodynamics was evaluated by hydrodynamic simulation using a mechanical mock circulation system, assuming the weight of the fetus to be 2 kg. The AP system was introduced to two fetal goats at a gestational age of 135 days. The general conditions of the experimental animals were evaluated. The mock simulation showed that in an AP system with ECMO in the form of an umbilical arterial-venous connection in series, it could be difficult to maintain fetal hemodynamics when high ECMO flow was applied. The developed AP system could have high ECMO flow with less umbilical blood flow; however, the possibility of excessive load on the fetal right-sided heart should be noted. In the animal experiment, kid 1 (1.9 kg) was maintained on the AP system for 12 days and allowed to grow to term. In kid 2 (1.6 kg), the AP system could not be established because of the occlusion of the system by a thrombus. The developed AP system was feasible under both in vitro and in vivo conditions. Improvements in the AP system and management of the general fetal conditions are essential., (© 2022. The Japanese Society for Artificial Organs.)

Cancer cells exhibit an altered redox balance and aberrant redox signaling due to genetic, metabolic, and microenvironment-associated reprogramming. Persistently elevated levels of reactive oxygen species (ROS) contribute to many aspects of tumor development and progression. Emerging studies demonstrated the vital role of apurinic/apyrimidinic endonuclease 1 or reduction/oxidation (redox) factor 1(APE1/Ref-1) in the oxidative stress response and survival of cancer cells. APE1/Ref-1 is a multifunctional enzyme involved in the DNA damage response and functions as a redox regulator of transcription factors. We herein demonstrated that basal hydrogen peroxide (H 2 O 2 ) and APE1/Ref-1 expression levels were markedly higher in cancer cell lines than in non-cancerous cells. Elevated APE1/Ref-1 levels were associated with shorter survival in liver cancer patients. Mechanistically, we showed that H 2 O 2 activated nuclear factor-κB (NF-κB). RelA/p65 inhibited the expression of the E3 ubiquitin ligase Parkin, possibly by interfering with ATF4 activity. Parkin was responsible for the ubiquitination and proteasomal degradation of APE1/Ref-1; therefore, the H 2 O 2 -induced suppression of Parkin expression increased APE1/Ref-1 levels. The probability of survival was lower in liver cancer patients with low Parkin and high RelA expression levels. Additionally, Parkin and RelA expression levels negatively and positively correlated with APE1/Ref-1 levels, respectively, in the TCGA liver cancer cohort. We concluded that increases in APE1/Ref-1 via the NF-κB and Parkin pathways are critical for cancer cell survival under oxidative stress. The present results show the potential of the NF-κB-Parkin-APE1/Ref-1 axis as a prognostic factor and therapeutic strategy to eradicate liver cancer.

Although pregnant women's fish consumption is beneficial for the brain development of the fetus due to the DHA in fish, seafood also contains methylmercury (MeHg), which adversely affects fetal brain development. Epidemiological studies suggest that high DHA levels in pregnant women's sera may protect the fetal brain from MeHg-induced neurotoxicity, but the underlying mechanism is unknown. Our earlier study revealed that DHA and its metabolite 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) produced by cytochrome P450s (P450s) and soluble epoxide hydrolase (sEH) can suppress MeHg-induced cytotoxicity in mouse primary neuronal cells. In the present study, DHA supplementation to pregnant mice suppressed MeHg-induced impairments of pups' body weight, grip strength, motor function, and short-term memory. DHA supplementation also suppressed MeHg-induced oxidative stress and the decrease in the number of subplate neurons in the cerebral cortex of the pups. DHA supplementation to dams significantly increased the DHA metabolites 19,20-epoxydocosapentaenoic acid (19,20-EDP) and 19,20-DHDP as well as DHA itself in the fetal and infant brains, although the expression levels of P450s and sEH were low in the fetal brain and liver. DHA metabolites were detected in the mouse breast milk and in human umbilical cord blood, indicating the active transfer of DHA metabolites from dams to pups. These results demonstrate that DHA supplementation increased DHA and its metabolites in the mouse pup brain and alleviated the effects of MeHg on fetal brain development. Pregnant women's intake of fish containing high levels of DHA (or DHA supplementation) may help prevent MeHg-induced neurotoxicity in the fetus., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Rehabilitation is usually provided to patients with chronic foot wounds (CFWs) after surgery. This study aimed to assess whether early postoperative rehabilitation could maintain walking independence in hospitalized patients with CFWs. This single-blind, randomized clinical trial was performed between September 10, 2018 and March 2019, involving 60 patients who underwent both surgical procedures and rehabilitation. Participants were randomly allocated into the early rehabilitation (EG, n = 30) or the control (CG, n = 30) groups. EG received early rehabilitation immediately after surgery, while CG received late rehabilitation after wound closure. Both groups received rehabilitation sessions 5 times per week until discharge. The primary outcome was walking independence, measured via Functional Independence Measure (FIM)-gait scores. Secondary outcomes included health-related quality of life (HRQoL) using EuroQol 5 dimensions 5-level (EQ-5D-5L) and the presence of rehabilitation-related adverse events, including dehiscence of wounds and falls. Differences in intervention timing effects were analyzed using nonparametric split-plot factorial design analysis, including Fisher's exact test, Mann-Whitney U test, and Wilcoxon signed-rank test ( P  < .05). Out of the 60 participants, 53 patients completed the discharge follow up. Three participants (10.0%) from the EG and 4 (13.3%) from the CG dropped out due to postoperative complications unrelated to rehabilitation intervention. No rehabilitation-related adverse events were found. Participants in the EG maintained greater FIM-gait scores during hospitalization than the CG (difference, -1; P  = .0001), with a difference of 0 ( P  = .109) at discharge. EQ-5D-5L significantly improved in both groups (EG: difference, 0.13 [ P  = .014], CG: difference, 0.17 [ P  = .0074]). The EG intervention was associated more with maintaining walking independence at discharge than CG intervention. Postoperative rehabilitation improved HRQoL without adverse events, indicating that clinicians should recommend early rehabilitation for patients with CFW to enhance walking independence.

