Epigenetic mutagen-like environmental chemicals alter neural differentiation of human induced pluripotent stem cells.

Various chemicals, including pesticides, heavy metals, and metabolites of tobacco, have been detected in fetal environment. Fetuses are exposed to these chemicals at relatively low concentrations; however, their risk of developing neurological and behavioral disorders increases after birth. We aimed to evaluate the effects of five chemicals (diethylphosphate, cotinine, octachlorodipropyl ether, mercury, and selenium) detected in the serum of pregnant mothers on neural development using human neurospheres (NSphs) differentiated from induced pluripotent stem cells. Exposure to each chemical at serum concentrations revealed no effects on NSph development. However, combined exposure to the five chemicals caused a significant decrease in NSph size and altered gene expression and neural differentiation. Thus, we next focused on DNA methylation to investigate changes in NSph properties caused by chemical exposure. Combined exposure to chemicals had extremely small effects on the DNA methylation status of NSphs at individual gene loci. However, stochastic changes in methylation status caused by chemical exposure were significantly accumulated throughout the entire genome. These results suggest that the five chemicals acted as epimutagens that alter the epigenetic status during human neural development at the biological level. Taken together, we showed for the first time, the epimutagen-induced alterations in neural differentiation at serum concentrations using an in vitro human neuronal model.

Internal Trapping of a Growing Ruptured Dissecting Aneurysm of the A1 Segment: A Case Report and Literature Review.

A 47-year-old man presented with sudden-onset headache and Fisher group 3 subarachnoid hemorrhage. The World Federation of Neurological Surgeons grade was II. Digital subtraction angiography (DSA) only showed a vessel wall irregularity in the A1 segment of the right anterior cerebral artery (ACA), but an obvious bleeding source was not detected. Repeat angiography showed a tiny aneurysmal dilatation in the A1 segment with an intimal flap. The aneurysm enlarged on subsequent angiograms. Dissecting aneurysm was diagnosed, and the patient underwent internal trapping of the A1 segment to prevent rerupture. Postoperative DSA showed complete obliteration of the dissected segment. Magnetic resonance imaging showed a clinically silent cerebral infarction in the territory of the A1 segment perforators. Parent vessel occlusion for a dissected A1 segment can be effective, provided that sufficient collateral blood flow from the contralateral ACA is observed. We recommend endovascular trapping in this setting and hope that fellow clinicians select this approach for this rare pathology.

Factors affecting reasonable duration of continuous electrocardiographic monitoring to detect atrial fibrillation in acute ischemic stroke.

BACKGROUND: To examine the reasonable duration of continuous electrocardiographic monitoring (CEM) to detect AF at acute ischemic stroke. MATERIALS AND METHOD: 811 consecutive patients admitted to Tsuruga Municipal Hospital by acute ischemic stroke between April 2013 and December 2021 were enrolled in this study. Excluding 78 patients, 733 patients were analyzed by cluster analysis with SurvCART algorithm, followed by Kaplan-Meier analysis. RESULTS: The analysis provided step graphs for 8 subgroups. The duration of CEM to achieve the sensitivity of 0.8, 0.9, and 0.95 in each could be calculated. The duration of CEM to achieve the sensitivity of 0.8 are 18 days in female patients with heart failure (HF) (subgroup 1), 24 days in male patients with HF (subgroup 2), 22 days in patients without HF with arterial occlusion and pulse rate (PR) more than 91 (subgroup 3), 24 days in patients without HF with occlusion with PR less than 91 (subgroup 4), 18 days in patients without HF without occlusion with lacuna (subgroup 5), 26 days in patients without HF, occlusion, and lacuna, with arterial stenosis (subgroup 6), 15 days in patients without HF, occlusion, lacuna, and stenosis with BMI more than 21%(subgroup 7), and 44 days in patients without HF, occlusion, lacuna, stenosis and with BMI less than 21% (subgroup 8). CONCLUSIONS: Duration of CEM with the sensitivity of 0.8, 0.9, and 0.95 could be determined by presence of HF, female sex, arterial occlusion, PR more than 91/minute, presence of lacuna, presence of stenosis, and BMI more than 21%. (250).

Variation of DNA methylation on the IRX1/2 genes is responsible for the neural differentiation propensity in human induced pluripotent stem cells.

