Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation.

Cohesin is important for 3D genome organization. Nevertheless, even the complete removal of cohesin has surprisingly little impact on steady-state gene transcription and enhancer activity. Here we show that cohesin is required for the core transcriptional response of primary macrophages to microbial signals, and for inducible enhancer activity that underpins inflammatory gene expression. Consistent with a role for inflammatory signals in promoting myeloid differentiation of hematopoietic stem and progenitor cells (HPSCs), cohesin mutations in HSPCs led to reduced inflammatory gene expression and increased resistance to differentiation-inducing inflammatory stimuli. These findings uncover an unexpected dependence of inducible gene expression on cohesin, link cohesin with myeloid differentiation, and may help explain the prevalence of cohesin mutations in human acute myeloid leukemia.

Endothelial sensing of AHR ligands regulates intestinal homeostasis.

Endothelial cells line the blood and lymphatic vasculature, and act as an essential physical barrier, control nutrient transport, facilitate tissue immunosurveillance and coordinate angiogenesis and lymphangiogenesis1,2. In the intestine, dietary and microbial cues are particularly important in the regulation of organ homeostasis. However, whether enteric endothelial cells actively sense and integrate such signals is currently unknown. Here we show that the aryl hydrocarbon receptor (AHR) acts as a critical node for endothelial cell sensing of dietary metabolites in adult mice and human primary endothelial cells. We first established a comprehensive single-cell endothelial atlas of the mouse small intestine, uncovering the cellular complexity and functional heterogeneity of blood and lymphatic endothelial cells. Analyses of AHR-mediated responses at single-cell resolution identified tissue-protective transcriptional signatures and regulatory networks promoting cellular quiescence and vascular normalcy at steady state. Endothelial AHR deficiency in adult mice resulted in dysregulated inflammatory responses and the initiation of proliferative pathways. Furthermore, endothelial sensing of dietary AHR ligands was required for optimal protection against enteric infection. In human endothelial cells, AHR signalling promoted quiescence and restrained activation by inflammatory mediators. Together, our data provide a comprehensive dissection of the effect of environmental sensing across the spectrum of enteric endothelia, demonstrating that endothelial AHR signalling integrates dietary cues to maintain tissue homeostasis by promoting endothelial cell quiescence and vascular normalcy.

Sex-specific chromatin remodelling safeguards transcription in germ cells.

Stability of the epigenetic landscape underpins maintenance of the cell-type-specific transcriptional profile. As one of the main repressive epigenetic systems, DNA methylation has been shown to be important for long-term gene silencing; its loss leads to ectopic and aberrant transcription in differentiated cells and cancer1. The developing mouse germ line endures global changes in DNA methylation in the absence of widespread transcriptional activation. Here, using an ultra-low-input native chromatin immunoprecipitation approach, we show that following DNA demethylation the gonadal primordial germ cells undergo remodelling of repressive histone modifications, resulting in a sex-specific signature in mice. We further demonstrate that Polycomb has a central role in transcriptional control in the newly hypomethylated germline genome as the genetic loss of Ezh2 leads to aberrant transcriptional activation, retrotransposon derepression and dramatic loss of developing female germ cells. This sex-specific effect of Ezh2 deletion is explained by the distinct landscape of repressive modifications observed in male and female germ cells. Overall, our study provides insight into the dynamic interplay between repressive chromatin modifications in the context of a developmental reprogramming system.

An intestinal zinc sensor regulates food intake and developmental growth.

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis.

Dysregulated RNA polyadenylation contributes to metabolic impairment in non-alcoholic fatty liver disease.

Pre-mRNA processing is an essential mechanism for the generation of mature mRNA and the regulation of gene expression in eukaryotic cells. While defects in pre-mRNA processing have been implicated in a number of diseases their involvement in metabolic pathologies is still unclear. Here, we show that both alternative splicing and alternative polyadenylation, two major steps in pre-mRNA processing, are significantly altered in non-alcoholic fatty liver disease (NAFLD). Moreover, we find that Serine and Arginine Rich Splicing Factor 10 (SRSF10) binding is enriched adjacent to consensus polyadenylation motifs and its expression is significantly decreased in NAFLD, suggesting a role mediating pre-mRNA dysregulation in this condition. Consistently, inactivation of SRSF10 in mouse and human hepatocytes in vitro, and in mouse liver in vivo, was found to dysregulate polyadenylation of key metabolic genes such as peroxisome proliferator-activated receptor alpha (PPARA) and exacerbate diet-induced metabolic dysfunction. Collectively our work implicates dysregulated pre-mRNA polyadenylation in obesity-induced liver disease and uncovers a novel role for SRSF10 in this process.