Partial foot amputation (PFA) is generally planned to minimize the amputation level; nonetheless, the effect of PFA levels on gait independence in amputees remains unclear. This study aimed to investigate the impact of PFA levels of the forefoot on gait independence in patients with chronic lower extremity (LE) wounds. This multicenter retrospective cohort study included 232 hospitalized Japanese patients treated and rehabilitated for chronic LE wounds. A multivariate analysis based on PFA levels was conducted for gait independence at discharge, with age and comorbidities as independent variables. Patients with Lisfranc amputation had significantly less independent gait than patients with more distal amputation and those without amputation (<22% vs >40%; P  = .027; Fisher's exact test). Logistic regression analysis revealed that Lisfranc amputation (odds ratio [OR]: 0.257, P  = .047), age (OR: 0.559, P  = .043), and chronic limb-threatening ischemia (OR: 0.450, P  = .010) were independent factors associated with gait independence. Additionally, the regression model confirmed discrimination performance using the C index (0.691, P  < .001) with receiver operating characteristic analysis. In patients with chronic LE wounds undergoing PFA, Lisfranc amputation was negatively associated with gait independence.

[Purpose] The recurrence rate of diabetic foot ulcers is high and is related to kinematic factors. Achilles tendon lengthening has been shown to reduce the recurrence rate of foot ulcers by increasing the range of motion in the ankle joint and decreasing the plantar load. However, there are few reports on the effects of Achilles tendon lengthening in Japanese patients, but the results are yet to be clarified. This study aims to investigate the effects of Achilles tendon lengthening on physical function and ambulatory state in patients with diabetic foot ulcers. [Participants and Methods] This study initially included 10 patients with diabetic ulcers who had undergone Achilles tendon lengthening between April 2013 and March 2020. We retrospectively evaluated the factors available from the medical records. [Results] The dorsiflexion range of motion in the ankle joint increased by 10.5 degrees on average after surgery, while the plantar load decreased by 19.1 percent, while gait speed and stride length remained unchanged. [Conclusion] Achilles tendon lengthening for diabetic foot ulcers increased the range of motion in the ankle joint and decreased the plantar load without changing the ambulatory state., Competing Interests: There are no conflicts of interest., (2023©by the Society of Physical Therapy Science. Published by IPEC Inc.)

