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In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Objectives The aim of this AFIRE (Atrial Fibrillation and Ischemic Events With Rivaroxaban in Patients With Stable Coronary Artery Disease) trial subgroup analysis was to examine rivaroxaban monotherapy benefits and their relation to the time between stenting and enrollment among patients after coronary stenting. Background Of 2,215 patients with atrial fibrillation and stable coronary artery disease in the AFIRE trial, rivaroxaban monotherapy was noninferior to rivaroxaban plus antiplatelet therapy (combination therapy) in terms of efficacy and superior for safety endpoints. However, thrombotic risk after antiplatelet therapy cessation remained a concern among 1,444 patients who had undergone coronary stenting >1 year before enrollment. Methods The benefits of rivaroxaban monotherapy in coronary stenting subgroups were assessed for efficacy (a composite of stroke, systemic embolism, myocardial infarction, unstable angina requiring revascularization, or death of any cause), safety (major bleeding defined according to International Society on Thrombosis and Haemostasis criteria), ischemic endpoints, net adverse clinical event, and time between stenting and enrollment. Results Efficacy and safety endpoints for monotherapy were superior to combination therapy, with HRs of 0.70 for efficacy (95% CI: 0.50-0.98; P = 0.036) and 0.55 for safety (95% CI: 0.33-0.92; P = 0.019). For ischemic endpoints, the HR was 0.82 (95% CI: 0.58-1.15; P = 0.240). The HR became smaller with longer time between stenting and enrollment (efficacy, P for interaction = 0.158; safety, P = 0.097). Conclusions In patients with atrial fibrillation after coronary stenting, the benefits of rivaroxaban monotherapy for efficacy and safety endpoints were consistent with those in the whole AFIRE trial population. The benefits became apparent with longer time between stenting and enrollment. (Atrial Fibrillation and Ischemic Events With Rivaroxaban in Patients With Stable Coronary Artery Disease Study [AFIRE]; UMIN000016612 , NCT02642419 )

Introduction Hypophosphatasia (HPP) is caused by mutations in the ALPL gene encoding tissue nonspecific alkaline phosphatase (TNSALP) and inherited in either an autosomal recessive or autosomal dominant manner. It is characterized clinically by defective mineralization of bone, dental problems, and low serum ALP levels. In the current report, we demonstrate a novel mutation in the ALPL gene (c.244G > A p.Gly82Arg) in a Japanese family with low serum ALP levels. Materials and methods The ALPL gene analysis using hybridization capture-based next-generation sequencing was performed. The expression plasmids of the wild type and mutated TNSALP were introduced into COS-7 cells. The enzymatic activity of ALP in the cell lysates was measured using p-nitrophenylphosphate as a substrate. Results TNSALP with the novel ALPL mutation (c.244G > A p.Gly82Arg) completely lost its enzymatic activity and suppressed that of wild-type TNSALP, corroborating its dominant negative effect. The diagnosis of autosomal dominant HPP was confirmed in three members of the family. Conclusion Our approach would help to avoid the inappropriate use of bone resorption inhibitors for currently mis- or under-diagnosed HPP, given that the presence of further, yet undetected mutations of the ALPL gene are plausible.

Low forced vital capacity (FVC) is associated with decreased exercise capacity in CHD. Multiple prior cardiac surgeries have been associated with low FVC. We seek to understand the relationship between low FVC, number of cardiac surgeries and cardiopulmonary response leading to decreased exercise capacity. Retrospective chart review of demographics, surgical history and exercise testing including spirometry was performed in patients with CHD. Single ventricle patients were excluded. Low FVC was defined as a Z-score below the lower limit of normal. Exercise parameters were expressed as a percent of predicted. There were 93 patients with 2 ventricle CHD identified over 34 months with cardiopulmonary exercise testing. The FVC Z-score directly correlated with peak VO2% (R2 = 0.07, p

Recently, mepolizumab, an interleukin (IL)-5 inhibitor, has been indicated for the treatment of eosinophilic granulomatosis with polyangiitis (EGPA) refractory to standard therapies. However, no reports have compared the efficacy of mepolizumab according to symptoms and organ lesions. Herein, we report two cases in which mepolizumab was highly effective in the management of EGPA with lung lesions and otitis media refractory to treatment with multiple immunosuppressive agents. These two cases suggest that mepolizumab is effective in treating pulmonary and ear lesions in EGPA.

