Japan Society For The Promotion Of Science Research Articles

In vivo dynamics of hydrogen peroxide and regulation of antioxidant gene expression in mouse skeletal muscle under cooling stimulus

Introduction: The dynamics of reactive oxygen species (ROS) in the cytosol and mitochondria function as signaling factors that regulates diverse cellular adaptations. Hydrogen peroxide (H2O2) is considered one of the most influential ROS in regulating physiological function; being a ubiquitous molecule with a long half-life and high diffusing capacity compared to other ROS (e.g., superoxide and hydroxyl radicals). Temperature changes within skeletal muscle myocytes constitute an environmental factor that modulates the expression of antioxidant-related genes. Although muscle temperature reduction by icing is a typical physical therapy modality, the relationship between cooling stimuli and in vivo H2O2 dynamics remains unclear. The purpose of this investigation was to elucidate the H2O2 dynamics in the cytosol and mitochondria induced by cooling stimuli and to determine the relationship between these dynamics and the expression of antioxidant-related genes. Specifically, we tested the hypothesis that cooling would reduce [H2O2] in intracellular compartments (cytosol and mitochondria) and suppress antioxidant-related gene expression levels. Methods: Experiment 1. in vivo H2O2 dynamics observation: Cytosolic and mitochondrial H2O2-sensitive fluorescent proteins (HyPer7, n = 6 and MLS-HyPer7, n = 9) were expressed by electroporation in the anterior tibialis muscle (TA) of male C57/BL6 mice (9-14 week of age). The TA was exposed under anesthesia, and cytosolic and mitochondrial [H2O2] were measured when the muscle temperature was reduced from 35 °C to 0 °C using in vivo bioimaging techniques. Experiment 2. Mice (9-12 week of age) were divided into an icing (ICE) group that received cooling stimulation (muscle temperature reduced from 35 °C to 13 °C and back to 35°C, total 6 sets for 60 min, n = 6) and a control (CONT) group (muscle temperature 35 °C for 60 min, n = 6). At 3 hours after temperature intervention, the TA was removed and mRNA levels were measured by Real-time PCR. Results: Experiment 1. In vivo [H2O2] in each compartment were significantly lower in the cytosol (over the range 28-6°C) and mitochondria (over the range 29-1°C) compared to 35 °C conditions. A decreasing phase of [H2O2] in both compartments was observed from 35 °C to around 13 °C, but, subsequently increasing [H2O2] were observed in the lower temperature range (i.e., 12 to 0 °C). Experiment 2: The ICE group significantly increased mRNA levels of antioxidant enzyme-related genes Nfe2l2 and HSF1 compared to CONT (1.2 ± 0.1-fold and 1.1 ± 0.1-fold, respectively, P < 0.01). On the other hand, mRNA levels of mitochondria-related genes such as PGC1α and COX4 were unchanged. Conclusions: This investigation is the first to reveal the H2O2 and related gene expression impact of cooling stimuli in an in vivo model. Cooling mouse skeletal muscle produced temperature-dependent decreases in [H2O2] that reversed with more severe cooling. In both cytosolic and mitochondrial compartments, the lowest point for [H2O2] was found to be 13°C. Repeated cooling cycles (between 35 and 13 °C) with their attendant decreasing [H2O2] elevated antioxidant-related mRNA levels. These results support that icing therapy may improve the antioxidant capacity of muscle despite decreasing muscle ROS levels. This study was supported in part by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) for Grant-in-Aid for JSPS Fellows (no. JP23KJ0967) and JSPS KAKENHI Grant (No. JP20H04074). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of endurance training under calorie restriction on the metabolic enzyme activity and transporter levels in mouse skeletal muscle and liver

Although endurance training (ET) and calorie restriction (CR) induce metabolic adaptations in skeletal muscle, the interaction of them have yet to be clarified. This study investigated the effects of endurance training with calorie restriction on the metabolic enzyme activity and transporter levels in mouse skeletal muscle and liver. Ten-week-old male ICR mice were fed ad libitum or subjected to 30% CR. The animals were subdivided into sedentary and ET groups. The animals in the ET group performed treadmill running (20 m/min, 30 min) five days a week for five weeks. Respiratory gas analysis during 24 h of rest showed that CR, but not ET, reduced carbohydrate consumption. While ET enhanced hexokinase (HK) activity in the plantaris and soleus muscles, CR decreased HK, phosphofructokinase, and lactate dehydrogenase activities and monocarboxylate transporter (MCT) 4 protein content. Although ET and CR alone increased citrate synthase (CS) activity and MCT1 protein content in the plantaris muscle, no additive or synergistic effects were observed. In the soleus muscle, ET increased CS, β-hydroxyacyl-CoA dehydrogenase (β-HAD), and total carnitine palmitoyltransferase (CPT) activities; however, CR significantly decreased CS and β-HAD activities but enhanced total CPT activity. Additionally, CR enhanced the glucose transporter 4 protein content in the plantaris and soleus muscles. In the liver, CR enhanced the protein levels of glucose 6-phosphatase, fructose 1,6-bisphosphatase, and mitochondrial phosphoenolpyruvate carboxykinase. Moreover, ET partially normalized the CR-induced increase in fatty acid synthase and acetyl-CoA carboxylase. These observations suggest that CR enhances the capacity for glucose and fatty acid synthesis in the liver as well as the glucose and lactate uptake capacity in skeletal muscle while reducing carbohydrate consumption in skeletal muscle. It is suggested that CR partially interferes with skeletal muscle adaptations to ET. This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant numbers 20H04071, 21K21249, and 23K16718). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Morphometric evaluation of mitochondrial networks in regenerating myofibers using photothermal microscopy