Aims: The aim of the present study is to gain insight into the biology of Parkinson's disease (PD) and cancer to drive translational advances enabling more effective prevention and/or potential treatments., Background: The expression of Cytochrome P450 2D6 (CYP2D6) is correlated with various diseases such as PD and cancer; therefore, exploring its regulatory mechanism at transcriptional levels is of interest. NF-E2-related factor 2 (Nrf2) has been known to be responsible for regulating phase II and phase III drug-metabolizing genes., Objectives: The objectives of this study are to investigate the transcriptional regulation of CYP2D6 by Nrf2 and to analyze its role in PD and cancer., Methods: Nrf2 was transiently expressed in human hepatoma Hep3B cells, and the expression of CYP2D6 was examined by RT-qPCR. The promoter activity of CYP2D6 and the DNA binding of Nrf2 were examined by luciferase and ChIP assay, respectively. We then investigated the expression and correlation of Nrf2 and CYP2D6 in the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets., Results: In the present study, we demonstrated that Nrf2 down-regulated CYP2D6 mRNA expression in hepatoma Hep3B cells. Mechanistically, Nrf2 binds to the antioxidant responsive element (ARE) in the proximity of krüppel- like factor 9 (KLF9)-binding site within the -550/+51 of CYP2D6 promoter. The inhibition and activation of Nrf2 enhanced and suppressed KLF9 effects on CYP2D6 expression, respectively. The expression levels of Nrf2 and CYP2D6 were upregulated and downregulated in the PD patient GEO datasets compared to the healthy control tissues, and Nrf2 was negatively correlated with CYP2D6 . In liver cancer patients, decreased CYP2D6 levels were apparent and associated with a lower probability of survival., Conclusion: Our work revealed the inhibitory role of Nrf2 in regulating CYP2D6 expression. Moreover, Nrf2- dependent regulation of CYP2D6 can be used as a prognostic factor and therapeutic strategy in PD and liver cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: A heart failure (HF) model using coronary microembolization in large animals is indispensable for medical research. However, the heterogeneity of myocardial response to microembolization is a limitation. We hypothesized that adjusting the number of injected microspheres according to coronary blood flow could stabilize the severity of HF. This study aimed to evaluate the effect of microsphere injection based on the left coronary artery blood flow in an animal model., Methods: Microembolization was induced by injecting different numbers of microspheres (polystyrene, diameter: 90 μm) into the left descending coronary artery of the two groups of sheep (400 and 600 times coronary blood flow [ml/min]). Hemodynamic parameters, the pressure-volume loop of the left ventricle, and echocardiography findings were examined at 0.5, 1.5, 3.5, and 6.5 h after microembolization., Results: End-diastolic pressure and normalized heart rate increased over time, and were significantly higher in 600 × coronary blood flow group than those in 400 × coronary blood flow group (p = 0.04 and p < 0.01, respectively). The maximum rate of left-ventricular pressure rise and normalized stroke volume decreased over time, and were significantly lower in 600 × coronary blood flow group than those in 400 × coronary blood flow group (p < 0.01 and p < 0.01, respectively). The number of microspheres per coronary blood flow was significantly correlated with the decrease in stroke volume and the maximum rate of left ventricular pressure rise in 6.5 h (r = 0.74, p = 0.01 and r = 0.71, p = 0.02, respectively)., Conclusions: Adjusting the number of injected microspheres based on the coronary blood flow enabled the creation of HF models with different degrees of severity., (© 2022 International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

Objectives: Medical facilities have been required to effectively utilize insufficient human resources in many countries. Therefore, we qualitatively and quantitively compared physicians' working burden, and assessed advantages and disadvantages of the single- and the multiple-attending physicians systems in inpatient care., Methods: In this cross-sectional study, we extracted electronic health record of patients from a hospital in Japan from April 2017 to October 2018 to compare anonymous statistical data between the single-attending and multiple-attending-physicians system. Then, we conducted a questionnaire survey for all physicians of single and multiple-attending systems, asking about their physical and psychiatric workload, and their reasons and comments on their working styles., Results: The average length of hospital stay was significantly shorter in the multiple-attending system than in the single-attending system, while patients' age, gender, and diagnoses were similar. From the questionnaire survey, no significant difference was found in all categories although physical burden in multiple-attending system tended to be lower than that in single-attending system. Advantages of multiple-attending system extracted from qualitative analysis are (1) improvement of physicians' quality of life (QOL), (2) lifelong-learning effect, and (3) improving the quality of medical care, while disadvantages were (1) risk of miscommunications, (2) conflicting treatment policies among physicians, and (3) patients' concern., Conclusions: The multiple-attending physician system in the inpatient setting can reduce the average length of stay for patients and also reduce the physical burden on physicians without compromising their clinical performance.

Solid tumors often contain regions with very low oxygen concentrations or hypoxia resulting from altered metabolism, uncontrolled proliferation, and abnormal tumor blood vessels. Hypoxia leads to resistance to both radio- and chemotherapy and a predisposition to tumor metastases. Under hypoxia, sequestosome 1 (SQSTM1/p62), a multifunctional stress-inducible protein involved in various cellular processes, such as autophagy, is down-regulated. The hypoxic depletion of p62 is mediated by autophagic degradation. We herein demonstrated that hypoxia down-regulated p62 in the hepatoma cell line Hep3B at the transcriptional and post-translational levels. At the transcriptional level, hypoxia down-regulated p62 mRNA by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2). The overexpression of Nrf2 and knockdown of Siah2, a negative regulator of Nrf2 under hypoxia, diminished the effects of hypoxia on p62 mRNA. At the post-translational level, the proteasome inhibitor MG132, but not the lysosomal inhibitors ammonium chloride and bafilomycin, prevented the hypoxic depletion of p62, suggesting the involvement of the proteasome pathway. Under hypoxia, the expression of the E3 ubiquitin ligase Parkin was up-regulated in a hypoxia-inducible factor 1α-dependent manner. Parkin ubiquitinated p62 and led to its proteasomal degradation, ensuring low levels of p62 under hypoxia. We demonstrated that the effects of Parkin on p62 required heat shock cognate 71 kDa protein (Hsc70). We also showed that the overexpression of Nrf2 and knockdown of Parkin or Hsc70 induced the accumulation of p62 and reduced the viability of cells under hypoxia. We concluded that a decrease in p62, which involves regulation at the transcriptional and post-translational levels, is critical for cell survival under hypoxia. The present results show the potential of targeting Nrf2/Parkin-Hsc70-p62 as a novel strategy to eradicate hypoxic solid tumors., (© 2022. The Author(s).)

