Identification of Surface Antigens That Define Human Pluripotent Stem Cell-Derived PRRX1+Limb-Bud-like Mesenchymal Cells

Stem cell-based therapies and experimental methods rely on efficient induction of human pluripotent stem cells (hPSCs). During limb development, the lateral plate mesoderm (LPM) produces limb-bud mesenchymal (LBM) cells that differentiate into osteochondroprogenitor cells and form cartilage tissues in the appendicular skeleton. Previously, we generated PRRX1-tdTomato reporter hPSCs to establish the protocol for inducing the hPSC-derived PRRX1(+) LBM-like cells. However, surface antigens that assess the induction efficiency of hPSC-derived PRRX1(+) LBM-like cells from LPM have not been identified. Here, we used PRRX1-tdTomato reporter hPSCs and found that high pluripotent cell density suppressed the expression of PRRX1 mRNA and tdTomato after LBM-like induction. RNA sequencing and flow cytometry suggested that PRRX1-tdTomato(+) LBM-like cells are defined as CD44(high) CD140B(high) CD49f(-). Importantly, other hPSC lines, including four human induced pluripotent stem cell lines (414C2, 1383D2, HPS1042, HPS1043) and two human embryonic stem cell lines (SEES4, SEES7), showed the same results. Thus, an appropriate cell density of hPSCs before differentiation is a prerequisite for inducing the CD44(high) CD140B(high) CD49f(-) PRRX1(+) LBM-like cells

サリチル酸誘発耳鳴に対する牛車腎気丸の抑制効果の行動学的および免疫組織化学的な根拠

Many people are affected by tinnitus, a sensation of ringing in the ear despite the absence of external sound. Goshajinkigan (GJG) is one of the formulations of Japanese traditional herbal medicine and is prescribed for the palliative treatment of patients with tinnitus. Although GJG is clinically effective in these patients, its behavioral effects and the underlying neuroanatomical substrate have not been modeled in animals. We modeled tinnitus using salicylate-treated rats, demonstrated the effectiveness of GJG on tinnitus, and examined the underlying neuronal substrate with c-Fos expression. Intraperitoneal injection of sodium salicylate (400 mg/kg) into rats for three consecutive days significantly increased false positive scores, which were used to assess tinnitus behavior. When GJG was orally administered one hour after each salicylate injection, the increase in tinnitus behavior was suppressed. The analysis of c-Fos expression in auditory-related brain areas revealed that GJG significantly reduced the salicylate-induced increase in the number of c-Fos-expressing cells in the auditory cortices, inferior colliculus, and dorsal cochlear nucleus. These results suggest a suppressive effect of GJG on salicylate-induced tinnitus in animal models.博士（医学）・甲第851号・令和4年9月28日Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wildtype strain required approximately 1 mu g of biotin per liter for normal growth, the bioY disruptant (Delta bioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The Delta bioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the Delta bioY strain. By selectively using the resulting two strains (Delta bioB and Delta bioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 mu g to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 mu g per liter).ArticleAPPLIED AND ENVIRONMENTAL MICROBIOLOGY. 79(15):4586-4594 (2013)journal articl

Neutrophil S100A9 supports M2 macrophage niche formation in granulomas

慢性炎症「肉芽腫」における好中球の新しい炎症制御系の解明 --M2マクロファージの新たな誘導メカニズム解明--. 京都大学プレスリリース. 2023-02-17.In search of inflammatory Achilles heel. 京都大学プレスリリース. 2023-03-10.Mycobacterium infection gives rise to granulomas predominantly composed of inflammatory M1-like macrophages, with bacteria-permissive M2 macrophages also detected in deep granulomas. Our histological analysis of Mycobacterium bovis bacillus Calmette-Guerin-elicited granulomas in guinea pigs revealed that S100A9-expressing neutrophils bordered a unique M2 niche within the inner circle of concentrically multilayered granulomas. We evaluated the effect of S100A9 on macrophage M2 polarization based on guinea pig studies. S100A9-deficient mouse neutrophils abrogated M2 polarization, which was critically dependent on COX-2 signaling in neutrophils. Mechanistic evidence suggested that nuclear S100A9 interacts with C/EBPβ, which cooperatively activates the Cox-2 promoter and amplifies prostaglandin E2 production, followed by M2 polarization in proximal macrophages. Because the M2 populations in guinea pig granulomas were abolished via treatment with celecoxib, a selective COX-2 inhibitor, we propose the S100A9/Cox-2 axis as a major pathway driving M2 niche formation in granulomas