Background: Mitochondria constitute their interconnected network through dynamic mitochondrial processes such as fission and fusion in response to changes in their intracellular environment, such as energy demand. Transmission electron microscopy is the standard method for evaluating mitochondrial morphology. We have developed a simple method to evaluate mitochondrial morphology using tissue sections for bright-field observation. In this study, we attempted to image light-absorbing molecules (cytochrome c) in mitochondria using photothermal (PT) microscopy. Purpose: This method can observe an entire myocyte, so this study attempted to evaluate the entire mitochondrial network of immature (regenerating) myocytes during the skeletal muscle regeneration process. Methods: The gastrocnemius muscle of anesthetized male Wistar rats (13 weeks old) was subjected to 300 controlled eccentric contractions (ECC) using electrical stimulation. 7 days after ECC loading, in situ perfusion fixation was performed, and transverse sections (1 μm) from red (deep regions) and white (superficial regions) were prepared after Epon embedding. The sections were observed by PT microscopy, and quantitative analysis of mitochondrial morphology was performed. Mitochondrial content was expressed as a percentage of the fiber total area in transverse sections. The PT imaging system uses lasers with wavelengths of 515 nm and 638 nm constituting the PT pump and the probe beams, respectively. Results: After 7 days of ECC, many myofibers with small fiber diameters and central nuclei, which are characteristic of the recovery process after muscle damage, were observed. The cross-sectional area of the regenerating muscle (superficial region 389.9 ± 15.2 μm2, deep region 366.2 ± 17.5 μm2) was significantly smaller than that of the normal muscle (superficial region 2211.1 ± 127.3 μm2, deep region 1396.1 ± 72.9 μm2). Mitochondrial content of the regenerating muscle (superficial region 26.1 ± 1.1%, deep region 22.7 ± 0.8%) was significantly lower than that of the normal muscle (superficial region 31.8 ± 1.9%, deep region 34.9 ± 1.8%). In normal muscle, a linear network of mitochondria along myofibrils was observed. On the other hand, no spatially distributed mitochondria were observed in the cytoplasm of the regenerating muscle. Furthermore, a mitochondrial network was formed around the periphery of the central nucleus. Conclusion: In muscle tissue imaging using PT microscopy, myofibers in the regenerative process have less mitochondrial content than mature muscle fibers and lack the mitochondrial network along myofibrils that characterizes mature muscle. On the other hand, the central nucleus of immature muscle was surrounded by highly developed mitochondria. The mitochondrial morphological characteristics of regenerating muscle fibers may offer a unique window through which to better understand mitochondrial design and function and their plasticity during maturation. This study was supported in part by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) KAKENHI Grant (No. JP20H04074, 21K19703). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Spatial and single-cell colocalisation analysis reveals MDK-mediated immunosuppressive environment with regulatory T cells in colorectal carcinogenesis

Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival. We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients. CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients. MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC. Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund.

Cultural engagement and prevalence of pain in socially isolated older people: a longitudinal modified treatment policy approach

It remains uncertain whether cultural engagement positively influences the reduction of pain risk, particularly depending on the social isolation status. The aim of this study was to examine the impact of cultural engagement on the reduction of pain prevalence over a 6-year follow-up period among older people, particularly those experiencing different dimensions of social isolation. This study was a prospective longitudinal study. We analysed the English Longitudinal Study of Ageing cohort, consisting of 6468 community-dwelling adults aged ≥50 years old who provided data in waves 6 (2012-2013), 7 (2014-2015), 8 (2016-2017), and 9 (2018-2019). Self-reported cultural engagement (going to museums, art galleries, exhibitions, the theatre, concerts, or the opera) measured in waves 6-8 was used as the exposure variable. Meanwhile self-reported moderate-to-severe pain in wave 9 was used as the outcome variable. Social isolation was considered in waves 6-8, and the possibility of effect modification was captured by assessing each component of the social isolation index: not married or cohabiting with a partner, fewer than monthly contact with children/other immediate family/friends, and not engaging in any organisations, religious groups, or committees. The estimated pain prevalence was 29.2% (95% confidence interval, 28.1-30.3; reference) after adjusting for time-variant, time-invariant, and loss to follow-up factors. Cultural engagement led to a reduction in pain prevalence to 24.1% for all individuals, representing a decrease of 5.1% (95% confidence interval, 0.6-9.6; P-value, 0.03). In older people who were not married or cohabiting, cultural engagement resulted in a decrease in pain prevalence to 25.8%, a reduction of 3.4% (95% confidence interval, 0.4-6.4; P-value, 0.01). For those with less frequent contact with close family members, the pain prevalence decreased to 25.3%, a reduction of 3.9% (95% confidence interval, 0.2-7.6; P-value, 0.03). Meanwhile, other dimensions of social isolation did not show a significant reduction in pain prevalence. Cultural engagement may help to reduce the risk of pain in socially isolated older adults. Those who were single or living alone and had less frequent contact with immediate family were particularly vulnerable. While cultural engagement might help certain socially isolated older people feel better, its effectiveness varies, highlighting the need for targeted interventions. The Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number (22K17648, Ikeda).

Design of Ni–Fe Hybrid Metal Hydroxide Nanomaterials As Self-Repairing Anode Catalysts For Alkaline Water Electrolysis

Introduction To use renewable energy efficiently, the conversion of renewable energy into hydrogen by alkaline water electrolysis has attracted much attention. When an alkaline water electrolyzer is powered by renewable energy, electrodes degrade due to reverse current generated on shutdown. To address this problem, we have reported the usefulness of a hybrid cobalt hydroxide nanosheet (Co-ns) as a self-repairing catalyst that keeps the oxygen evolution reaction (OER) ability under frequent potential fluctuations.[1] However, the OER activity of Co-ns is relatively lower than the state-of-the-art catalysts, such as Ni–Fe layered double hydroxides (LDHs). In this study, we synthesized hybrid Ni–Fe hydroxides with various compositions and structures, and their correlations with the OER activity. Experimental Hybrid Ni–Fe hydroxides were synthesized by mixing nickel chloride, iron chloride, and tris(hydroxymethyl)aminomethane (Tris-NH2) similar to the method to synthesize Co-ns.[2] Electrochemical measurements were performed in 1.0 M KOH, using a three-electrode cell made of PFA. A nickel plate, a nickel coil, and a reversible hydrogen electrode (RHE) were used as the working, counter, and reference electrodes, respectively. The chronoamperometry –0.5 V vs. RHE for 3 min was repeated to remove oxides from the Ni substrate surface. A dispersion of hybrid Ni–Fe hydroxide was added in the electrolyte to adjust the total metal concentration (Ni + Fe) at 40 ppm. Catalysts were deposited by repeating the constant current electrolysis at 800 mA cm- 2 for 30 min, cyclic voltammetries (CVs) and electrochemical impedance spectroscopy for the evaluation of OER activities for 8 times (total electrolysis time is 240 min). Results and discussion The synthesis of hybrid Ni–Fe hydroxides were confirmed by XRD, FTIR, elemental analyses, and XAFS. The structure of products were layered, amorphous, and tunnel structures, depending on the metal composition (represented by Ni/(Ni + Fe)) and the degree of modification (represented by Tris-NH2/(Ni + Fe)). Ni/(Ni + Fe) tends to be high when the concentration of metal salts was low and the concentration of Tris-NH2 was high in the preparation solution. The oxidation states of Ni and Fe in all samples were +2.1 and +3.0, respectively.The amount of catalyst deposited on the electrode by the electrolysis was confirmed by elemental analyses. The amount of deposited catalyst increased along with Ni/(Ni + Fe) (Fig. 1). The lowest deposition amount was 2.74 µg cm- 2 (Ni/(Ni + Fe)=0) and the highest one was 41.5 µg cm– 2 (Ni/(Ni + Fe)=0.82), whereas the values were still lower than that of Co-ns (88.7 µg cm– 2). In our previous study, the oxidation of Co2+ to Co3+ on an electrode surface is crucial for the electrochemical deposition of Co-ns.[3] Thus, it is reasonable that the content of divalent cation (Ni2+) is related with the deposition amount.From the OER measurements, the mass activity of catalysts was defined as the current at 1.53 V vs. RHE normalized by the amount of deposited catalyst (i m,1.53). i m,1.53 decreased along with the increase in Ni content (Fig. 2). The increase in Ni content is beneficial to the electrochemical deposition, though it is negatively correlated with the OER activity. Therefore, there is a trade-off between the amount of deposited catalyst and mass activity. The highest geometrical OER current density was obtained for Ni/(Ni+Fe) = 0.82 and i 1.53 = 296 mA cm–2 (Fig. 3). The deposited amount of catalyst significantly contributed to the highest activity of the electrode. Conclusion In this study, we synthesized Ni–Fe hybrid metal hydroxides and demonstrated that the Ni content of the catalysts affect both its deposition behavior and oxygen evolution reaction activity. The highest Ni content catalyst showed the highest activity, indicating a trade-off between the amount of catalyst deposited and mass activity. Acknowledgement A part of this study was supported by KAKENHI (Grant-in-Aid for Scientific Research 20H02821) from Japan Society for the Promotion of Science (JSPS) Reference [1] Y. Kuroda, T. Nishimoto, S. Mitsushima, Electrochim. Acta, 323, 134812, (2019).[2] Y. Kuroda, T. Koichi, K. Muramatsu, K. Yamaguchi, N. Mizuno, A. Shimojima, H. Wada, K. Kuroda, Chem. Eur. J., 23, 5023, (2017).[3] R. Nakajima, T. Taniguchi, Y. Sasaki, Y. Nishiki, Z. Awaludin, T. Nakai, A. Kato, S. Mitsushima, Y. Kuroda, submitted. Figure 1