Chlorogenic acid (CGA) is the most abundant polyphenol in coffee. It has been widely reported to exhibit antioxidant activity by activating nuclear factor erythroid 2-related factor 2 (Nrf2) potentially via the canonical Kelch-like-ECH-associated protein 1 (Keap1)-Nrf2 pathway. We herein demonstrated that the knockdown of WD40 repeat protein 23 (WDR23), but not Keap1, abolished the effects of CGA on the activation of Nrf2. CGA decreased the expression of DDB1, an adaptor for WDR23-Cullin 4A-RING ligase (CRL4AWDR23). FOXO3, a major target for inactivation by the PI3K/Akt pathway, was identified as the transcription factor responsible for the basal and CGA-inhibited expression of the DDB1 gene. CGA blocked FOXO3 binding to importin-7 (IPO7), thereby inhibiting the nuclear accumulation of FOXO3, down-regulating the expression of DDB1, inhibiting the activity of CRL4WDR23, and ultimately increasing that of Nrf2. This pathway was conserved in Caenorhabditis elegans, and CGA extended the lifespan partly through this pathway. We found that in C. elegans, the isoform DAF-16a, but not DAF-16f, regulated the expression levels of ddb-1 mRNA and SKN-1 protein. CGA prolonged the mean lifespan of DAF-16a- and DAF-16f-rescued worms by 24% and 9%, respectively, suggesting that both isoforms involve in lifespan-extending effects of CGA, with DAF-16a being more important than DAF-16f. Based on these results, we established a novel Akt-FOXO3/DAF16a-DDB1 axis that links nutrient sensing and oxidative stress response pathways. Our results also provide a novel molecular mechanism for Nrf2/SKN-1 activation by CGA and the increased lifespan of C. elegans by CGA via this pathway., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pericytes (PCs) are a mural support cell population elongated at intervals along the walls of capillaries. Recent studies reported that PCs are multipotent cells that are activated in response to tissue injury and contribute to the regenerative process. Using a C.B-17 mouse model of ischemic stroke, it has been proposed that normal brain pericytes (nPCs) are converted to ischemic pericytes (iPCs), some of which function as multipotent stem cells. Furthermore, oxygen-glucose deprivation (OGD) promoted mesenchymal-epithelial transition in nPCs; however, nestin was not induced under OGD conditions. Therefore, further studies are needed to elucidate the PC reprogramming phenomenon. We herein isolated nPCs from the cortex of C.B-17 mice, and compared the traits of iPCs and nPCs. The results obtained showed that nPCs and iPCs shared common pericytic markers. Furthermore, intercellular levels of reactive oxygen species and the nuclear accumulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a key player in antioxidant defenses, were higher in iPCs than in nPCs. OGD/reoxygenation and a treatment with tBHQ, an Nrf2 inducer, increased nestin levels in nPCs. Moreover, epithelial marker levels, including nestin, Sox2, and CDH1 (E-cadherin) mRNAs, were elevated in Nrf2-overexpressing PCs, which formed neurosphere-like cell clusters that differentiated into Tuj1-positive neurons. The present results demonstrate that oxidative stress and Nrf2 are required for the generation of stem cells after stroke and will contribute to the development of novel therapeutic strategies for ischemic stroke., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aberrant right subclavian artery is a common aortic arch anomaly that can cause dysphagia as a result of compression by the aberrant artery. For patients with an aneurysm associated with an aberrant right subclavian artery, surgical or endovascular intervention is a well-described treatment. However, for patients with a nonaneurysmal aberrant right subclavian artery, treatment with thoracic endovascular aortic repair has been limited. We describe the use of thoracic endovascular aortic repair and subclavian revascularization to treat esophageal stricture in a patient with a symptomatic nonaneurysmal aberrant right subclavian artery. The patient's dysphagia was successfully relieved after the operation., (© 2022 by the Texas Heart® Institute, Houston.)