Study question In the management of male infertility, we investigated whether urological consultation could improve the live birth rate, and who should visit urologists in the setting of IVF clinic. Summary answer Urologic consultation resulted in improvement of semen quality and live birth rate with more IVF use in those with adverse semen parameters. What is known already Male factor infertility exists in about a half of infertility couples. This accounts for about 8% in male reproductive age. Therefore, ideally every male partner of infertility couples attempting conception should have a urological evaluation. However, it is not very easy to access urologists who specialized in reproductive medicine in Japan because we have very few of such urologists. One the other hand, a certain number of couples are wasting their time during IVF failure without urological evaluation. Study design, size, duration This is a single-institution retrospective study. We enrolled male partners of infertility couples who visited Kameda IVF clinic Makuhari, Chiba, Japan, between May 2016 and December 2020 and followed at least one year. Live birth rate and the frequency of IVF use were investigated according to semen quality and urological consultation status. Chi-square tests and T tests were used to compare the results between groups. Participants/materials, setting, methods Among 2225 couples who visited Kameda IVF clinic Makuhari, 803 male partners (Group A, 36.0%) were evaluated by urologists who were specialized in male reproductive medicine. Remaining 1422 patients did not (Group B, 64.0%). Lifestyle evaluation, physical examination, semen analyses, scrotal ultrasonography, blood test including sexual hormones and zinc concentration were performed in Group A. Semen analyses and lifestyle evaluation were performed in Group B. Urological treatments were done according to factors of male infertility. Main results and the role of chance Semen quality was worse in Group A as compared to Group B (sperm motility, 28.5±16.9% vs. 46.0±17.0%; total sperm count, 105±108 million/mL vs. 176±155; total motile sperm count, 34±49 vs.87±98; mean±S.D.; p = 0.0001, 0.0001, 0.0001, A vs. B, respectively). After urologic consultation and managements, sperm motility was improved to 34±18% (p = 0.001). Live birth rate in groups A and B were similar (56.0% vs. 57.2%), however couples who obtained a child in Group A used IVF more often than those in Group B (70% vs. 49.9%, p 50 million, n = 900), 119 visited urologist (31.1%, Group 3) and 781 did not (Group 4). Live birth rate and the use of IVF were not different between Groups 3 and 4 (51.1% vs.60.9%; 50.4% vs. 62.9%). Limitations, reasons for caution This study is a single-institution, retrospective study in the setting of IVF clinic. There may be a selection bias since men first visit gynecologists. These could affect the study results. Wider implications of the findings In the setting of IVF clinic, urologic consultation resulted in improved semen quality and better live birth rate with the use of IVF, especially in those who have adverse semen parameters. The results of this study encourage patients to see urologists and physicians to introduce urologist to patients. Trial registration number not applicable