Male applicants are more likely to be awarded fellowships than female applicants: A case study of a Japanese national funding agency.

Scientific grant applications are subjected to scholarly peer review. Studies show that the success rates of grant applications are often higher for male than for female applicants, suggesting that gender bias is common in peer review. However, these findings mostly come from studies in Europe, North America and Australia. Here we report the analyses of gender-specific success rates of applications to the fellowships offered by Japan Society for the Promotion of Science (JSPS). Because we analyze the observational data (i.e., not experimental), our aim here is to describe the possible gender gaps in the success rates, rather than the examination of gender bias per se. Results show that the success rates are consistently higher for male applicants than for female applicants among five different fellowship categories. The gender gaps in the success rates varied significantly between research fields in some Fellowship categories. Furthermore, in some fellowship categories, the gender gaps were significantly associated with the representation of female applicants (both positive and negative correlations were found). Though the causes of the gender gaps are unknown, unintentional gender bias during the review process is suggested. Pre-application gender gaps may also be contributing to the gender gaps in success rates. At least some of the observed gender gaps were relatively small, which may be partly explicable by the designs of the review process. However, gender gaps or biases acting prior to the application, such as self-selection bias, may have reduced the superficial gender gaps in the success rates. Further investigations that control for the effects of covariates (e.g., scientific merits of each applicant, which were not accessible to us) and those of other funding agencies, especially of non-Western countries, are warranted.

Design of Self-Repairing Anode Catalysts for Alkaline Water Electrolysis with Both OER Activity and Durability Via Colloidal Self-Assembly

INTRODUCTION The demand of renewable energy is increasing, yet fluctuating power supply is one of the problems. Therefore, technology for energy storage such as alkaline water electrolysis is required. When an alkaline water electrolyzer is powered by renewable energy, electrodes degrade due to reverse current generated on shutdown.[1] We have reported self-repairing catalysts based on hybrid cobalt hydroxide nanosheets (Co-ns), and β-FeOOH nanorods (FeOOH-nr).[2,3] Our previous studies showed that Co-ns formed thick catalyst layer with high oxygen evolution reaction (OER) activity, whereas FeOOH-nr formed a thin layer with much higher OER activity than that of Co-ns. If the thickness of FeOOH-nr increases, the OER activity and durability are expected to be improved. Here, we demonstrate the design of composite catalysts, consisting of both Co-ns and FeOOH-nr. Co-ns promotes stacking of the catalysts on the electrode, and FeOOH-nr forms active sites. EXPERIMENTAL Co-ns[2] and FeOOH-nr[3] were synthesized according to the literature. Electrochemical tests were performed in a 1.0 M KOH, using a PFA three-electrode cell. A nickel plate, a nickel coil, and a reversible hydrogen electrode were used as the working, counter, and reference electrodes, respectively. The Co-ns dispersion, FeOOH-nr dispersion, or their mixture (total concentration of catalyst weights = 80 ppm) was added in the electrolyte. Catalysts were deposited by repeating the following processes 10 times according to our previous report:[2] i) chronopotentiometry at 800 mA cm– 2 for 30 min, ii) cyclic voltammetry (CV) between 0.5 and 1.8 V vs. RHE at 5 mV s– 1, iii) CV between 0.5 and 1.6 V vs. RHE at 50 mV s– 1, and iv) electrochemical impedance spectroscopy at 1.6 V vs. RHE. Furthermore, an accelerated durability test (ADT)[4] was conducted 2000 cycles to examine the degradation. RESULTS AND DISCUSSION The synthesis of Co-ns and FeOOH-nr was confirmed by XRD, FTIR, elemental analyses, and TEM. Co-ns is modified with tripodal ligand, whereas FeOOH-nr is non-modified. The electrophoretic mobilities of Co-ns and FeOOH-nr were 1.98 and –2.80 µm cm V– 1 s– 1 at pH 9.7. Because Co-ns and FeOOH-nr are oppositely charged, they are expected to be self-assembled into a composite in the electrolyte.The OER polarization curves during the repeated electrolysis are shown in the Fig. 1. The OER activity of the electrode prepared from the mixture of Co-ns and FeOOH-nr (Co-ns/FeOOH-nr molar ratio = 10) was higher than those of the electrodes prepared from only one component. The OER overpotential at 100 mA cm–2 (η 100) of the composite catalyst was 291 mV after 300 min of electrolysis. The η 100 of Co-ns and FeOOH-nr were 310 and 353 mV, respectively.The deposited amount of Fe (M Fe) and the η 100 as a function of the deposited amount of Co (M Co) is shown in the Fig. 2. The M Fe using the composite catalysts was much larger than that using FeOOH-nr only. The M Fe was 48.5 µg cm–2 when the Co-ns/FeOOH-nr ratio is 10, although it was 2.00 µg cm–2 when only FeOOH-nr was used. M Fe is correlated with the OER activity. The highest OER activity was obtained at Co-ns/FeOOH-nr = 10.The assembled structure of FeOOH-nr and Co-ns in the deposited catalyst layer was observed by TEM (Fig. 3). A rod-like FeOOH-nr was surrounded by sheet-like Co-ns. Thus, it was suggested that Co-ns was assembled with FeOOH-nr by the electrostatic interactions.The results of ADT with the composite catalyst dispersed in the electrolyte for self-repairing (Fig. 4) shows that there was little degradation after 2000 cycles of start-up and shut-down. Therefore, these composite catalysts have excellent stability. However, when no composite catalyst was dispersed in the electrolyte, meaning that no self-repairing occurred, η 100 gradually increased in the 2000 cycles. The degradation behavior was milder than that of the FeOOH-nr only; thus, the composite catalyst exhibited the improved stability of the FeOOH-based catalyst layer.In conclusion, the deposited amount of FeOOH-nr was successfully increased by the addition of Co-ns in the electrolyte to form composite catalysts. The electrostatic self-assembly is useful to combine the properties of different catalysts for a self-repairing system. ACKNOWLEDGEMENT A part of this study was supported by KAKENHI (Grant-in-Aid for Scientific Research 20H02821) from Japan Society for the Promotion of Science (JSPS). REFERENCE [1] Y. Uchino et al., Electrocatalysis 9, 67 (2018).[2] Y. Kuroda et al., Electrochim. Acta. 323, 1348122 (2019).[3] Y. Kuroda et al., J. Sol-Gel Sci. Technol., 104, 647-658 (2022).[4] A. Haleem et al., Electrochemistry. 89(2), 186 (2021). Figure 1