Multicenter registry of multisystem inflammatory syndrome in children (MIS-C) and Paired comparison with Kawasaki disease.

OBJECTIVES: This study aimed to identify clinical characteristics to differentiate multisystem inflammatory syndrome in children (MIS-C) and Kawasaki disease (KD) in Taiwan, an island with a delayed cluster of MIS-C and a high incidence of KD. Additionally, we studied risk factors for developing severe complications in patients with MIS-C. METHODS: We conducted a retrospective, multicenter, cohort, and observational study that linked data on patients with MIS-C between May and December 2022 and patients with KD between 2019 and 2021 from 12 medical centers. Hemodynamic compromise, defined as the need for inotropic support or fluid challenge, was recorded in patients with MIS-C. We also evaluated maximal coronary Z-scores before treatment and one month after disease onset. RESULTS: A total of 83 patients with MIS-C and 466 patients with KD were recruited. A 1:1 age and gender-matched comparison of 68 MIS-C and KD pairs showed that MIS-C patients had a lower percentage of positive BCG red halos, lower leukocyte/platelet counts, more gastrointestinal symptoms, and a higher risk of hemodynamic compromise. In Taiwan, 38.6% of MIS-C patients experienced hemodynamic compromise, with presence of conjunctivitis and elevated levels of procalcitonin (>1.62 ng/mL) identified as independent risk factors. CONCLUSIONS: We identified two independent risk factors associated with hemodynamic compromise in MIS-C patients. The comparison between matched MIS-C and KD patients highlighted significant differences in clinical presentations, like BCG red halos, which may aid in the differential diagnosis of the two disease entities, especially in regions with a high incidence rate of KD.

RUNX1 Regulates a Transcription Program That Affects the Dynamics of Cell Cycle Entry of Naive Resting B Cells.

RUNX1 is a transcription factor that plays key roles in hematopoietic development and in hematopoiesis and lymphopoiesis. In this article, we report that RUNX1 regulates a gene expression program in naive mouse B cells that affects the dynamics of cell cycle entry in response to stimulation of the BCR. Conditional knockout of Runx1 in mouse resting B cells resulted in accelerated entry into S-phase after BCR engagement. Our results indicate that Runx1 regulates the cyclin D2 (Ccnd2) gene, the immediate early genes Fosl2, Atf3, and Egr2, and the Notch pathway gene Rbpj in mouse B cells, reducing the rate at which transcription of these genes increases after BCR stimulation. RUNX1 interacts with the chromatin remodeler SNF-2-related CREB-binding protein activator protein (SRCAP), recruiting it to promoter and enhancer regions of the Ccnd2 gene. BCR-mediated activation triggers switching between binding of RUNX1 and its paralog RUNX3 and between SRCAP and the switch/SNF remodeling complex member BRG1. Binding of BRG1 is increased at the Ccnd2 and Rbpj promoters in the Runx1 knockout cells after BCR stimulation. We also find that RUNX1 exerts positive or negative effects on a number of genes that affect the activation response of mouse resting B cells. These include Cd22 and Bank1, which act as negative regulators of the BCR, and the IFN receptor subunit gene Ifnar1 The hyperresponsiveness of the Runx1 knockout B cells to BCR stimulation and its role in regulating genes that are associated with immune regulation suggest that RUNX1 could be involved in regulating B cell tolerance.

Kawasaki disease in children with Bacillus Calmette-Guérin scar reactivity: Focus on coronary outcomes.

