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Local anesthetics (LAs) inhibit endoplasmic reticulum-associated protein degradation, however the mechanisms remain elusive. Here, we show that the clinically used LAs pilsicainide and lidocaine bind directly to the 20S proteasome and inhibit its activity. Molecular dynamic calculation indicated that these LAs were bound to the β5 subunit of the 20S proteasome, and not to the other active subunits, β1 and β2. Consistently, pilsicainide inhibited only chymotrypsin-like activity, whereas it did not inhibit the caspase-like and trypsin-like activities. In addition, we confirmed that the aromatic ring of these LAs was critical for inhibiting the proteasome. These LAs stabilized p53 and suppressed proliferation of p53-positive but not of p53-negative cancer cells.

Background:The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). Methods: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. Results: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. Conclusions: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.

[Background] Subcutaneous injection of tumescent solution, which contains local anesthetic, adrenaline, and saline, before split-thickness skin graft harvesting, shows a significant hemostatic effect. This method can reduce the initial bleeding from the donor site. The aim of this study is to assess the benefits of controlling the bleeding from donor sites by tumescent injection. A randomized, controlled trial was performed to compare the wound healing of split-thickness skin graft donor sites treated with or without tumescent injection. [Methods] This randomized, controlled trial examined donor site healing days as the main measure of outcome. postoperative pain, donor site ulceration, and scar quality were evaluated as secondary outcome measures. Patients planned for split-thickness skin graft harvest were randomly assigned to receive either pre-harvest subcutaneous injection of local anesthetic, adrenaline, and saline solution (tumescent solution) (Group 1) or post-harvest application of adrenaline solution-soaked gauze to the skin graft donor sites (Group 2). Donor sites were treated with calcium alginate dressings after graft harvesting. On the 10th postoperative day, the dressings were removed and donor site healing were measured. Follow-up evaluation of scar quality was performed 6 months after surgery. Postoperative pain was evaluated on the 1st day after operating. [Results] Forty-five patients (26 males; average age 61.8 years) completed the late follow-up evaluation (6 months postoperatively), with 26 patients in group 1 and 19 in group 2. There were no significant differences between the two groups in any of the outcome measures. [Conclusion] Tumescent technique provides sufficient hemostasis in split skin graft donor sites, especially the initial bleeding just after graft harvesting, without any negative effects. Larger series should be studied to evaluate the effect in donor site wound healing.

Background: We examined whether a vascular smooth muscle cell (SMC) sheet is effective in the treatment of a rat myocardial infarction (MI) model. Methods: We examined the effect of SMC sheet on the cardiac function and cardiac remodeling in a rat MI model in comparison with their effect of dermal fibroblast (DFB) sheet in vivo. Furthermore, we estimated the apoptosis and secretion of angiogenic factor of SMC under hypoxic condition in comparison with DFB. Seven days after MI, monolayer cell sheets were transplanted on the infarcted area (SMC transplantation group, SMC-Tx; DFB transplantation group, DFB-Tx; no cell sheet transplantation group, Untreated; neither MI nor cell sheet transplantation group, Sham). We evaluated cardiac function by echocardiogram, degree of cardiac remodeling by histological examination, and secretion of angiogenic growth factor by enzyme immunoassay. Results: Twenty-eight days after transplantation, SMC-Tx showed the following characteristics compared with the other groups: 1) significantly greater fractional area shortening (SMC-Tx, 32.3 ± 2.1 %; DFB-Tx, 23.3 ± 2.1 %; untreated, 25.1 ± 2.6 %), 2) suppressed left ventricular dilation, smaller scar expansion, and preserved wall thickness of the area at risk and the posterior wall, 3) decreased fibrosis, preserved myocardium in the scar area, and greater number of arterioles in border-zone, 4) tight attachment of SMC sheets on the scarred myocardium, and less apoptotic cell death. In in vitro experiments, SMCs secreted higher amounts of basic fibroblast growth factor (SMC, 157.7 ± 6.4 pg/ml; DFB, 3.1 ± 1.0 pg/ml), and showed less apoptotic cell death under hypoxia. Conclusions: Our results illustrate that transplantation of SMC sheets inhibited the progression of cardiac remodeling and improve cardiac function. These beneficial effects may be due to superior SMC survival.