Tight junctions in Schwann cells of peripheral myelinated axons: a lesson from claudin-19–deficient mice

Tight junction (TJ)–like structures have been reported in Schwann cells, but their molecular composition and physiological function remain elusive. We found that claudin-19, a novel member of the claudin family (TJ adhesion molecules in epithelia), constituted these structures. Claudin-19–deficient mice were generated, and they exhibited behavioral abnormalities that could be attributed to peripheral nervous system deficits. Electrophysiological analyses showed that the claudin-19 deficiency affected the nerve conduction of peripheral myelinated fibers. Interestingly, the overall morphology of Schwann cells lacking claudin-19 expression appeared to be normal not only in the internodal region but also at the node of Ranvier, except that TJs completely disappeared, at least from the outer/inner mesaxons. These findings have indicated that, similar to epithelial cells, Schwann cells also bear claudin-based TJs, and they have also suggested that these TJs are not involved in the polarized morphogenesis but are involved in the electrophysiological “sealing” function of Schwann cells

Pterygoid Muscle Necrosis Caused by Radiation and Intra-Arterial Cisplatin Infusion Chemotherapy (RADPLAT): A Case Report

Introduction: Radiation and intra-arterial cisplatin infusion chemotherapy (RADPLAT) for advanced maxillary sinus cancer has accumulated evidence as a treatment with fewer complications and better 5-year survival rates. In this study, we report a case in which pterygoid muscle necrosis occurred 6 months following RADPLAT treatment for maxillary sinus cancer. Case Presentation: The 45-year-old woman had a long history of taking immunosuppressants against rheumatoid arthritis (RA) prior to treatment. Although achieving complete response (CR) to RADPLAT, the patient developed trismus (1 fingerbreadth or less) 6 months following treatment. Abscess formation and recurrence were suspected from the imaging findings; however, the biopsy with endoscopy indicated necrotic tissue. Currently, 18 months have passed without cancer recurrence. Although trismus temporarily improved with rehabilitation, the width of the mouth opening is currently a few millimeters, so the patient can only take liquid food. Conclusion: Pterygoid muscle necrosis should be recognized as a new major complication

Predictors of Survival in Patients With Ischemic Stroke and Active Cancer: A Prospective, Multicenter, Observational Study

BACKGROUND: Limited data exist on the prognostic factors for patients with ischemic stroke and active cancer. METHODS AND RESULTS: We conducted a prospective, multicenter, observational study in Japan, including patients with acute ischemic stroke and active cancer, to investigate the prognostic factors. We followed up the patients for 1 year after stroke onset. The patients were divided into 2 groups according to cryptogenic stroke and known causes (small-vessel occlusion, large-artery atherosclerosis, cardioembolism, and other determined cause), and survival was compared. The hazard ratios (HRs) and 95% CIs for mortality were calculated using Cox regression models. We identified 135 eligible patients (39% women; median age, 75 years). Of these patients, 51% had distant metastasis. A total of 65 (48%) and 70 (52%) patients had cryptogenic stroke and known causes, respectively. Patients with cryptogenic stroke had significantly shorter survival than those with known causes (HR [95% CI], 3.11 [1.82–5.32]). The multivariable Cox regression analysis revealed that distant metastasis, plasma D-dimer levels, venous thromboembolism (either deep venous thrombosis or pulmonary embolism) complications at stroke onset were independent predictors of mortality after adjusting for potential confounders. Cryptogenic stroke was associated with prognosis in univariable analysis but was not significant in multivariable analysis. The plasma D-dimer levels stratified the prognosis of patients with ischemic stroke and active cancer. CONCLUSIONS: The prognosis of patients with acute ischemic stroke and active cancer varied considerably depending on stroke mechanism, distant metastasis, and coagulation abnormalities. The present study confirmed that coagulation abnormalities were crucial in determining the prognosis of such patients.Gon Y., Sakaguchi M., Yamagami H., et al. Predictors of Survival in Patients With Ischemic Stroke and Active Cancer: A Prospective, Multicenter, Observational Study. Journal of the American Heart Association 12, e029618 (2023); https://doi.org/10.1161/JAHA.123.029618