Etoposide is regarded as one of the main standard cytotoxic drugs for lung cancer. However, mutations in Kelch-like ECH-associated protein 1 ( Keap1 ), the main regulator of nuclear factor erythroid 2-related factor 2 (Nrf2), are often detected in lung cancer and lead to chemoresistance. Since the aberrant activation of Nrf2 enhances drug resistance, the suppression of the Nrf2 pathway is a promising therapeutic strategy for lung cancer. We herein used the human lung adenocarcinoma cell line A549 because it harbors a Keap1 loss-of-function mutation. A treatment with β -glucan, a major component of the fungal cell wall, reduced Nrf2 protein levels; downregulated the expression of cytochrome P450 3A5 , UDP glucuronosyltransferase 1A1, and multidrug resistance protein 1; and increased etoposide sensitivity in A549 cells. Furthermore, the ephrin type-A receptor 2 (EphA2) receptor was important for the recognition and biologic activity of β -glucan in A549 cells. EphA2 signaling includes nuclear factor kappa B (NF- κ B), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). However, treatment of cells with stattic (STAT3 inhibitor) or SB203580 (p38 MAPK inhibitor) did not diminish the effects of β -glucan. In contrast, knockdown of v-rel reticuloendotheliosis viral oncogene homolog B (RelB) abolished the effects of β -glucan, suggesting the involvement of the noncanonical NF- κ B pathway. The β -glucan effects were also attenuated by the knockdown of WD40 Repeat protein 23 (WDR23). The β -glucan treatment and RelB overexpression induced the expression of Cullin-4A ( CUL4A ), which increased WDR23 ligase activity and promoted the subsequent depletion of Nrf2. These results revealed a novel property of β -glucan as a resistance-modifying agent in addition to its widely reported immunomodulatory effects for lung cancer therapy via the EphA2-RelB-CUL4A-Nrf2 axis. SIGNIFICANCE STATEMENT: Chemotherapeutic resistance remains a major obstacle in cancer therapy despite extensive efforts to elucidate the underlying molecular mechanisms and overcome multidrug resistance. The present study revealed a novel resistance-modifying property of β-glucan, thereby expanding our knowledge on the beneficial roles of β-glucan and providing an alternative strategy to prevent drug resistance by cancer. The present results provide evidence for the involvement of a novel mode of NF-κB and Nrf2 crosstalk in the drug resistance phenotype., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Borophene has been recently proposed as a next-generation two-dimensional material with promising electronic and optical properties. However, its instability has thus far limited its large-scale applications. Here, we investigate a liquid-state borophene analogue with an ordered layer structure derived from two-dimensional borophene oxide. The material structure, phase transition features and basic properties are revealed by using X-ray analysis, optical and electron microscopy, and thermal characterization. The obtained liquid crystal exhibits high thermal stability at temperatures up to 350 °C and an optical switching behaviour driven by a low voltage of 1 V., (© 2022. The Author(s).)

[Purpose] Information about clinical trials related to physical therapy (CTPT) in Japan, which has the highest aging rate in the world, is essential for physical therapy education, research, and policymaking to change and strengthen the education system and promote research grants. This survey aimed to clarify the proportion of CTPT in the clinical registry and compare the proportion of CTPT in Japan with that in North America. [Participants and Methods] The ClinicalTrials.gov (CTG) and National Institute of Public Health (NIPH) Clinical Trials were used. The number and proportion of CTPT were compared each year. The analyzed data spanned 10 years from 2010 to 2019. [Results] A total of 222,821 trials were registered in CTG during the 10 years. In search of "physical therapy", 3,001 trials searched. The proportion of CTPT increased from 0.8% to 1.7%. In total 42,194 trials were registered in the NIPH Clinical Trials Search. From the CTPT, 141 trials were obtained. The proportion of CTPT increased from 0.05% to 0.5%. The proportion of CTPT in the NIPH Clinical Trials Search was one-third or less than that in the CTG. The proportion of CTPT in CTG increased yearly, but the proportion of CTPT in NIPH Clinical Trials Search has not increased since 2016. [Conclusion] The proportion of CTPT is relatively low in Japan, compared with that in North America, and it showed no increasing trend. It is important to provide education and support for clinical trials in an aging country such as Japan., Competing Interests: There are no conflicts of interest., (2021©by the Society of Physical Therapy Science. Published by IPEC Inc.)

Chemo-enzymatic synthesis of lacto-N-biose I (LNB) catalyzed by β-1,3-galactosidase from Bacillus circulans (BgaC) has been developed using 4,6-dimethoxy-1,3,5-triazin-2-yl β-galactopyranoside (DMT-β-Gal) and GlcNAc as the donor and acceptor substrates, respectively. BgaC transferred the Gal moiety to the acceptor, giving rise to LNB. The maximum yield of LNB was obtained at the acceptor : donor substrate ratio of 1:30., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Background: Cardiogenic shock due to acute severe mitral regurgitation is characterized by multiple organ failure and acute pulmonary edema, leading to a high risk of mortality. Methods and Results: We report on a patient with acute, severe mitral regurgitation complicated by cardiogenic shock, refractory to both inotrope treatment and intra-aortic balloon pump support. The patient was successfully bridged to surgery with an Impella CP, a percutaneous left ventricular assist device. Conclusions: Mechanical support using an Impella CP can stabilize hemodynamics and may be used as a bridge to elective surgery for patients with mitral regurgitation with cardiogenic shock., Competing Interests: Y. Sawa is a member of Circulation Reports’ Editorial Board., (Copyright © 2021, THE JAPANESE CIRCULATION SOCIETY.)