Serum uric acid (UA) is taken up by endothelial cells and reduces the level of nitric oxide (NO) by inhibiting its production and accelerating its degradation. Cytosolic and plasma xanthine oxidase (XO) generates superoxide and also decreases the NO level. Thus, hyperuricemia is associated with impaired endothelial function. Hyperuricemia is often associated with vascular diseases such as chronic kidney disease (CKD) and cardiovascular disease (CVD). It has long been debated whether hyperuricemia is causally related to the development of these diseases. The 2020 American College of Rheumatology Guideline for the Management of Gout (ACR2020) does not recommend pharmacological treatment of hyperuricemia in patients with CKD/CVD. In contrast, the Japanese Guideline on Management of Hyperuricemia and Gout (JGMHG), 3rdedition, recommends pharmacological treatment of hyperuricemia in patients with CKD. In a FREED study on Japanese hyperuricemic patients with CVD, an XO inhibitor, febuxostat, improved the primary composite endpoint of cerebro-cardio-renovascular events, providing a rationale for the use of urate-lowering agents (ULAs). Since a CARES study on American gout patients with CVD treated with febuxostat revealed increased mortality, ACR2020 recommends switching to different ULAs. However, there was no difference in the mortality of Japanese patients between the febuxostat-treated group and the placebo or allopurinol-treated groups in either the FEATHER or FREED studies.

【Background】Peripheral artery disease（ PAD） caused the poor prognosis of the patients. The life style intervention is necessary to protect patients from PAD. Fucoidan is the member of the polysaccharide originated from Mozuku sea weed, which is useful supplement for life style related disease. 【Purpose】We examined whether fucoidan could improve limb ischemic lesions in mice and rats. 【Method and Result】We created the ischemic limb model of mice and rats and evaluated their blood flow using the laser Doppler blood perfusion. We separated them into three groups, control group （without any treatment），post-treatment group （treated with fucoidan after ischemia for 4 weeks），and pre- and post-treatment group （treated with fucoidan before ischemia for 1 week and after ischemia for 4 weeks）．Fucoidan significantly improved the blood perfusion of ischemic limb in pre- and post-treatment group compared to those in control group. Neovascularization was remarkably increased in muscle of ischemic limb in pre- and post-treatment group compared to those in control group. Fucoidan did not improve either the blood perfusion or neovascularization in post-treatment group. 【Conclusion】Pretreatment with fucoidan through oral administration would be a promising supplement for protection from PAD.

[Background] Technical issues in free flap transfer, such as the selection of recipient vessels and the positioning and method of anastomosis of the vascular pedicle, have been the subject of vigorous debate. Recent developments in computational fluid dynamics (CFD) have enabled the analysis of blood flow within microvessels. In this study, CFD was used to analyze hemodynamics in a microanastomosis. [Methods] In the fluid calculation process, the fluid domain modelizes microvessels with anastomosis. The inlet flow conditions were measured as venous waveform, and the fluid is simulated as blood. Streamlines (SL), wall shear stress (WSS), and oscillatory shear index (OSI) at the anastomosis were visualized and analyzed for observing effects from the flow field. [Results] Some flow disruption was evident as the SL passed over the sutures. The maximum recorded WSS was 13.37 Pa where the peak of a suture was exposed in the lumen. The local maximum value of the OSI was 0.182, recorded at the base of the anastomosis on the outflow side. [Conclusion] In the ideal anastomosis, the SL is disrupted as little as possible by the sutures. The WSS indicated that thrombus formation is unlikely to occur at suture peaks, but more likely to occur at the base of sutures, where the OSI is high. Tight suture knots are important in microanastomosis.

The optimal range of serum uric acid (urate) associated with the lowest risk for developing cardiometabolic diseases is unknown in a generally healthy population. This 5-year cohort study is designed to identify the optimal range of serum urate. The data were collected from 13,070 Japanese between ages 30 and 85 at the baseline (2004) from the Center for Preventive Medicine, St. Luke's International Hospital, Tokyo. We evaluated the number of subjects (and prevalence) of those free of the following conditions: hypertension, diabetes, dyslipidemia, and chronic kidney disease (CKD) over 5 years for each 1 mg/dL of serum urate stratified by sex. Furthermore, the odds ratios (ORs) for remaining free of these conditions were calculated with multiple adjustments. Except for truly hypouricemic subjects, having lower serum urate was an independent factor for predicting the absence of hypertension, dyslipidemia, and CKD, but not diabetes. The OR of each 1 mg/dL serum urate decrease as a protective factor for hypertension, dyslipidemia, and CKD was 1.153 (95% confidence interval, 1.068-1.245), 1.164 (1.077-1.258), and 1.226 (1.152-1.306) in men; 1.306 (1.169-1.459), 1.121 (1.022-1.230), and 1.424 (1.311-1.547) in women, respectively. Moreover, comparing serum urate of 3-5 mg/dL in men and 2-4 mg/dL in women, hypouricemia could be a higher risk for developing hypertension (OR: 4.532; 0.943-21.78) and CKD (OR: 4.052; 1.181-13.90) in women, but not in men. The optimal serum urate range associated with the lowest development of cardiometabolic diseases was less than 5 mg/dL for men and 2-4 mg/dL for women, respectively., National Endowment for the Humanities; National Science Foundation; Netherlands Organization for Scientific Research (NWO); Institute for Aegean Prehistory; Loeb Classical Library Foundation; Merops Foundation; Boston University Vecchiotti Archaeology Fund; Catharine van Tussenbroek/Anneke Clason Zooarchaeology Grant