P-706 Non-invasive analysis of mitochondrial DNA mutation for embryo evaluation.

Abstract Study question Is it possible to amplify whole mitochondrial DNA (mtDNA) and detect all mtDNA mutation in human embryo from spent media after embryo culture? Summary answer We were able to amplify whole mtDNA from 43% of spent medium after 24-hour culture. The most of mtDNA mutation in embryo could be detected. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) with embryo biopsy is now commonly used to improve pregnancy rate. However, invasive embryo biopsy requires highly trained embryologist and induced higher risk of pre-eclampsia. Therefore, non-invasive methods with cell-free DNA in spent medium have been studied. Our group previously showed euploid or viable embryos at post-implantation stage have lower number of non-synonymous mtDNA mutations and this may help to select the best blastocyst to transfer. Although cell-free DNA is reported to contain mtDNA, it remains unclear whether mtDNA mutations number can be assessed from cell-free DNA. Study design, size, duration Donated low grade blastocysts were cultured for additional 24 hours with new media, cell-free mtDNA were obtained from the spent culture medium (non-invasive samples). For comparison, trophectoderm cells were biopsied from each embryo (invasive sample). The mtDNA selective amplifications were done to both samples. We performed mtDNA sequencing by next generation sequencing (NGS) and compared mtDNA mutations between non-invasive and invasive samples to clarify the capability of non-invasive assessment for mtDNA mutation. Participants/materials, setting, methods Under the ethical review of Yokohama City University and informed consent with patients, we collected human blastocysts which were discarded because of slow development or morphologically low-grade. We extracted whole DNA from biopsy and medium samples and selectively amplified mtDNA by long-range PCR for two overlapping amplicons. The mtDNA sequencing with NGS was performed to successfully amplified mtDNA. The 90% or more heteroplasmy level non-synonymous mutations were investigated. Main results and the role of chance We collected 34 discarded embryos from 13 patients. All embryos were cultured individually for additional 24 hours with 20 μl drop of newly prepared media. Amplified mtDNA were visualized by the presence of 8 kb band following agarose gel-apheresis for two fragments PCR. Both fragments were detected in 41% (14/34) of non-invasive samples and all invasive samples. Only one fragment was detected in 14% (5/34) of non-invasive samples. The completely amplified non-invasive samples and corresponding invasive samples were performed mtDNA sequencing with NGS. In results, 13 non-synonymous mutations were identified in invasive samples. All of these mutations were also detected in non-invasive samples. Although the number and location of mutations completely matched between the invasive and non-invasive samples in 78.5% (11/14) embryos, in other 3 embryos, mtDNA mutation numbers were higher in non-invasive samples even though no mutation existed in invasive samples. This means, if selective mtDNA amplification is completed, mtDNA mutations can be evaluated from the non-invasive samples with a probability of about 80% but include 20% false positive mtDNA mutations. To realize this, we need to improve the mtDNA amplification method. Limitations, reasons for caution We can used only clinically discarded embryos because ethical restriction. And it was necessary to additional 24-hours culture to obtain individually cultured spent media because embryos are co-cultured until they are donated. Because of small number of samples available, we keep collecting more samples for more accurate evaluation. Wider implications of the findings If the mtDNA mutations can be analyzed from spent medium, high-quality embryos can be selected without invasive embryo biopsy and complicated procedure. If this non-invasive mtDNA analysis can be performed to clinical embryo for embryo transfer, it may contribute to further improvement of pregnancy rate without any invasion to embryo. Trial registration number the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research C (Grant Number JP21K09474) and Early-Career Scientists (Grant Number JP20K18169)

CD8 +T-cell memory induced by SARS-CoV-2 mRNA vaccination is maintained by clonal replenishment

Abstract mRNA vaccines against the Spike glycoprotein of severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2) elicit strong CD8 +T-cell responses. However, in repetitive vaccination, it is unknown whether T-cell clones induced by the first dose respond repeatedly or new clones are mobilized and respond after each dose. Here, we temporally tracked the CD8 +T-cell repertoire in individuals who received three doses of the BNT162b2 mRNA vaccine. CD8 +T-cell clones that expanded after the first, second, or third dose were identified, and their kinetic profiles throughout the vaccination were analyzed. The extent of clonal expansion after the first vaccination was significantly less than that after the second or third vaccination. After the second vaccination, clones that had expanded after the first dose (“first responders”) were less responsive, while a new set of clones (“second responders”) greatly expanded and surpassed the first responders. After the third vaccination, the second responders expanded again, while the first responders did not. Moreover, we observed expansion of another set of clones that had not responded to the previous doses (“third responders”). Single-cell mRNA/protein/TCR analysis after the second vaccination revealed that the first-responder clones exhibited a terminally differentiated phenotype, whereas the second-responder clones exhibited an actively proliferating phenotype. These results suggest that CD8 +T-cell memory induced by repetitive mRNA vaccination is maintained by the mobilization of newly generated clones with proliferative potential. This work was supported by Japan Society for the Promotion of Science (JSPS) under Grant Number 17929397 and 20281832, and Japan Agency for Medical Research and Development (AMED) under Grant Number JP22fk0310509, JP22ama221306, and P21gm6210025.