Background: Diabetes-related foot lesions are a major cause of non-traumatic lower limb amputations and are associated with a high re-amputation rate. Lesions can cause hindrance in activities of daily living, reduce physical function, and lower a patient's quality of life. Physical therapy is necessary to prevent these limitations. Thus far, there has been limited investigation into the re-amputation rate in patients who have undergone physical therapy. This study aimed to elucidate modifiable risk factors for re-amputation in patients with minor amputations who were treated with physical therapy during their hospitalization., Methods: This was a retrospective cohort study of 245 consecutive hospitalized patients who presented to our Wound Care Center between January 2015 and February 2018 and received physical therapy after a minor amputation. Participants were identified from admission records to surgical and physical therapy units stored in the electronic medical records. We examined re-amputations that occurred in the ipsilateral lower extremity during the 1-year post-discharge outpatient period. The maximum follow-up period was set at 1 year. We used Cox proportional hazards analysis to examine factors affecting the risk of re-amputation., Results: Of the 129 patients enrolled, 42 patients (32.5%) underwent re-amputations during an average observation period of 6.2 months (range, 2.1 to 10.9 months). The factors associated with re-amputation were a requirement for hemodialysis, ankle dorsiflexion angle, and the Functional Independence Measure (FIM) ambulation score., Conclusions: In diabetes patients with minor amputations, a requirement for hemodialysis, ankle dorsiflexion angle, and the FIM ambulation score were shown to be modifiable risk factors for re-amputation. This emphasizes that maintaining vascular endothelial function through lower limb muscle exercises for hemodialysis, improving ankle mobility, and relieving plantar pressure during walking are necessary to reduce the risk of re-amputation. Patients with these risk factors should be encouraged to participate in physical therapy.

Hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor, plays a critical role in adaption to hypoxia, which is a major feature of diseases, including cancer. Protein disulfide isomerase (PDI) is up-regulated in numerous cancers and leads to cancer progression. PDI, a member of the TRX superfamily, regulates the transcriptional activities of several transcription factors. To investigate the mechanisms by which PDI affects the function of HIF-1alpha, the overexpression or knockdown of PDI was performed. The overexpression of PDI decreased HIF-1alpha expression in the human hepatocarcinoma cell line, Hep3B, whereas the knockdown of endogenous PDI increased its expression. NH4Cl inhibited the decrease in HIF-1alpha expression by PDI overexpression, suggesting that HIF-1alpha was degraded by the lysosomal pathway. HIF-1alpha is transferred to lysosomal membranes by heat shock cognate 70 kDa protein (HSC70). The knockdown of HSC70 abolished the decrease, and PDI facilitated the interaction between HIF-1alpha and HSC70. HIF-1alpha directly interacted with PDI. PDI exists not only in the endoplasmic reticulum (ER), but also in the cytosol. Hypoxia increased cytosolic PDI. We also investigated changes in the redox state of HIF-1alpha using PEG-maleimide, which binds to thiols synthesized from disulfide bonds by reduction. An up-shift in the HIF-1alpha band by the overexpression of PDI was detected, suggesting that PDI formed disulfide bond in HIF-1alpha. HIF-1alpha oxidized by PDI was not degraded in HSC70-knockdown cells, indicating that the formation of disulfide bond in HIF-1alpha was important for decreases in HIF-1alpha expression. To the best of our knowledge, this is the first study to show the regulation of the expression and redox state of HIF-1alpha by PDI. We also demonstrated that PDI formed disulfide bonds in HIF-1alpha 1-245 aa and decreased its expression. In conclusion, the present results showed that PDI is a novel factor regulating HIF-1alpha through lysosome-dependent degradation by changes in its redox state., Competing Interests: The authors have declared that no competing interests exist.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is important for adaptation to hypoxia. Hypoxia is a common feature of cancer and inflammation, by which HIF-1alpha increases. However, prolonged hypoxia decreases HIF-1alpha, and the underlying mechanisms currently remain unclear. Cellular reactive oxygen species (ROS) increases in cancer and inflammation. In the present study, we demonstrated that prolonged hypoxia increased ROS, which induced prolyl hydroxylase domain-containing protein 2 (PHD2) and factor inhibiting HIF-1 (FIH-1), major regulators of HIF-1alpha. Cellular stress response (CSR) increased HIF-1alpha transcriptional activity by scavenging endogenous ROS. PHD2 and FIH-1 were induced by external hydrogen peroxide (H 2 O 2 ) but were suppressed by ROS-scavenging catalase. We investigated the mechanisms by which PHD2 and FIH-1 are regulated by ROS. The knockdown of HIF-1alpha decreased PHD2 and FIH-1 mRNA levels, suggesting their regulation by HIF-1alpha. We then focused on redox factor-1 (Ref-1), which is a regulator of HIF-1alpha transcriptional activity. The knockdown of Ref-1 decreased PHD2 and FIH-1. Ref-1 was regulated by ROS. Prolonged hypoxia and the addition of H 2 O 2 induced the expression of Ref-1. Furthermore, the knockdown of p65, a component of kappa-light-chain enhancer of activated B cells (NF-κB), efficiently inhibited the induction of Ref-1 by ROS. Collectively, the present results showed that prolonged hypoxia or increased ROS levels induced Ref-1, leading to the activation of HIF-1alpha transcriptional activity, while the activation of HIF-1alpha via Ref-1 induced PHD2 and FIH-1, causing the feedback of HIF-1alpha. To the best of our knowledge, this is the first study to demonstrate the regulation of HIF-1alpha via Ref-1 by ROS.