BACKGROUND: /Purpose: Reactivity at the Bacillus Calmette-Guérin (BCG) scar is a pathognomonic feature of Kawasaki disease (KD). However, its value in predicting KD outcomes has not been emphasized. This study explored the clinical significance of BCG scar redness with respect to coronary artery outcomes. METHODS: This retrospective study collected data on children with KD from 13 hospitals in Taiwan during 2019-2021. Children with KD were categorized into four groups based on the KD type and BCG scar reactivity. Risk factors of coronary artery abnormalities (CAA) were analyzed in all groups. RESULTS: BCG scar redness occurred in 49% of 388 children with KD. BCG scar redness was associated with younger age, early intravenous immunoglobulin (IVIG) treatment, hypoalbuminemia, and CAA at the first echocardiogram (p < 0.01). BCG scar redness (RR 0.56) and pyuria (RR 2.61) were independent predictors of any CAA within 1 month (p < 0.05). Moreover, pyuria (RR 5.85, p < 0.05) in children with complete KD plus BCG scar redness was associated with CAA at 2-3 months; first IVIG resistance (RR 15.2) and neutrophil levels ≥80% (RR 8.37) in children with complete KD plus BCG scar non-redness were associated with CAA at 2-3 months (p < 0.05). We failed to detect any significant risk factors of CAA at 2-3 months in children with incomplete KD. CONCLUSION: BCG scar reactivity contributes to diverse clinical features in KD. It can be effectively applied to determine the risk factors of any CAA within 1 month and CAA at 2-3 months.

Dosimetric assessment of patient dose calculation on a deep learning-based synthesized computed tomography image for adaptive radiotherapy.

PURPOSE: Dose computation using cone beam computed tomography (CBCT) images is inaccurate for the purpose of adaptive treatment planning. The main goal of this study is to assess the dosimetric accuracy of synthetic computed tomography (CT)-based calculation for adaptive planning in the upper abdominal region. We hypothesized that deep learning-based synthetically generated CT images will produce comparable results to a deformed CT (CTdef) in terms of dose calculation, while displaying a more accurate representation of the daily anatomy and therefore superior dosimetric accuracy. METHODS: We have implemented a cycle-consistent generative adversarial networks (CycleGANs) architecture to synthesize CT images from the daily acquired CBCT image with minimal error. CBCT and CT images from 17 liver stereotactic body radiation therapy (SBRT) patients were used to train, test, and validate the algorithm. RESULTS: The synthetically generated images showed increased signal-to-noise ratio, contrast resolution, and reduced root mean square error, mean absolute error, noise, and artifact severity. Superior edge matching, sharpness, and preservation of anatomical structures from the CBCT images were observed for the synthetic images when compared to the CTdef registration method. Three verification plans (CBCT, CTdef, and synthetic) were created from the original treatment plan and dose volume histogram (DVH) statistics were calculated. The synthetic-based calculation shows comparatively similar results to the CTdef-based calculation with a maximum mean deviation of 1.5%. CONCLUSIONS: Our findings show that CycleGANs can produce reliable synthetic images for the adaptive delivery framework. Dose calculations can be performed on synthetic images with minimal error. Additionally, enhanced image quality should translate into better daily alignment, increasing treatment delivery accuracy.

Evolution of an Amniote-Specific Mechanism for Modulating Ubiquitin Signaling via Phosphoregulation of the E2 Enzyme UBE2D3.

Genetic variation in the enzymes that catalyze posttranslational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signaling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme, which acts as an ubiquitin donor for E3 ligases that catalyze ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologs to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse embryonic stem cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal mutation in mice. When mutant S138A ESCs were differentiated into extraembryonic primitive endoderm, levels of the PDGFRα and FGFR1 receptor tyrosine kinases were reduced and primitive endoderm differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the receptor tyrosine kinases. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution.

Fiducial detection and registration for 3D IMRT QA with organ-specific dose information.