Study question What are the similarities and differences between the morphokinetics of abnormal, one- (1PN) and three-pronuclear (3PN) and normal bi-pronuclear (2PN) fertilisation? Summary answer The morphokinetic analysis of 1PN/3PN fertilisation reveals novel aspects of abnormal early development. What is known already Assisted reproduction technology has allowed the observation of early human development. Initially assessed statically at a single time point, fertilization has revealed its complexity once observed by Time-Lapse Microscopy (TLM). Detailed morphokinetic analysis of fertilisation has been reported in the last few years, unveiling previously unknown cytoplasmic phenomena (e.g. the cytoplasmic wave and halo) and the importance of cell symmetry for embryo development. At present, abnormal fertilization remains neglected, despite potential for understanding the physiology and pathology of early human development. Study design, size, duration This retrospective study involved TLM observation of normally (2PN, n = 2,685) and abnormally (1PN, n = 41; 3PN, n = 127) fertilised oocytes generated in ICSI cycles. Oocyte retrievals were carried out after the clomiphene citrate-based minimal ovarian stimulation, between October 2019 and December 2020. Oocytes of patients with different diagnoses of infertility were included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. Participants/materials, setting, methods Microinjected oocytes were assessed by a combined TLM-culture system (Embryoscope). Oocytes not suitable for TLM assessment, due to excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena, relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage pattern, and embryo quality, were annotated and compared between groups. Main results and the role of chance Second polar body (PBII) extrusion was observed in all 1PN- and in a majority of 3PN-zygotes (92.1%). A 0.3-hour delay in PBII extrusion was confirmed in 3PN-zygotes (P = 0.0439). In a significant proportion of 3PN-zygotes, a third (female) PN formed from reabsorption of the PBII. The cytoplasmic wave was observed not only in 2PN- and 3PN-, but also in 1PN-zygotes. The presence and position of cytoplasmic halo were comparable among the three classes of zygotes. However, the duration of the cytoplasmic halo was prolonged in 1PN-zygotes (P < 0.0001). PN juxtaposition immediately before PN breakdown was less frequent in 3PN- compared with 2PN-zygotes (P = 0.0159). Furthermore, asynchronous PN breakdown was increased in 3PN- compared with 2PN-zygotes (P = 0.0026). The PN area of 1PN- was larger than that of 2PN-zygotes; however, the PN area of 3PN-zygotes was smaller than that of 2PN-zygotes. In 1PN-zygotes, a developmental delay was observed starting from the disappearance of the cytoplasmic halo, reaching 9 hours at the time of cleavage (P < 0.0001). A higher incidence of abnormal cleavage (P = 0.0019) and blastomere fragmentation (P < 0.0001) was observed in 1PN-zygotes. Cleavage progression was increasingly affected especially in 1PN-zygotes, resulting in blastocyst formation rates of 70.2%, 12.2% and 53.5% in 2PN-, 1PN- and 3PN-zygotes, respectively (P < 0.0001). Limitations, reasons for caution The study data derive from treatments carried out in a single centre. The study findings therefore require independent verification from other research groups. Wider implications of the findings These observations suggest that 1PN and 3PN fertilisation follow the general pattern of normal fertilization. Crucially, they also shed light on diverse and previously undescribed phenomena - e.g. reabsorption of the PBII in 3PN zygotes - underpinning the origins of abnormal fertilization and potentially clinically relevant. Trial registration number not applicable

Collagen and Polylactic Acid (PLA) as biomaterials in the tissue engineering have been investigated for the structure and function in considerably large progress. From a viewpoint of its electronic structures in the subnano-meter range, we investigate the bonding nature for five models of the type I collagen, PLA models and the bimolecular interaction between the models by using Density Functional Theory (DFT) calculations with Frontier MO theory. In order to confirm the accuracy of the models, we compare the simulated IR and C1s X-ray photoelectron spectra with experimental results. Especially, the interaction between PLA and (AspHypGly)-collagen models was obtained as the chemical bond energy (1540 kJ/mol), and the results of PLA-other four collagen bimolecular models were given as intermolecular bond energies of 30 ∼ 81 kJ/mol. The intermolecular interaction is due to inter-H-bond of –OH---O=, -NH---O=, and –CH---O= functional groups, respectively, using MO calculations.

IntroductionHigh levels of treatment adherence are critical for achieving optimal treatment outcomes among patients with tuberculosis (TB), especially for drug-resistant TB (DR TB). Current tools for identifying high-risk non-adherence are insufficient. Here, we apply trajectory analysis to characterize adherence behavior early in DR TB treatment and assess whether these patterns predict treatment outcomes.MethodsWe conducted a retrospective analysis of Philippines DR TB patients treated between 2013 and 2016. To identify unique patterns of adherence, we performed group-based trajectory modelling on adherence to the first 12 weeks of treatment. We estimated the association of adherence trajectory group with six-month and final treatment outcomes using univariable and multivariable logistic regression. We also estimated and compared the predictive accuracy of adherence trajectory group and a binary adherence threshold for treatment outcomes.ResultsOf 596 patients, 302 (50.7%) had multidrug resistant TB, 11 (1.8%) extremely drug-resistant (XDR) TB, and 283 (47.5%) pre-XDR TB. We identified three distinct adherence trajectories during the first 12 weeks of treatment: a high adherence group (n=483), a moderate adherence group (n=93) and a low adherence group (n=20). Similar patterns were identified at 4 and 8 weeks. Being in the 12-week moderate or low adherence group was associated with unfavorable six-month (adjusted OR [aOR] 3.42, 95% CI 1.90 - 6.12) and final (aOR 2.71, 95% 1.73 - 4.30) treatment outcomes. Adherence trajectory group performed similarly to a binary threshold classification for the prediction of final treatment outcomes (65.9 % vs. 65.4 % correctly classified), but was more accurate for prediction of six-month treatment outcomes (79.4% vs. 60.0% correctly classified).ConclusionsAdherence patterns are strongly predictive of patient treatment outcomes. Trajectory-based analyses represent an exciting avenue of research into TB patient adherence behavior seeking to inform interventions which rapidly identify and support patients with high-risk adherence patterns.