Enrichement of type I interferon signalling in the colonic ILC2 under inflammatory condition

Abstract Background Group2 innate lymphoid cells (ILC2s) are known to contribute to a frontline defence against parasites and formation of allergic diseases, but little is known about the roles of ILC2s under intestinal inflammation including intestinal bowel diseases (IBD). In this study, we investigate the characteristics of ILC2s in health and colitis. Methods ILC2s were analysed in the mice with or without treatment of dextran sulphate sodium (DSS)-induced colitis, where surface markers and cytokine production was assessed by flow cytometry. The global gene expression of ILC2 in DSS-induced colitis and control mice were analysed by bulk RNA-sequencing (RNA-seq). Lastly, the mice was treated with anti-IFN-alpha/beta receptor1 (IFNAR1) antibody and then challenged to DSS-induced colitis. Results The number of ILC2s in the gastrointestinal tract was larger than that of spleen and liver in the healthy mice. At day 7 of DSS-induced colitis, IL-5 production from ILC2s was significantly reduced in flow cytometry data, and this was accompanied by enrichment of type I interferon (T1IFN) signalling in colitic ILC2 in RNA-seq data. Neutralisation of T1IFN signalling by anti-IFNAR antibody significantly increased severity of colitis. Conclusion Our data suggest that downregulation of Th2 cytokines and enrichment of T1IFN signatures in colitic ILC2s. T1IFN may stimulate ILC2 to induce colitis suppressive functions. This study was funded by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Transformative Research Areas(B): 21H05123; Advanced Research and Development Programs for Medical Innovation (AMED-CREST: 21gm1510002h0001, and 20gm1210001h0001; the Practical Research Project for Rare/Intractable Disease: 21ek0109556h0001)

MP05-11 INNOVATIVE URETERAL STENT DEVELOPMENT USING NOVEL BIO-LIKE MATERIALS WITH ABILITY TO PREVENT CRYSTAL ADHESION IN VITRO AND IN VIVO

You have accessJournal of UrologyCME1 Apr 2023MP05-11 INNOVATIVE URETERAL STENT DEVELOPMENT USING NOVEL BIO-LIKE MATERIALS WITH ABILITY TO PREVENT CRYSTAL ADHESION IN VITRO AND IN VIVO Kana Hayashi, Katsumi Shigemura, Kazuo Kumagai, Ralph Rolly Gonzales, Hiroshi Tanimoto, Young-Min Yang, Maeda Koki, Hideto Matsuyama, and Masato Fujisawa Kana HayashiKana Hayashi More articles by this author , Katsumi ShigemuraKatsumi Shigemura More articles by this author , Kazuo KumagaiKazuo Kumagai More articles by this author , Ralph Rolly GonzalesRalph Rolly Gonzales More articles by this author , Hiroshi TanimotoHiroshi Tanimoto More articles by this author , Young-Min YangYoung-Min Yang More articles by this author , Maeda KokiMaeda Koki More articles by this author , Hideto MatsuyamaHideto Matsuyama More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003216.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Ureteral stents used to prevent ureteral obstruction in urological patients present the challenge of ureteral stent obstruction due to urinary crystal adhesion. There is active research into the development of ureteral stents with surface coatings and smoothing of the surface to reduce foreign body adhesion. In this study, the novel ureteral stent coated with polydopamine (PDA), which is formed by the polymerization of dopamine and close to the substances existed in human body and so has a low recognition reaction as a foreign substance, were investigated for ability to prevent crystal (Calcium: Ca) adhesion. METHODS: The ureteral stent was incubated in artificial urine at 37°C for 3 weeks, and the amount of Ca deposition on the surface of the ureteral stent was compared between the PDA-coated stent and commercially available stents by microscopic observation and Inductively Coupled Plasma (ICP) analysis. As to in vivo study, in rat model of hypercalciuria induced by drinking ethylene glycol and ammonium chloride, stents were implanted in the bladder for one month and Ca deposition on the surface of the ureteral stent was determined. RESULTS: After immersion in artificial urine for 3 weeks, the PDA-coated ureteral stent showed less Ca deposition on the stent surface than the commercially available ureteral stent (p=0.0468). There was no significant difference in the increase in pH of the artificial urine in which the ureteral stent was immersed between the PDA-coated and commercially available ureteral stents. With respect to the rat experimental model with Ca including-urine, the PDA-coated ureteral stent had less Ca deposition on the stent surface than the commercially available stent (p=0.0412). No apparent safety-related adverse events were observed. CONCLUSIONS: Our novel PDA-coated ureteral stent showed less amount of Ca deposition on the stent surface than commercial stents in artificial urine. The experimental rat model with Ca including-urine also showed a similar data of less Ca deposition on the stent surface than commercial stent, suggesting that this novel stent is less likely to cause stent occlusion (WO/2022/176834). Source of Funding: This study was supported by the Uehara Memorial Foundation, a Grant-in-Aid for Research Activity Start-up (20K22979) from the Japan Society for the Promotion of Science (JSPS) and Suzuken Memorial Foundation (20-014) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e47 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kana Hayashi More articles by this author Katsumi Shigemura More articles by this author Kazuo Kumagai More articles by this author Ralph Rolly Gonzales More articles by this author Hiroshi Tanimoto More articles by this author Young-Min Yang More articles by this author Maeda Koki More articles by this author Hideto Matsuyama More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement PDF downloadLoading ...

