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Exposure tests of an oxide dispersion strengthened martensitic type steel (ODS) were performed in stagnant lead bismuth eutectic (LBE) containing 10−4, 10−6 and 10−8 wt% of oxygen at 500–650 °C up to 10000 h. It resulted that the base metal was protected from corrosion by the formation of a magnetite and spinel layer in liquid Pb45Bi55 containing 10−6 wt% of oxygen at 550 °C or less, not however, at higher temperatures. At 650 °C and 10−8 wt% of oxygen, the ODS steel showed good compatibility with LBE by formation of a thin mixed high chromium spinel layer, while at 10−4 wt% multilayers of magnetite and spinel develop at this temperature which break off but are renewed by oxide layers which protect the steel again. [Copyright &y& Elsevier]

Sonic hedgehog (Shh) is expressed in the oropharyngeal epithelium, including the frontonasal ectodermal zone (FEZ), which is defined as the boundary between Shh and Fgf8 expression domains in the frontonasal epithelium. To investigate the role of SHH signaling from the oropharyngeal epithelium, we generated mice in which Shh expression is specifically deleted in the oropharyngeal epithelium (Isl1-Cre; Shh f/f ). In the mutant mouse, Shh expression was excised in the oropharyngeal epithelium as well as FEZ and ventral forebrain, consistent with the expression pattern of Isl1. Isl1-Cre; Shh f/f mice exhibited a complete loss of lower jaw components and a malformed upper jaw with defects in the cranial base and secondary palate. Massive cell death was observed in the mandibular process at embryonic day (E) 9.5 and E10.5, while mild cell death was observed in the lambdoidal region (the fusion area in the maxillary, lateral nasal, and medial nasal processes) at E10.5. An RNA-seq analysis revealed that Satb2, a gene involved in cell survival during jaw formation, was downregulated in the lambdoidal region in Isl1-Cre; Shh f/f mice. These results suggest that Shh expression in the FEZ is required for cell survival and skeletogenesis in the lambdoidal region during the development of the upper jaw and that the developmental control governed by SHH signaling is different between upper and lower jaws., (© 2024 Japanese Teratology Society.)