PURPOSE: Two-dimensional (2D) IMRT QA has been widely performed in Radiation Oncology clinic. However, concerns regarding its sensitivity in detecting delivery errors and its clinical meaning have been raised in publications. In this study, a robust methodology of three-dimensional (3D) IMRT QA using fiducial registration and structure-mapping was proposed to acquire organ-specific dose information. METHODS: Computed tomography (CT) markers were placed on the PRESAGE dosimeter as fiducials before CT simulation. Subsequently, the images were transferred to the treatment planning system to create a verification plan for the examined treatment plan. Patient's CT images were registered to the CT images of the dosimeter for structure mapping according to the positions of the fiducials. After irradiation, the 3D dose distribution was read-out by an optical-CT (OCT) scanner with fiducials shown on the OCT dose images. An automatic localization algorithm was developed in MATLAB to register the markers in the OCT images to those in the CT images of the dosimeter. SlicerRT was used to show and analyze the results. Fiducial registration error was acquired by measuring the discrepancies in 20 fiducial registrations, and thus the fiducial localization error and target registration error (TRE) was estimated. RESULTS: Dosimetry comparison between the calculated and measured dose distribution in various forms were presented, including 2D isodose lines comparison, 3D isodose surfaces with patient's anatomical structures, 2D and 3D gamma index, dose volume histogram and 3D view of gamma failing points. From the analysis of 20 fiducial registrations, fiducial registration error was measured to be 0.62 mm and fiducial localization error was calculated to be 0.44 mm. Target registration uncertainty of the proposed methodology was estimated to be within 0.3 mm in the area of dose measurement. CONCLUSIONS: This study proposed a robust methodology of 3D measurement-based IMRT QA for organ-specific dose comparison and demonstrated its clinical feasibility.

MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is widely prevalent in Taiwan, and high metastatic spread of ESCC leads to poor survival rate. Fibronectin (FN) assembly on the cell membrane may induce ESCC mobility. MicroRNAs (MiRNAs) are abundant in and participate in tumorigenesis in many cancers. However, the role of MiRNA in FN assembly-related ESCC mobility remains unexplored. METHODS: We divided ESCC CE81T cells into high-FN assembly (CE81FN+) and low-FN assembly (CE81FN-) groups by flow cytometry. MiRNA microarray analysis identified miR-146a expression as the most down-regulated miRNA in comparison of CE81FN+ and CE81FN- cells. RESULTS: Cell proliferation and migration were decreased when CE81FN+ cells overexpressed transgenic miR-146a compared to the parental cells, indicating an inverse correlation between low miR-146a expression and high proliferation as well as motility of FN assembly ESCC cells. Furthermore, vimentin is the target gene of miR-146a involved in ESCC tumorigenesis. MiR-146a suppressed cell proliferation, migration and invasion of CE81FN+ cells through the inhibition of vimentin expression, as confirmed by real-time PCR, Western blotting and Transwell™ assay. Analysis of one hundred and thirty-six paired ESCC patient specimens revealed that low miR-146a and high vimentin levels were frequently detected in tumor, and that the former was associated with late tumor stages (III and IV). Notably, either low miR-146a expression or high vimentin level was significantly associated with poor overall survival rate among ESCC patients. CONCLUSIONS: This is the first report to link FN assembly in the cell membrane with miR-146a, vimentin and ESCC tumorigenesis both in vitro and in ESCC patients.

Cascade In Situ Phosphorylation and One-Pot Glycosylation for Rapid Synthesis of Heptose-Containing Oligosaccharides.

We report a one-pot glycosylation strategy for achieving rapid syntheses of heptose (Hep)-containing oligosaccharides. The reported procedure was designed to incorporate an in situ phosphorylation step into an orthogonal one-pot glycosylation. Hep-containing oligosaccharides were assembled directly from building blocks with minimal effort expended on manipulation of protecting and aglycone leaving groups. The utility of our one-pot procedure was illustrated by synthesizing partial core oligosaccharide structure present in the lipopolysaccharide of Ralstonia solanacearum.

Dosimetric characterization of a body-conforming radiochromic sheet.