Docosahexaenoic acid (DHA) has been shown to have neuroprotective effects in Parkinson's disease, but the underlying mechanism has not been fully elucidated. DHA is metabolized to DHA epoxides (EDPs) and hydroxides by cytochrome P450s (P450s), and EDPs are further hydroxylated to the corresponding diols, dihydroxydocosapentaenoic acids (DHDPs) by soluble epoxide hydrolase (sEH). In the present study, we investigated the roles of these DHA metabolites in the beneficial effects of DHA supplementation on a rotenone-induced rat model of Parkinson's disease. Metabolite analysis by LC-MS revealed that CYP2A1, 2C11, 2C13, 2C23, and 2E1 contributed to the formation of EDPs, and these P450s and sEH were expressed in the rat brain. We found that DHA supplementation in rats improved the motor dysfunction induced by rotenone. In addition, DHA reversed the decrease in tyrosine hydroxylase and the increase in lipid peroxidation generated by rotenone in the striatum. DHA supplementation also induced mRNA expression of antioxidant genes, such as sod1 and catalase, and Nrf2 protein expression in the striatum. However, these effects of DHA supplementation were eliminated by cosupplementation with the sEH inhibitor TPPU. Supplementation with DHA increased the amount of 19,20-DHDP in the rat brain, while the amount of EDPs was not significantly increased. In addition, TPPU suppressed the increase in DHDPs and increased EDPs in the brain. In PC12 cells, 19,20-DHDP increased the mRNA levels of sod1 and catalase along with Nrf2 induction. This study suggests that DHA metabolites-DHDPs generated by P450s and sEH-have an important role in improving rotenone-induced Parkinson's disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical transcription factor that orchestrates cellular responses to oxidative stress. Because the dysregulation of Nrf2 has been implicated in many diseases, precise regulation of its protein level is crucial for maintaining homeostasis. Kelch-like-ECH-associated protein 1 (Keap1) and WD40 repeat protein 23 (WDR23) directly regulate Nrf2 levels via similar but distinct proteasome-dependent pathways. WDR23 forms a part of the WDR23-Cullin 4A-RING ubiquitin ligase complex (CRL4A WDR23 ), whereas Keap1 serves as a substrate adaptor for the Cullin 3-containing ubiquitin ligase complex. However, the mechanisms underlying crosstalk between these Keap1 and WDR23 pathways for the regulation of Nrf2 levels have not been investigated. Here, we showed that knockdown (KD) of Keap1 upregulated the expression of Cullin4A (CUL4A) in a specificity protein 1 (Sp1)-dependent manner. We also revealed that Sp1 interacted with Keap1, leading to ubiquitination of Sp1. Increases in Sp1 by Keap1 KD triggered Sp1 binding to the fourth Sp1 binding site (Sp1&#95;M4) within the -230/+50 region of the CUL4A gene. We also demonstrated that the overexpression and KD of Sp1 reduced and increased Nrf2 protein levels, respectively. These effects were abrogated by the WDR23 KD, suggesting that Sp1 also regulates Nrf2 levels via the ubiquitin ligase complex CRL4A WDR23 . In conclusion, we discovered Sp1 as a novel substrate of Keap1 and provided evidence that Sp1 regulates the expression of CUL4A. We revealed a novel role for Sp1 in mediating crosstalk between two independent regulators of Nrf2 protein levels., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bisphenol A (BPA) is an endocrine-disrupting chemical used in polycarbonate and epoxy resins. Previously, we found that BPA stabilized the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) by inducing Ca 2+ efflux into the cytosol, followed by nitric oxide synthase activation, resulting in the enhanced nitrosylation of Keap1, which is a negative regulator of Nrf2. However, the mechanisms behind the stimulation of Ca 2+ efflux by BPA remain unknown. In the present study, we found that BPA stimulated Ca 2+ efflux into the cytosol from the ER, but not from outside of cells through the plasma membrane in Hep3B cells. Ca 2+ efflux and Nrf2 stabilization by BPA were inhibited by an inhibitor of the inositol 1,4,5-trisphosphate (IP 3 ) receptor, 2-aminoethoxydiphenylborane, in the endoplasmic reticulum. IP 3 is produced by activation of phospholipase C (PLC) from a membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP 2 ). The induction of Nrf2 by BPA was not inhibited by a PLC inhibitor, U-73122, suggesting that BPA does not induce the production of IP 3 via PLC activation. We found that BPA bound directly to the IP 3 binding core domain of the IP 3 receptor, and BPA competed with IP 3 on this site. In addition, overexpression of this domain of the IP 3 receptor in Hep3B cells inhibited the stabilization of Nrf2 by BPA. These results clarified that the IP 3 receptor is a new target of BPA, and that BPA induces Ca 2+ efflux from the endoplasmic reticulum via activation of the IP 3 receptor.