MP01-10 A NOVEL AND FEASIBLE METHOD FOR QUANTIFYING TESTICULAR SPERAMATOGENESIS BY USING CREATINE CHEMICAL EXCHANGE SATURATION TRANSFER IMAGING

You have accessJournal of UrologyCME1 Apr 2023MP01-10 A NOVEL AND FEASIBLE METHOD FOR QUANTIFYING TESTICULAR SPERAMATOGENESIS BY USING CREATINE CHEMICAL EXCHANGE SATURATION TRANSFER IMAGING Sohei Kuribayashi, Shinichiro Fukuhara, Go Tsujimura, Takahiro Imanaka, Koichi Okada, Norichika Ueda, Kentaro Takezawa, Shigeyoshi Saito, Yusuke Takahashi, Hidetaka Kioka, and Norio Nonomura Sohei KuribayashiSohei Kuribayashi More articles by this author , Shinichiro FukuharaShinichiro Fukuhara More articles by this author , Go TsujimuraGo Tsujimura More articles by this author , Takahiro ImanakaTakahiro Imanaka More articles by this author , Koichi OkadaKoichi Okada More articles by this author , Norichika UedaNorichika Ueda More articles by this author , Kentaro TakezawaKentaro Takezawa More articles by this author , Shigeyoshi SaitoShigeyoshi Saito More articles by this author , Yusuke TakahashiYusuke Takahashi More articles by this author , Hidetaka KiokaHidetaka Kioka More articles by this author , and Norio NonomuraNorio Nonomura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The sperm retrieval rate in non-obstructive azoospermia (NOA) patients is still insufficient. To overcome this problem, an accurate noninvasive method of evaluating testicular spermatogenesis is needed. Chemical exchange saturation transfer (CEST) imaging is a new magnetic resonance imaging (MRI) technique that can image the distribution of trace substances in vivo. We focused on the potential role of creatine (Cr) in testes and hypothesized that Cr-CEST could indicate intratesticular spermatogenesis noninvasively. METHODS: We investigated the Cr-CEST signal in the testes of the testicular ischemia model and the partial testicular irradiation model. In the testicular ischemia model, we clamped the right spermatic cord from 6–42 hours. To evaluate spatial resolution, we performed Cr-CEST for a partial testicular irradiation model in which the lower half of the testes was irradiated with a single 6 Gy dose. RESULTS: In the testicular ischemia models, eight testicular ischemia models were serially imaged at two or three-time points and a total of 26 images of ischemic testis were obtained. The signal intensity of ischemic testis was significantly lower than that of controls (4.0±2.0 vs. 8.9±0.6, p<0.01, Figure 1). Correlation analysis revealed a strong linear correlation between the signal intensity decline and the duration of ischemia (r=0.96, p<0.01, Figure 1). In the partial irradiation model, the signal intensity in the irradiated area was significantly lower than that in the non-irradiated area (5.3± 1.4 vs 7.6±0.3, p<0.05, Figure 1). CONCLUSIONS: Cr-CEST noninvasively evaluate intratesticular spermatogenesis both qualitatively and spatially from this study. Cr-CEST would be used as feasible preoperative imaging for evaluating intratesticular spermatogenesis and a navigation system during microscopic intratesticular sperm extraction. Source of Funding: This research was supported by the Japan Agency for Medical Research and Development (AMED) (grant number: J210705035) and Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (grant number: A21H030670) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e5 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sohei Kuribayashi More articles by this author Shinichiro Fukuhara More articles by this author Go Tsujimura More articles by this author Takahiro Imanaka More articles by this author Koichi Okada More articles by this author Norichika Ueda More articles by this author Kentaro Takezawa More articles by this author Shigeyoshi Saito More articles by this author Yusuke Takahashi More articles by this author Hidetaka Kioka More articles by this author Norio Nonomura More articles by this author Expand All Advertisement PDF downloadLoading ...

Liver autophagy-induced valine and leucine in plasma reflect the metabolic effect of sodium glucose co-transporter 2 inhibitor dapagliflozin.

Sodium glucose co-transporter 2 (SGLT2) inhibitors are anti-diabetic drugs for type 2 diabetes that lower blood glucose levels and body weight. It is of special interest that SGLT2 inhibitors also improve liver metabolism and fatty liver. Liver is an important organ in regulation of energy metabolism, but the metabolic action of SGLT inhibitors in liver remains unclear. We investigated the factors associated with the beneficial effects of dapagliflozin, a SGLT2 inhibitor, in the liver after confirming its glucose-lowering and weight loss effects using an obesity and diabetes mouse model. We also performed clinical study of patients with type 2 diabetes to explore candidate biomarkers that reflect the beneficial action of dapagliflozin in the liver. In animal study, dapagliflozin induced autophagy in the liver (LC3-II to LC3-I expression ratio: P<0·05 vs. control), and valine and leucine levels were increased in plasma (P<0·01 vs. control) as well as in liver (P<0·05 vs. control). Thus, increased plasma valine and leucine levels are potential biomarkers for improved liver metabolism. Clinical study found that valine and leucine levels were markedly higher in patients treated with dapagliflozin (valine: P<0·05 vs. control, leucine: P<0·01 vs. control) than those not treated after one week intervention. Dapagliflozin improves liver metabolism via hepatic autophagy, and plasma valine and leucine levels may reflect its metabolic effect. AstraZeneca K.K., Ono Pharmaceutical Co., Ltd., Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan Society for the Promotion of Science (JSPS), Japan Agency for Medical Research and Development (AMED), Novo Nordisk Pharma Ltd., and Japan Foundation for Applied Enzymology, and MSD Life Science Foundation International.

Impact of the new 12C+12C reaction rate on presupernova nucleosynthesis* *Supported by the National Natural Science Foundation of China (11988101, 11890694), and the National Key R&D Program of China (2019YFA0405502). K. Nomoto is supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan, and the Japan Society for the Promotion of Science (JSPS) KAKENHI grant (JP17K05382, JP20K04024, JP21H04499)

The 12C+12C reaction rate plays an essential role in stellar evolution and nucleosynthesis. Nevertheless, the uncertainties of this reaction rate are still large. We calculate a series of stellar evolution models with the near solar abundance from the zero-age main-sequence through presupernova stages for initial masses of 20 M to 40 M . The 12C+12C reaction rates from two different studies are used in our investigation. One is the rate obtained using the Trojan Horse Method (THM) by Tumino et al. [Nature 557(7707), 687 (2018)], and the other was obtained by Mukhamedzhanov et al. [Physical Review C 99(6), 064618 (2019)] (Muk19). Then, comparisons of the nucleosynthesis and presupernova isotopic abundances are conducted. In particular, we find that in the C burning shell, models with the THM produce a smaller amount of 23Na and some neutron-rich isotopes than Muk19. The difference in the abundance ratios of Na/Mg, S/Mg, Ar/Mg, and K/Mg between the two models are apparent. We compare Na/Mg obtained from our theoretical presupernovae models with Na/Mg in stellar atmospheres observed with high-resolution spectra as well as from the latest galactic chemical evolution model. Although Na/Mg obtained using the THM is within 2σ of the observed stellar ratio, the theoretical uncertainty on Na/Mg introduced by the uncertainty of the 12C+12C reaction rate is almost equivalent to the standard deviation of astronomical observations. Therefore, a more accurate 12C+12C reaction rate is crucial.