PURPOSE: A novel radiochromic PRESAGE sheet (Heuris Inc.) with 3 mm thickness has been developed as a measurement tool for 2D dosimetry. Its inherent ability to conform to irregular surfaces makes this dosimeter advantageous for patient surface dosimetry. This study is a comprehensive investigation into the PRESAGE sheet's dosimetric characteristic, accuracy and its potential use as a dosimeter for clinical applications. METHODS: The characterization of the dosimeter included evaluation of the temporal stability of the dose linearity, reproducibility, measurement uncertainties, dose rate, energy, temperature and angular dependence, lateral response artifacts, percent depth dose curve, and 2D dose measurement. Dose distribution measurements were acquired for regular square fields on a flat and irregular surface and an irregular modulated field on the smooth surface. All measurements were performed using an Epson 11000XL high-resolution scanner. RESULTS: The examined dosimeters exhibit stable linear response, standard error of repeated measurements within 2%, negligible dose rate, energy, and angular dependence. The same linear dose response was measured while the dosimeter was in contact with a heated water surface. Gamma test and histogram analysis of the dose difference between PRESAGE and EBT3 film, PRESAGE and the treatment planning system (TPS) were used to evaluate the measured dose distributions. The PRESAGE sheet dose distributions showed good agreement with EBT3 film and TPS. A discrepancy smaller than the statistical error of the two dosimeters was reported. CONCLUSIONS: This study established a full dosimetric characterization of the PRESAGE sheets with the purpose of laying the foundation for future clinical uses. The results presented here for the comparison of this novel dosimeter with those currently in use reinforce the possibility of using this dosimeter as an alternative for irregular surface dose measurements.

Isolated Compounds from Turpinia formosana Nakai Induce Ossification.

Bone metabolism is a homeostatic process, imbalance in which leads to the onset of diseases such as osteoporosis and osteopenia. Although several drugs are currently available to treat such conditions, they are associated with severe side effects and do not enhance bone formation. Thus, identifying alternative treatment strategies that focus on enhancing bone formation is essential. Herein, we explored the osteogenic potential of Turpinia formosana Nakai using human osteoblast (HOb) cells. The plant extract was subjected to various chromatographic techniques to obtain six compounds, including one new compound: 3,3'-di-O-methylellagic acid-4-O-α-l-arabinofuranoside (1). Compounds 3,3'-di-O-methylellagic acid-4-O-α-l-arabinofuranoside (1), gentisic acid 5-O-ß-d-(6'-O-galloyl) glucopyranoside (2), strictinin (3), and (-)-epicatechin-3-O-ß-d-allopyranoside (6) displayed no significant cytotoxicity toward HOb cells, and thus their effects on various osteogenic markers were analyzed. Results showed that 1-3 and 6 significantly increased alkaline phosphatase (ALP) activity up to 120.0, 121.3, 116.4, and 125.1%, respectively. Furthermore, 1, 2, and 6 also markedly enhanced the mineralization process with respective values of up to 136.4, 118.9, and 134.6%. In addition, the new compound, 1, significantly increased expression levels of estrogen receptor-α (133.4%) and osteogenesis-related genes of Runt-related transcription factor 2 (Runx2), osteopontin (OPN), bone morphogenetic protein (BMP)-2, bone sialoprotein (BSP), type I collagen (Col-1), and brain-derived neurotropic factor (BDNF) by at least 1.5-fold. Our results demonstrated that compounds isolated from T. formosana possess robust osteogenic potential, with the new compound, 1, also exhibiting the potential to enhance the bone formation process. We suggest that T. formosana and its isolated active compounds deserve further evaluation for development as anti-osteoporotic agents.

Treatment of Lumbar Tuberculosis by Mini-Open Anterior Approach Focal Cleaning Combined with Posterior Internal Fixation.

BACKGROUND The aim of this study was to evaluate the efficiency and clinical outcomes of mini-open anterior approach focal cleaning combined with posterior internal fixation compared to conventional anterior extraperitoneal approach focal cleaning combined with posterior internal fixation in the treatment of lumbar tuberculosis (TB). MATERIAL AND METHODS Medical records from 124 patients were collected from February 2010 to April 2015; patients were divided into two groups: group A (mini-open anterior approach focal cleaning combined with posterior internal fixation) and group B (conventional anterior extraperitoneal approach focal cleaning combined with posterior internal fixation in period I). The data on postoperative mechanical ventilation time, preoperative, postoperative, and last follow-up Cobb angle, visual analog scale (VAS), erythrocyte sedimentation rate (ESR), and Frankel classification were collected and analyzed. Operative complications, internal stability, and bone graft fusion were also observed. RESULTS All patients were followed-up for 12 to 36 months (average 22.5 months). Seven cases (five in group A and two in group B) had side psoas abscess and were cured after secondary drainage surgery. The rest of the cases were all cured after primary surgery, with no formation of sinus, incisional hernia, cerebrospinal fluid leakage, or recurrence of spinal TB, with no TB symptoms. Bone graft fusion ranged from 3 to 8 months (average 4.7 months). Compared to group B, group A, which had less time on postoperative mechanical ventilation, had a higher VAS score. Both groups had distinct improvements in Cobb angle, ESR, and Frankel classification after surgery. CONCLUSIONS Treating lumbar TB by mini-open anterior approach focal cleaning combined with posterior internal fixation was safe and effective.