The renal glomerulus produces primary urine from blood plasma by ultrafiltration. The ultrastructure of the glomerulus is closely related to filtration function and disease development. The ultrastructure of glomeruli has mainly been evaluated using transmission electron microscopy; however, the volume that can be observed using transmission electron microscopy is extremely limited relative to the total volume of the glomerulus. Consequently, observing structures that exist in only one location in each glomerulus, such as the vascular pole, and evaluating low-density or localized lesions are challenging tasks. Array tomography (AT) is a technique used to analyze the ultrastructure of tissues and cells via scanning electron microscopy of serial sections. In this study, we present an AT workflow that is optimized for observing complete serial sections of the whole glomerulus, and we share several analytical examples that use the optimized AT workflow, demonstrating the usefulness of this approach. Overall, this AT workflow can be a powerful tool for structural and pathological evaluation of the glomerulus. This workflow is also expected to provide new insights into the ultrastructure of the glomerulus and its constituent cells., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Male infertility can be caused by chromosomal abnormalities, mutations and epigenetic defects. Epigenetic modifiers pre-program hundreds of spermatogenic genes in spermatogonial stem cells (SSCs) for expression later in spermatids, but it remains mostly unclear whether and how those genes are involved in fertility. Here, we report that Wfdc15a, a WFDC family protease inhibitor pre-programmed by KMT2B, is essential for spermatogenesis. We found that Wfdc15a is a non-canonical bivalent gene carrying both H3K4me3 and facultative H3K9me3 in SSCs, but is later activated along with the loss of H3K9me3 and acquisition of H3K27ac during meiosis. We show that WFDC15A deficiency causes defective spermiogenesis at the beginning of spermatid elongation. Notably, depletion of WFDC15A causes substantial disturbance of the testicular protease-antiprotease network and leads to an orchitis-like inflammatory response associated with TNFα expression in round spermatids. Together, our results reveal a unique epigenetic program regulating innate immunity crucial for fertility., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Intra-articular platelet-rich plasma (PRP) injections have been proposed for the treatment of knee osteoarthritis (OA); however, their effectiveness in Japanese patients remains unclear., Purpose: To investigate whether 3 intra-articular injections of leukocyte-poor PRP (LP-PRP) improve symptoms and joint function in symptomatic Japanese patients with mild to moderate knee OA., Study Design: Randomized controlled trial; Level of evidence, 1., Methods: Of 72 patients screened, 30 were included and randomized to receive LP-PRP (n = 15) or saline (placebo; n = 15) injections between March 2019 and February 2023. Patients attended a screening visit and 3 treatment visits at 1 week apart, followed by 3 follow-up visits (at 4, 12, and 24 weeks) after the initial treatment visit. The primary efficacy outcome measure was the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, whereas the secondary efficacy outcome measures were the proportion of patients showing a visual analog scale (VAS) improvement of ≥50%. Magnetic resonance imaging was performed to evaluate joint effusion and bone marrow lesions using the Whole-Organ Magnetic Resonance Imaging Score. Patients were followed for 24 weeks., Results: Patients in the PRP group (mean age, 65.9 years) had a mean hip-knee-ankle angle of 5.1°, with 7 and 8 patients demonstrating Kellgren-Lawrence grade 2 and 3 knee OA, respectively. Patients in the placebo group (mean age, 67.9 years) had a mean hip-knee-ankle angle of 3.8°, with 6 and 9 patients showing Kellgren-Lawrence grade 2 and 3 knee OA, respectively. No significant differences were identified in any baseline factors. The percentage change in Western Ontario and McMaster Universities Osteoarthritis Index scores from baseline to 24 weeks was significantly different (P= .032) between the PRP (median, 75.9%; quantile 1 [Q1], 49.6; quantile 3 [Q3], 94.1]) and placebo (median, 27.7%; Q1, -9.4; Q3, 80.9]) groups. Overall, 73.3% and 28.6% of the PRP group and placebo group, respectively, exhibited an improvement in visual analog scale scores of ≥50%, with a significant improvement observed in the PRP group ( P = .027). Changes in bone marrow lesions from baseline to 24 weeks, as assessed on magnetic resonance imaging, significantly differed between groups ( P = .017), with no significant differences in other secondary endpoints., Conclusion: In Japanese patients with knee OA, 3 intra-articular LP-PRP injections led to clinical improvements at 24-week follow-up and significant functional improvements and pain relief after 24 weeks., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: This study was funded by a grant for the implementation of advanced medicine from the University of Tsukuba Hospital (No. TRM2019-06). AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Introduction: Carbapenems and piperacillin/tazobactam (PIPC/TAZ) are commonly used as the initial therapy to treat extended-spectrum β-lactamase (ESBL)-producing Enterobacterales in acute cholangitis. However, the overuse of these antibiotics contributes to the spread of antimicrobial resistance. Cefmetazole (CMZ) is stable to hydrolysis by ESBLs, so it may be an alternative to carbapenems and PIPC/TAZ. However, the effectiveness of CMZ compared with that of carbapenems and PIPC/TAZ as the initial therapy for acute cholangitis is unknown., Methods: We conducted a retrospective cohort study at a university hospital between April 1, 2014, and December 31, 2022. Patients with bacteremic acute cholangitis who received CMZ, carbapenems, or PIPC/TAZ as the initial therapy were included. The patients were divided into a CMZ group and a carbapenems or PIPC/TAZ (CP) group to compare patient outcomes., Results: A total of 99 patients (54 in the CMZ group and 45 in the CP group) were analyzed. The baseline characteristics of the patients were similar and 30-day mortality did not differ between groups (4% vs. 7%, P = 0.66). However, the CMZ group had a shorter length of stay (LOS) (8 days vs. 15 days, P < 0.001) and lower mean antibiotic cost (98.92 USD vs. 269.49 USD, P < 0.001) than the CP group., Conclusions: In bacteremic acute cholangitis, initial therapy with CMZ may contribute to a shorter LOS and lower antibiotic costs than treatment with carbapenems and PIPC/TAZ, without worsening patient outcomes., Competing Interests: Declarations of competing interest All authors have no conflicts of interest to declare., (Copyright © 2023 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Impaired development and maintenance of Schlemm's canal (SC) are associated with perturbed aqueous humor outflow and intraocular pressure. The angiopoietin (ANGPT)/TIE2 signaling pathway regulates SC development and maintenance, whereas the molecular mechanisms of crosstalk between SC and the neural crest (NC)-derived neighboring tissue, the trabecular meshwork (TM), are poorly understood. Here, we show NC-specific forkhead box ( Fox)c2 deletion in mice results in impaired SC morphogenesis, loss of SC identity, and elevated intraocular pressure. Visible-light optical coherence tomography analysis further demonstrated functional impairment of the SC in response to changes in intraocular pressure in NC- Foxc2 -/- mice, suggesting altered TM biomechanics. Single-cell RNA-sequencing analysis identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM cell clusters, including increased matrix metalloproteinase expression, which can cleave the TIE2 ectodomain to produce soluble TIE2. Moreover, endothelial-specific Foxc2 deletion impaired SC morphogenesis because of reduced TIE2 expression, which was rescued by deleting the TIE2 phosphatase VE-PTP. Thus, Foxc2 is critical in maintaining SC identity and morphogenesis via TM-SC crosstalk., (© 2023 Ujiie et al.)