Investigation of Band Structure in Strained Single Crystalline Si1-X Sn X

1. Background and purpose Silicon tin (SiSn) alloys are attractive candidate for the next-generation group-IV semiconductors. It is well known that Si and Ge change from indirect to direct transition types with the addition of Sn. Among them, SiSn alloys are expected to be applied for the near-infrared optical devices because it is predicted to become a direct band-gap appropriate for the optical communication with sufficient Sn content [1]. Although the Sn composition that changes from indirect to direct band-gap has been reported using several calculations, there are few reports of experimental results. In addition, the optical properties of single crystalline Si1-x Sn x have not been sufficiently clarified owing to the difficulty of crystal growth. In this study, we evaluated the optical properties to clarify the band structure of single crystalline Si1-x Sn x . 2. Experimental method Strained 30 nm-thick Si1-x Sn x films were grown on Si substrate by molecular beam epitaxy (MBE), and epitaxial growth was confirmed by X-ray diffraction (XRD) two-dimensional reciprocal space mapping (2DRSM) [2]. The Sn composition was determined by 2DRSM. The Sn fraction x of Si1-x Sn x samples used in this study were 0.005, 0.009, 0.018, 0.022, and 0.06. The spectroscopic ellipsometry measurements were performed with an incident angle of 70°, using a 70 W xenon lamp with a wavelength range of 200 to 1600 nm for the light source. The functions used in the analysis are the Tauc-Lorentz and Lorentz function. 3. Results and Discussion Figure 1(a) and (b) show real part and imaginary part of the complex dielectric functions for Si1-x Sn x (x=0.005 - 0.06) and pure-Si, respectively. From Fig. 1, it can be confirmed that the spectra shift with increasing Sn composition and the peaks appear around 1.8 eV and 2.8 eV for the samples with high Sn composition (2.2% and 6.0%). The peak shift characteristic with the addition of Sn is similar to the results of germanium tin (GeSn) [3].The complex dielectric functions of the semiconductor include much information about the electronic band structure. In Fig. 1, the sharp peaks that are due to direct band transitions show what is known as critical points (CPs). In the case of Si, E'0 CP (Γ) and E 1 CP (L) energy are around 3.2 eV [4]. Therefore, it can be considered that the peak at 2.8 eV indicates the separation of the E'0 and E 1 CP energies due to the change in the band gap at the Γ point with the addition of Sn. The peak at 1.8 eV is unique to Si1-x Sn x , which is not found in Si. We consider that this peak is due to the split-off valence band caused by the Sn addition. The behavior of the valence band is strongly affected by strain. These results suggest a reduction of the band gap at the Γ point and the formation of an optical transition region due to the Sn addition, and reveal important findings for the application of SiSn for the near-infrared devices. Acknowledgment This work was partly supported by the Japan Society for the Promotion of Science (JSPS) (17J08240, 19K21971, and 21H01366).

(Invited) Effect of Water Management in Membrane and Cathode Catalyst Layers on Suppressing the Performance Hysteresis Phenomenon in Anion-Exchange Membrane Fuel Cells

Anion exchange membrane fuel cells (AEMFCs) are vulnerable to water management problems, since water is produced at the anode and consumed at the cathode. Previously we found severe voltage losses when increasing the current density in an AEMFC with a commercial Fe–N–Cp cathode catalyst and an in-house-developed anion exchange ionomer (quaternized poly(arylene perfluoroalkylene), i.e., QPAF-4, developed in this laboratory (Figure 1)1,2. In the present work,3 we have clearly identified the problem as being related to water management and developed two approaches to alleviating the problem: i) by use of a thin hydrophilized membrane, the diffusivity of water at the surface was improved, and the severe I–V hysteresis was suppressed, despite the cell using an Fe–N–Cp cathode catalyst with high-water absorption (Figs. 2a and 3a); ii) the voltage loss was also alleviated by the use of a recently developed Fe–N–Cc catalyst with higher hydrophobicity, which decreased the absorption of back-diffusing water into the catalyst layer and increased the amount of water supplied to the reaction sites. These improvements have demonstrated that water transport is the main limitation for the previously reported hysteresis and provide strategies to achieve higher performance AEMFCs through proper water management and formation of water transport pathways (Figs. 2b and 3b). Acknowledgements This project was partly supported by the New Energy and Industrial Technology Development Organization (NEDO) Japan through funds for the “Advanced Research Program for Energy and Environmental Technologies,” by the Japan Society for the Promotion of Science (JSPS) and the the Swiss National Science Foundation (SNSF) under the Joint Research Projects (JRPs) program, and by the Japan Science and Technology (JST) through the Strategic International Collaborative Research Program (SICORP). We are grateful to Pajarito Powder supplying the Fe–N-Cp catalyst. Also, we are deeply grateful to Dr. Weilin Xu and Dr. Ping Song of Changchun Institute of Applied Chemistry and Dr. Shuanjin Wang of Sun Yat-sen University for supplying the Fe–N-Cc catalyst, and to Takahata Precision Co. Ltd. for kindly providing the QPAF-4 membrane and ionomer. References H. Ono, T. Kimura, A. Takano, K. Asazawa, J. Miyake, J. Inukai, K. Miyatake, Robust anion conductive polymers containing perfluoroalkylene and pendant ammonium groups for high performance fuel cells, J. Mater. Chem. A 5 (2017) 24804–24812, https://doi.org/10.1039/c7ta09409d.K. Otsuji, N. Yokota, D. A. Tryk, K. Kakinuma, K. Miyatake, M. Uchida, Performance hysteresis phenomena of anion exchange membrane fuel cells using an Fe–N–C cathode catalyst and an in-house developed polymer electrolyte, J. Power Sources, 487 (2021) 229407, https://doi.org/10.1016/j.jpowsour.2020.229407K. Otsuji, Y. Shirase, T. Asakawa, N. Yokota, K. Nagase, W. Xu, P. Song, S. Wang, D. A. Tryk, K. Kakinuma, J. Inukai, K. Miyatake, M. Uchida, J. Power Sources, 522 (2022) 230997, https://doi.org/10.1016/j.jpowsour.2022.230997 Figure 1

The impact of mitral regurgitation on the incidence of stroke in patients with atrial fibrillation