Perivascular epithelioid cell tumor (PEComa) expresses melanocytic and smooth muscle markers. A man with a primary malignant cutaneous (distal left forearm) PEComa is reported. Immunohistochemistry demonstrated MiTF, HMB-45, caldesmon, desmin, and smooth muscle actin, as well as BCL1, CD10, and CD68. Next generation sequencing showed four pathogenic genomic aberrations involving BIRC3, FANCC, TP53, and TSC1 genes. His work-up was negative for metastatic disease; a wide local excision was performed. Including the reported patient, cutaneous PEComa has been described in 65 individuals: primary benign (N=58), primary malignant (N=5), and metastatic malignant (N=2). Cutaneous PEComa typically presented as a painless, slowly growing nodule oflt;2 centimeters on the lower extremity of a woman in her fifth decade. The neoplasms consisted of epithelioid cells, spindle cells, or both. The most reliable markers were MiTF (100%), HMB45 (94%), and NKIC3 (94%) for melanocytes and smooth muscle actin (43%) and desmin (40%) for smooth muscle. There has been no reported recurrence of a primary cutaneous benign or malignant PEComa after complete excision. Genomic alterations in malignant PEComas frequently involve TSC1 and TSC2 genes (mTOR activators), as well as TFE3 fusions. In November 2021, the FDA approved nab-sirolimus (mTOR inhibitor) for PEComas.

This article proposes a method to improve the thermoelectric conversion efficiency by controlling the heat flow direction of a thermoelectric element (TE) with a trapezoidal section. A thermoelectric generator (TEG) generally consists of a rectangular parallelepiped element. In theory, the thermoelectromotive force E can be easily estimated from the Seebeck coefficient α and the temperature difference ΔT, namely, E = αΔT. Seebeck coefficient α and resistivity ρ are known to have temperature dependence. We fabricated a TEG with a trapezoidal section which modified temperature distribution by TE shapes. It also provided many high Seebeck coefficient regions. As a result, the output power of TEG with a trapezoidal section was increased by 63% due to heat flow directions. It was also increased by 10% higher than the rectangular parallelepiped TEG at 400°C.

In addition to fatty acids, glucose and lactate are important myocardial substrates under physiologic and stress conditions. They are metabolized to pyruvate, which enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle metabolism. In the present study, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1−/−) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate and glycogen, and displayed increased protein O-linked N-acetylglucosamine, which was prevented by increasing availability of non-glucose substrates in vivo by a ketogenic diet (KD) or a high-fat diet, which reversed the structural, metabolic and functional remodelling of non-stressed cMPC1−/− hearts. Although concurrent short-term KDs did not rescue cMPC1−/− hearts from rapid decompensation and early mortality after pressure overload, 3 weeks of a KD before transverse aortic constriction was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function. Metabolic substrate partitioning underlies myocardial homeostatic capacity and adaptation to stress. Zhang et al. show that decreasing mitochondrial pyruvate carrier expression redirects glycolytic intermediates and leads to heart failure, which is prevented or reversed with a high-fat or ketogenic diet.

Objective Hydroxychloroquine (HCQ) has been prescribed in Japan only relatively recently and is recommended for the treatment of skin lesions, arthritis and renal lesions according to the Japanese Guideline for the Management of systemic lupus erythematosus (SLE) (2019). However, the associations between the efficacy and safety and the HCQ dose in Japanese SLE patients remain unclear. We investigated the efficacy and safety of different HCQ doses in Japanese SLE patients with a low disease activity who were not receiving immunosuppressants. Methods The disease activity was evaluated using the SELENA-SLEDAI 2011 criteria, the Cutaneous Lupus Erythematous Disease Area and Severity Index (CLASI) and serum biomarkers. Safety was evaluated via the frequency of adverse events over a period of three months. Results We enrolled 61 SLE patients treated with HCQ and no additional immunosuppressive therapy for more than 3 months. HCQ was administered to 46 patients at the usual dose and to 15 cases at a lower than usual dose. Although the CLASI activity scores decreased significantly in both groups, the magnitude of this decrease was larger in the usual-dose HCQ group than in the low-dose HCQ group. SLEDAI scores and immunological activity were significantly improved only in the usual-dose HCQ group. In addition, changes in the serum complement levels in the usual-dose HCQ group were more dramatic than in the low-dose HCQ group six months after the initiation of HCQ administration. Adverse events were more frequent in the usual-dose HCQ group than in the low-dose HCQ group (30.4% and 13.3%, respectively). Conclusion HCQ therapy is effective for maintenance therapy of SLE patients. The usual dose of HCQ may have some advantage in ameliorating low complement levels.