Intestinal ischemia underlies several clinical conditions and can result in the loss of the intestinal mucosal barrier. Ischemia-induced damage to the intestinal epithelium is repaired by stimulation of intestinal stem cells (ISCs), and paracrine signaling from the vascular niche regulates intestinal regeneration. Here, we identify FOXC1 and FOXC2 as essential regulators of paracrine signaling in intestinal regeneration after ischemia-reperfusion (I/R) injury. Vascular endothelial cell (EC)- and lymphatic EC (LEC)-specific deletions of Foxc1, Foxc2, or both in mice worsen I/R-induced intestinal damage by causing defects in vascular regrowth, expression of chemokine CXCL12 and Wnt activator R-spondin 3 (RSPO3) in blood ECs (BECs) and LECs, respectively, and activation of Wnt signaling in ISCs. Both FOXC1 and FOXC2 directly bind to regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Treatment with CXCL12 and RSPO3 rescues the I/R-induced intestinal damage in EC- and LEC-Foxc mutant mice, respectively. This study provides evidence that FOXC1 and FOXC2 are required for intestinal regeneration by stimulating paracrine CXCL12 and Wnt signaling., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Identification of biallelic CNPY3 mutations in patients with epileptic encephalopathy and abnormal electroencephalography findings of Cnpy3 knock-out mice have indicated that the loss of CNPY3 function causes neurological disorders such as epilepsy. However, the basic property of CNPY3 in the brain remains unclear., New Method: We generated C-terminal 2xHA-tag knock-in Cnpy3 mice by i-GONAD in vivo genome editing system to investigate the expression and function of Cnpy3 in the mouse brain., Results: 2xHA-tagged Cnpy3 was confirmed by immunoblot analysis using anti-HA and CNPY3 antibodies, although HA tagging caused the decreased Cnpy3 protein level. Immunohistochemical analysis of Cnpy3 2xHA knock-in mice showed that Cnpy3-2xHA was predominantly expressed in the neuron. In addition, Cnpy3 and Cnpy3-2xHA were both localized in the endoplasmic reticulum and synaptosome and showed age-dependent expression changes in the brain., Comparison With Existing Methods: Conventional Cnpy3 antibodies could not allow us to investigate the distribution of Cnpy3 expression in the brain, while HA-tagging revealed the expression of CNPY3 in neuronal cells., Conclusions: Taken together, we demonstrated that Cnpy3 2xHA knock-in mice would be useful to further elucidate the property of Cnpy3 in brain function and neurological disorders., Competing Interests: Declaration of Competing Interest We have no conflicts of interest to declare., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Here we report a rare case of peritonitis caused by radiation enteritis. The 82-year-old woman who underwent surgery and radiotherapy for uterine cancer in her 30s. Emergency operation was performed for the perforation of the ileum. The small intestine showed changes of radiation enteritis extensively on macroscopy. The first surgery was performed to resect the perforated ileum and make intestinal anastomosis at the change of radiation enteritis. However, suture failure was occurred, reoperation was performed after conservative therapy. Reoperation was performed extensively resection of the intestinal tract and made anastomosis where was mild change of radiation enteritis. Pathological findings of the intestinal stump revealed that the arterial vessels of the submucosal layer were highly thicken and the lumen of artery was stenosis and occlusion with severe changes of radiation enteritis at the first operation. Blood flow disorders by irradiation were presumed to be the cause of suture failure. On the other hand, the intestinal stump did not indicate thickened of vascular wall and lumen stenosis of the vessels, only edematous changes in the submucosal layer were observed at the reoperation. It was important to determine the surgical procedure with the change of radiation enteritis for gastrointestinal operation with abdominal irradiation.