Abstract Background Stroke is a significant cause of mortality and a leading cause of disability in elderly patients. Atrial fibrillation (Af) is the most common cardiac disease with assumed embolic stroke. Therefore, patients with Af need anti-coagulant therapy with a Warfarin (vitamin K antagonist) or a DOAC (Direct Oral Anti-Coagulant), also considering the risk and benefit net balance. It has been suggested that significant mitral regurgitation (MR) and its vortex jet in the left atrium might reduce embolic stroke source in patients with Af. However, the effect of MR on the risk of stroke in patients with Af is still controversial. Therefore, we hypothesized whether MR might be associated with a lower risk of thromboembolic events in patients with Af. Purpose This study aims to investigate whether MR was associated with a decreased risk of stroke in patients with Af, especially in the elderly population. Methods We studied 1,714,955 patients in the LIFE Study database, comprising Japanese claims data for public insurance policyholders between 2014 and 2020. We identified 51,939 patients with new-onset Af. Among them, patients with pre-existing stroke patients (N=28,519) were excluded, and new-onset Af patients without stroke (N=23,420) were obtained. Based on the baseline information, the Af patients were stratified into the MR group (N=3,439) and Without-MR group (N=19,981). Propensity score matching (the nearest neighbor method with a caliper of 0.2) was performed to balance baseline features and stroke risk factors between the MR group (N=3439) and the Without-MR group (N=3439). Propensity scores were estimated using logistic regression with age, sex, and all factors in Elixhauser's comorbidity system. The incidence rate of the stroke (events per person-year) was calculated by the event numbers divided by the overall period for the observations months × 12. Kaplan-Meier curves of stroke-free survival were generated, and the two groups' comparisons were performed using the log-rank test. Results As expected, patients' characteristics were comparable between the two groups. The observation period was 3 years for both groups. In the MR group, 136 patients with stroke were detected. On the other hand, 122 patients with stroke were detected in the Without-MR group. The incidence rate of the stroke per person-year was 0.027 and 0.025 events per person-year in patients with the MR and Without-MR groups. In addition, the Kaplan-Meier analysis showed that the incidence rate of stroke was comparable between the two groups (P=0.43). Conclusions The frequency of stroke in patients with atrial fibrillation was similar, despite mitral regurgitation. Therefore, patients with atrial fibrillation should have anti-coagulation therapy, regardless of the presence or absence of mitral regurgitation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was supported by grants provided by the Japan Science and Technology Agency (JST) Fusion Oriented Research for disruptive Science and Technology (FOREST) Program (Grant Number: JPMJFR205J) and the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Program (Grant Numbers: JP20H00563 and JP19K21590).

Single-cell transcriptomics reveals an angiogenic cell population for therapeutic angiogenesis in adipose tissue

Abstract Background Therapeutic angiogenesis mediated by stem/progenitor cells is an attractive therapeutic option against cardiovascular disease (CVD). Adipose tissue (AT) can be safely obtained even in CVD patients with anti-platelet medications, and it is a readily available source of culture-expanded adipose-derived stem cells (ADSCs) for transplantation. Single-cell transcriptome enables us to screen all the surface markers at once, while conventional strategies have been limited for the number of target markers. Furthermore, gene profiling at single-cell resolution can be used for the quantification of each marker by how many favorable cells can be purified without mixing of detrimental cells. Purpose We aimed to identify and characterize a cell population with in vivo angiogenic potential by single-cell RNA sequencing (scRNA-seq) analysis and xenograft experiments. Methods We revisited scRNA-seq datasets of single cell fraction from AT, bone-marrow (BM), and umbilical-cord blood (UCB, n=6/organ) to find cell populations with pro-angiogenic potential. Next, we collected AT from CVD patients (n=23) and used multicolor flow cytometry to quantify and sort the specific populations. PBS, the specific marker-negative and unsorted ADSCs were used as controls. Xenograft models of PKH26 pre-labeled human ADSC transplantation in limb ischemia were used to evaluate the lectin capillary density, PKH+ engrafted ADSCs, and blood flow recovery. Results Clustering divided CD45–CD31–CD34+ progenitor fraction into 3 clusters. We identified pro-/anti-angiogenic clusters based on the expressions of well-known pro-/anti-angiogenic factors. All genes encoding cell-surface proteins were compared in this functional clustering, resulted in 17 markers screened (Fig. 1A, B). Taken together with enrichment analysis, CD271+ cells showed predominant and pro-angiogenic gene profile from the other top candidates including CD36 and CD54 (Fig. 1C, D). Next, we evaluated the number and gene profile of CD271+ cells in well-known stem cell sources including BM and UCB. Surprisingly, the number of CD271 expressing cells were significantly lower and did not show angiogenic gene profile in BM and UCB (Fig. 2A). In analysis of AT from 23 CVD patients, CD271+ cells were significantly decreased by donor insulin resistance (Fig. 2B). Cell therapy using CD271+ ADSCs demonstrated in vivo angiogenic capacity compared to those of CD271– ADSCs and PBS in limb ischemia model. Furthermore, CD271+ ADSC transplantation showed enhanced efficacy compared to unsorted ADSCs from the same donors (Fig. 2C–E). Conclusion In this study, we identified CD271+ cell population in AT as an angiogenic cell population through scRNA-seq analysis and cell therapy experiments. AT obtained from donors without insulin resistance would be the most suitable for CD271+ ADSC isolation. CD271+ ADSC transplantation with a promising angiogenic capacity could contribute better cell-based therapy tackling CVD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science (JSPS) KAKENHI (Tokyo, Japan)

Development of a new generation of ion beams

Ion beams have many applications. Junior Associate Professor Masahi Nojima, Research Institute of Science and Technology at the Tokyo University of Science, Japan, is working to create a new generation of ion beams. Already, he has successfully developed new techniques for producing focused ion beams (FIB). In his most recent work, Nojima has developed a metallic solution ion source that will be used to construct unknown functional materials by using ion beams made up of specific elements. The metallic solution ion source can generate a copy of an original structure using elementally selected materials. With support from the Japan Society for the Promotion of Science (JSPS), KAKENHI, the academic incentive system of the KIOXIA Corporation 2021 and the Toshiba Electric Devices &amp; Storage Corporation 2021, Nojima is seeking to open up new materials for use as ion sources utilising electrospray ionisation (ESI) methods. By harnessing the elements of FIBs primarily created from liquid metal ion source (LMIS) by field ionisation processes, Nojima hopes to create new generations of ion beams. To date, he and the team have displayed the result of mass separating metallic ion solution ion beams on random conditions and, looking ahead, intend to estimate the stopping range of mass selected ion beams in solid materials.