В статье рассматриваются проблемы в отношениях между Японией и Республикой Корея и, в частности, их спор относительно территориального суверенитета над островами Такэсима/Токто. Этот спор обострился с конца 1990-х годов, после того, как вступила в силу Конвенция ООН по морскому праву и страны Северо-Восточной Азии стали добиваться делимитации своих морских границ. Автор анализирует эволюцию проблемы Такэсимы/Токто в меняющихся международных условиях и в контексте двусторонних отношений с точки зрения объединяющих две страны общих интересов. С конца 1990-х годов Япония и Республика Корея пытаются сформировать порядок мирного совместного использования морских ресурсов, подписав в 1998 г. соглашение о рыболовстве. В 2015 г. страны также подписали двустороннее соглашение по обмену информацией военного характера с целью сотрудничества в противодействии угрозе, исходящей из Северной Кореи. Ни то, ни другое соглашение, однако, должным образом не действует ввиду эскалации напряженности между Японией и Республикой Кореей в 2010-е годы, которая вышла за рамки территориального спора за острова Такэсима/Токто. Произошедший в июле 2019 г. воздушный инцидент с участием китайских и российских самолетов показал, что нерешенная проблема морской границы может спровоцировать соперничество по поводу не только небольших островов и морских биоресурсов, но и контроля над воздушным пространством и зоной идентификации противовоздушной обороны, если он не регулируется общей правовой базой.

Vaso-occlusive crisis (VOC) is a hallmark of sickle cell disease (SCD) and occurs when deoxygenated sickled red blood cells occlude the microvasculature. Any stimulus, such as mental stress, which decreases microvascular blood flow will increase the likelihood of red cell entrapment resulting in local vaso-occlusion and progression to VOC. Neurally mediated vasoconstriction might be the physiological link between crisis triggers and vaso-occlusion. In this study, we determined the effect of mental stress on microvascular blood flow and autonomic nervous system reactivity. Sickle cell patients and controls performed mentally stressful tasks, including a memory task, conflict test and pain anticipation test. Blood flow was measured using photoplethysmography, autonomic reactivity was derived from electrocardiography and perceived stress was measured by the State-Trait Anxiety Inventory questionnaire. Stress tasks induced a significant decrease in microvascular blood flow, parasympathetic withdrawal and sympathetic activation in all subjects. Of the various tests, pain anticipation caused the highest degree of vasoconstriction. The magnitude of vasoconstriction, sympathetic activation and perceived stress was greater during the Stroop conflict test than during the N-back memory test, indicating the relationship between magnitude of experimental stress and degree of regional vasoconstriction. Baseline anxiety had a significant effect on the vasoconstrictive response in sickle cell subjects but not in controls. In conclusion, mental stress caused vasoconstriction and autonomic nervous system reactivity in all subjects. Although the pattern of responses was not significantly different between the two groups, the consequences of vasoconstriction can be quite significant in SCD because of the resultant entrapment of sickle cells in the microvasculature. This suggests that mental stress can precipitate a VOC in SCD by causing neural-mediated vasoconstriction.

Naive human induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with Sendai virus (SeV) vectors. However, only dermal fibroblasts have been successfully reprogrammed this way, and the process requires culture on feeder cells. Moreover, SeV vectors are highly persistent and inhibit subsequent differentiation of iPSCs. Here, we report a modified SeV vector system to generate transgene-free naive human iPSCs with superior differentiation potential. The modified method can be applied not only to fibroblasts but also to other somatic cell types. SeV vectors disappear quickly at early passages, and this approach enables the generation of naive iPSCs in a feeder-free culture. The naive iPSCs generated by this method show better differentiation to trilineage and extra-embryonic trophectoderm than those derived by conventional methods. This method can expand the application of iPSCs to research on early human development and regenerative medicine., 優れた多分化能を持つヒトのナイーブ型iPS細胞を迅速に作製する方法を発明. 京都大学プレスリリース. 2022-10-18., A novel method for generating naive human iPS cells with significantly higher differentiation potency. 京都大学プレスリリース. 2022-11-15.