Nrf2 plays a central role in the response to xenobiotics and oxidative stress. The activation of Nrf2 induces the expression of drug-metabolizing enzymes (DMEs) and is important for cytoprotection. Keap1 is a widely accepted proteasome-dependent regulator of Nrf2. Keap1 was reported to be absent in Caenorhabditis elegans, and the level of the Nrf2 ortholog SKN-1 was mainly regulated by WDR23. The WDR23 locus is highly conserved from C. elegans to humans. We investigated whether WDR23 regulates Nrf2 activity in mammalian cells, hepatocellular carcinoma cells (Hep3B) and human cervical carcinoma cells (HeLa). We found that WDR23 has two isoforms (1 and 2) and that knockdown of WDR23 was sufficient to stabilize Nrf2 and alter the expression of several DMEs. Keap1 knockdown resulted in higher Nrf2 levels than WDR23 knockdown, and their effects on DMEs differed. These results were consistent with Keap1 being a canonical regulator of Nrf2, and that WDR23 may assist in Nrf2 regulation. We confirmed that WDR23 physically interacted with Nrf2, suggesting that WDR23 directly regulates Nrf2-dependent DMEs. In immunostaining experiments, human WDR23 isoform 1 was localized to the cytoplasm, whereas isoform 2 mainly resided in the nucleus. Taken together, our results suggested WDR23 is a novel regulator of DME expression., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of protein disulfide isomerase (PDI) is increased in brains with sporadic Alzheimer's disease and Parkinson's disease. The aim of the present study was to clarify the role of nitric oxide (NO) in BPA-induced neurotoxicity. Since rotenone induces NO-mediated neurodegeneration through S-nitrosylation of PDI, it was used as a positive control. First, rats were treated with BPA and rotenone, and S-nitrosylation of PDI was detected in rat brain microsomes. BPA and rotenone decreased RNase oxidation activity of PDI concomitant with S-nitrosylation of PDI. Next, to clarify S-nitrosylation of PDI by BPA and rotenone in rat brains, we treated the rat pheochromocytoma cell line PC12 and primary cultured neuron cells from the rat hippocampus with BPA (5 and 10 μM) and rotenone (100 or 200 nM). BPA induced S-nitrosylation of PDI, while NG-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, exerted the opposite effects. Finally, to evaluate the toxicity of BPA in the CNS, we investigated its effects on neurite outgrowth of PC12 and primary cultured neuron cells. BPA inhibited neurite outgrowth of these cells, while L-NMMA reversed this inhibition. The involvement of PDI activity in neurite outgrowth was also examined, and bacitracin, a PDI inhibitor, is shown to decrease neurite outgrowth. Furthermore, the overexpression of PDI, but not a catalytically inactive PDI mutant, enhanced neurite outgrowth. These results suggested that S-nitrosylation of PDI induced by excessive NO caused BPA-induced neurotoxicity.

TMX2 is a thioredoxin family protein, but its functions have not been clarified. To elucidate the function of TMX2, we explored TMX2-interacting proteins by LC-MS. As a result, importin-β, Ran GTPase (Ran), RanGAP, and RanBP2 were identified. Importin-β is an adaptor protein which imports cargoes from cytosol to the nucleus, and is exported into the cytosol by interaction with RanGTP. At the cytoplasmic nuclear pore, RanGAP and RanBP2 facilitate hydrolysis of RanGTP to RanGDP and the disassembly of the Ran-importin-β complex, which allows the recycling of importin-β and reentry of Ran into the nucleus. Despite its interaction of TMX2 with importin-β, we showed that TMX2 is not a transport cargo. We found that TMX2 localizes in the outer nuclear membrane with its N-terminus and C-terminus facing the cytoplasm, where it co-localizes with importin-β and Ran. Ran is predominantly distributed in the nucleus, but TMX2 knockdown disrupted the nucleocytoplasmic Ran gradient, and the cysteine 112 residue of Ran was important in its regulation by TMX2. In addition, knockdown of TMX2 suppressed importin-β-mediated transport of protein. These results suggest that TMX2 works as a regulator of protein nuclear transport, and that TMX2 facilitates the nucleocytoplasmic Ran cycle by interaction with nuclear pore proteins.

Borophene and the analogs are attractive 2D-materials showing unique mechanical and electronic properties. In this study, the bottom-up synthesis of an atomic boron network possessing a completely planar skeleton was achieved from KBH 4 . The borophene-analog was stabilized by oxygen atoms positioned on the same plane, providing holes and the anionic state of the layer. Potassium cations between the layers enabled crystalline stacking of the layers, as well as dissolution in solvents as atomically thin layers. The conductivity measurements revealed the electronic feature. Unlike the interplane conducting property, almost zero activation energy like a metal was suggested from the in-plane measurement.