A novel function of calcitonin gene-related peptide in body fluid Cl- homeostasis.

Vertebrates need to maintain extracellular chloride (Cl(-)) concentrations to ensure the normal operation of physiological processes; the transition from aquatic to terrestrial environments necessitated the development of sophisticated mechanisms to ensure Cl(-) homeostasis in the face of fluctuating Cl(-) levels. Zebrafish calcitonin gene-related peptide (CGRP), unlike its splice variant calcitonin, does not respond to environmental Ca(2+) levels. This study aimed to test the hypothesis that CGRP is involved in the control of body fluid Cl(-) homeostasis. Acclimation to high-Cl(-) artificial water stimulated the mRNA expression of cgrp and the receptor (crlr1) when compared with low-Cl(-) CGRP knockdown induced upregulation of the Na(+)-Cl(-) co-transporter (ncc2b), while overexpression of CGRP resulted in the downregulation of ncc2b mRNA synthesis and a simultaneous decrease in Cl(-) uptake in embryos. Consistent with these findings, knockdown of either cgrp or crlr1 was found to increase the density of NCC2b-expressing cells in embryos. This is the first demonstration that CGRP acts as a hypochloremic hormone through suppressing NCC2b expression and the differentiation of NCC-expressing ionocytes. Elucidation of this novel function of CGRP in fish body fluid Cl(-) homeostasis promises to enhance our understanding of the related physiology in vertebrates.

Enhancing Safety in AI-Driven Cone Beam CT-based Online Adaptive Radiation Therapy: Development and Implementation of an Interdisciplinary Workflow.

Purpose: The emerging online adaptive radiation therapy (OART) treatment strategy based on cone beam computed tomography allows for real-time replanning according to a patient's current anatomy. However, implementing this procedure requires a new approach across the patient's care path and monitoring of the "black box" adaptation process. This study identifies high-risk failure modes (FMs) associated with AI-driven OART and proposes an interdisciplinary workflow to mitigate potential medical errors from highly automated processes, enhance treatment efficiency, and reduce the burden on clinicians. Methods and Materials: An interdisciplinary working group was formed to identify safety concerns in each process step using failure mode and effects analysis (FMEA). Based on the FMEA results, the team designed standardized procedures and safety checklists to prevent errors and ensure successful task completion. The Risk Priority Numbers (RPNs) for the top twenty FMs were calculated before and after implementing the proposed workflow to evaluate its effectiveness. Three hundred seventy-four adaptive sessions across 5 treatment sites were performed, and each session was evaluated for treatment safety and FMEA assessment. Results: The OART workflow has 4 components, each with 4, 8, 13, and 4 sequentially executed tasks and safety checklists. Site-specific template preparation, which includes disease-specific physician directives and Intelligent Optimization Engine template testing, is one of the new procedures introduced. The interdisciplinary workflow significantly reduced the RPNs of the high-risk FMs, with an average decrease of 110 (maximum reduction of 305.5 and minimum reduction of 27.4). Conclusions: This study underscores the importance of addressing high-risk FMs associated with AI-driven OART and emphasizes the significance of safety measures in its implementation. By proposing a structured interdisciplinary workflow and integrated checklists, the study provides valuable insights into ensuring the safe and efficient delivery of OART while facilitating its effective integration into clinical practice.

Determining the potency of primordial germ cells by injection into early mouse embryos.

Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency in vitro by forming embryonic germ (EG) cells and in vivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation in vivo establishes a latent form of pluripotency that restricts chimera contribution.