Background: We have previously confirmed the safety and feasibility of intra-articular (IA) platelet-rich plasma (PRP) injections in Japanese patients with osteoarthritis (OA) of the knee. This study aimed to investigate the clinical and radiological outcomes in patients who were followed up for three years., Methods: Nine patients were evaluated in this observational study. All the patients were women with a mean age of 60.6 years. PRP was prepared by single centrifugation and classified as leukocyte-poor (LP)-PRP, and was administered via three IA injections at weekly intervals. Patients were evaluated at baseline and final follow-up using the Kellgren-Lawrence (KL) grade and the Japanese Orthopedics Association (JOA) score. The need for additional treatment was also investigated at the final follow-up., Results: At a mean follow-up of three years, the JOA score improved from 75 points (p) to 83.8 p. The sub-categorical scores changed as follows: gait, 23.1-26.9 p; stairs, 12.5-17.5 p; range of motion, 30-30 p; and swelling, 9.4-10 p. While the KL grade was maintained in six patients, it progressed in two patients from I to II and II to III, respectively. Two patients received additional treatment at the final follow-up., Conclusions: At the final follow-up, the functional knee score, especially gait and the ability to go up and down the stairs, improved in six out of eight patients without additional treatment. These results suggest that LP-PRP injections produced safe outcomes without OA-worsening in most patients at three-year follow-up., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Taniguchi et al.)

Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIβ mice by combining CRISPR-Cas9 genome editing with electroporation ( i -GONAD). With i -GONAD, it is possible to insert a small fragment of up to 200 bp into the genome of the target gene, enabling efficient and convenient tagging of a small epitope. Experiments with commercially available anti-Flag antibodies could readily detect endogenous CaMKIIα and β proteins by Western blotting, immunoprecipitation, and immunohistochemistry. Our data demonstrated that the generation of Flag/DYKDDDDK tag knock-in mice by i -GONAD is a useful and convenient choice, especially if specific antibodies are unavailable.

Improved genome editing via oviductal nucleic acids delivery ( i -GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i -GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00-13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i -GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.

Exome sequencing and panel testing have improved diagnostic yield in genetic analysis by comprehensively detecting pathogenic variants in exonic regions. However, it is important to identify non-exonic pathogenic variants to further improve diagnostic yield. Here, we present a female proband and her father who is diagnosed with Marfan syndrome, a systemic connective tissue disorder caused by pathogenic variants in FBN1. There are also two affected individuals in the siblings of the father, indicating the genetic basis in this family. However, panel testing performed by two institutions reported no causal variants. To further explore the genetic basis of the family, we performed genome sequencing of the proband and RNA sequencing of urinary cells derived from urine samples of the proband and her father because FBN1 is strongly expressed in urinary cells though it is poorly expressed in peripheral blood mononuclear cells. Genome sequencing identified a rare intronic variant (c.5789-15G>A) in intron 47 of FBN1 (NM&#95;000138.4), which was transmitted from her father. RNA sequencing revealed allelic imbalance (monoallelic expression) of FBN1, retention of intron 47, and fewer aberrant transcripts utilizing new acceptor sites within exon 48, which were confirmed by RT-PCR. These results highlighted urinary cells as clinically accessible tissues for RNA sequencing if disease-causing genes are not sufficiently expressed in the blood, and the usefulness of multi-omics analysis for molecular diagnosis of genetic disorders., (© 2022. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)