Medium-chain fatty acids suppress lipotoxicity-induced hepatic fibrosis via the immunomodulating receptor GPR84

食事性肥満から肝炎発症に関わる制御因子の同定 --中鎖脂肪酸油による予防・GPR84標的NASH治療薬の可能性--. 京都大学プレスリリース. 2023-01-18.Medium-chain triglycerides (MCTs), which consist of medium-chain fatty acids (MCFAs), are unique forms of dietary fat with various health benefits. G protein–coupled 84 (GPR84) acts as a receptor for MCFAs (especially C10:0 and C12:0); however, GPR84 is still considered an orphan receptor, and the nutritional signaling of endogenous and dietary MCFAs via GPR84 remains unclear. Here, we showed that endogenous MCFA-mediated GPR84 signaling protected hepatic functions from diet-induced lipotoxicity. Under high-fat diet (HFD) conditions, GPR84-deficient mice exhibited nonalcoholic steatohepatitis (NASH) and the progression of hepatic fibrosis but not steatosis. With markedly increased hepatic MCFA levels under HFD, GPR84 suppressed lipotoxicity-induced macrophage overactivation. Thus, GPR84 is an immunomodulating receptor that suppresses excessive dietary fat intake–induced toxicity by sensing increases in MCFAs. Additionally, administering MCTs, MCFAs (C10:0 or C12:0, but not C8:0), or GPR84 agonists effectively improved NASH in mouse models. Therefore, exogenous GPR84 stimulation is a potential strategy for treating NASH

Dynamic Analysis of Photosynthate Translocation Into Strawberry Fruits Using Non-invasive 11C-Labeling Supported With Conventional Destructive Measurements Using 13C-Labeling

In protected strawberry (Fragaria × ananassa Duch.) cultivation, environmental control based on the process of photosynthate translocation is essential for optimizing fruit quality and yield, because the process of photosynthate translocation directly affects dry matter partitioning. We visualized photosynthate translocation to strawberry fruits non-invasively with 11CO2 and a positron-emitting tracer imaging system (PETIS). We used PETIS to evaluate real-time dynamics of 11C-labeled photosynthate translocation from a 11CO2-fed leaf, which was immediately below the inflorescence, to individual fruits on an inflorescence in intact plant. Serial photosynthate translocation images and animations obtained by PETIS verified that the 11C-photosynthates from the source leaf reached the sink fruit within 1 h but did not accumulate homogeneously within a fruit. The quantity of photosynthate translocation as represented by 11C radioactivity varied among individual fruits and their positions on the inflorescence. Photosynthate translocation rates to secondary fruit were faster than those to primary or tertiary fruits, even though the translocation pathway from leaf to fruit was the longest for the secondary fruit. Moreover, the secondary fruit was 25% smaller than the primary fruit. Sink activity (11C radioactivity/dry weight [DW]) of the secondary fruit was higher than those of the primary and tertiary fruits. These relative differences in sink activity levels among the three fruit positions were also confirmed by 13C tracer measurement. Photosynthate translocation rates in the pedicels might be dependent on the sink strength of the adjoining fruits. The present study established 11C-photosynthate arrival times to the sink fruits and demonstrated that the translocated material does not uniformly accumulate within a fruit. The actual quantities of translocated photosynthates from a specific leaf differed among individual fruits on the same inflorescence. To the best of our knowledge, this is the first reported observation of real-time translocation to individual fruits in an intact strawberry plant using 11C-radioactive- and 13C-stable-isotope analyses