Introduction: Human induced pluripotent stem cells (hiPSCs) are useful tools for reproducing neural development in vitro. However, each hiPSC line has a different ability to differentiate into specific lineages, known as differentiation propensity, resulting in reduced reproducibility and increased time and funding requirements for research. To overcome this issue, we searched for predictive signatures of neural differentiation propensity of hiPSCs focusing on DNA methylation, which is the main modulator of cellular properties. Methods: We obtained 32 hiPSC lines and their comprehensive DNA methylation data using the Infinium MethylationEPIC BeadChip. To assess the neural differentiation efficiency of these hiPSCs, we measured the percentage of neural stem cells on day 7 of induction. Using the DNA methylation data of undifferentiated hiPSCs and their measured differentiation efficiency into neural stem cells as the set of data, and HSIC Lasso, a machine learning-based nonlinear feature selection method, we attempted to identify neural differentiation-associated differentially methylated sites. Results: Epigenome-wide unsupervised clustering cannot distinguish hiPSCs with varying differentiation efficiencies. In contrast, HSIC Lasso identified 62 CpG sites that could explain the neural differentiation efficiency of hiPSCs. Features selected by HSIC Lasso were particularly enriched within 3 Mbp of chromosome 5, harboring IRX1, IRX2, and C5orf38 genes. Within this region, DNA methylation rates were correlated with neural differentiation efficiency and were negatively correlated with gene expression of the IRX1/2 genes, particularly in female hiPSCs. In addition, forced expression of the IRX1/2 impaired the neural differentiation ability of hiPSCs in both sexes. Conclusion: We for the first time showed that the DNA methylation state of the IRX1/2 genes of hiPSCs is a predictive biomarker of their potential for neural differentiation. The predictive markers for neural differentiation efficiency identified in this study may be useful for the selection of suitable undifferentiated hiPSCs prior to differentiation induction.

Lead optimization of pyrido[2,3-d][1]benzazepin-6-one derivatives leading to the discovery of a potent, selective, and orally available human parathyroid hormone receptor 1 (hPTHR1) antagonist (DS69910557).

We report the discovery of a series of novel zwitterionic hPTHR1 antagonists. Optimization of lead compound 2 led to 4-[[1-[4-(2,9-dichloro-5,5-dimethyl-6-oxo-pyrido[2,3-d][1]benzazepin-7-yl)phenyl]-3-fluoro-azetidin-3-yl]methylamino]cyclohexanecarboxylic acid (19e, DS69910557), a compound with excellent potency and selectivity over activity at the human ether-a-go-go-related-gene (hERG) channel. Compound 19e demonstrated in vivo potency to decrease the plasma calcium concentration in rats upon oral administration. 2022 Elsevier Ltd. All rights reserved.

Identification of an epigenetic signature in human induced pluripotent stem cells using a linear machine learning model.

The use of human induced pluripotent stem cells (iPSCs), used as an alternative to human embryonic stem cells (ESCs), is a potential solution to challenges, such as immune rejection, and does not involve the ethical issues concerning the use of ESCs in regenerative medicine, thereby enabling developments in biological research. However, comparative analyses from previous studies have not indicated any specific feature that distinguishes iPSCs from ESCs. Therefore, in this study, we established a linear classification-based learning model to distinguish among ESCs, iPSCs, embryonal carcinoma cells (ECCs), and somatic cells on the basis of their DNA methylation profiles. The highest accuracy achieved by the learned models in identifying the cell type was 94.23%. In addition, the epigenetic signature of iPSCs, which is distinct from that of ESCs, was identified by component analysis of the learned models. The iPSC-specific regions with methylation fluctuations were abundant on chromosomes 7, 8, 12, and 22. The method developed in this study can be utilized with comprehensive data and widely applied to many aspects of molecular biology research.

Discovery of novel, potent, and orally bioavailable pyrido[2,3-d][1]benzazepin-6-one antagonists for parathyroid hormone receptor 1.

Structural modification of a 1,4-benzodiazepin-2-one-based PTHR1 antagonist 5, a novel type of PTHR1 antagonist previously synthesized in our laboratories, yielded compound 10, which had better chemical stability than compound 5. Successive optimization of the lead 10 improved aqueous solubility, metabolic stability, and animal pharmacokinetics, culminating in the identification of DS37571084 (12). Our study paves the way for the discovery of novel and orally bioavailable PTHR1 antagonists.

Dog Steroidogenic Factor-1: Molecular cloning and analysis of epigenetic regulation.

Steroidogenic factor 1 (SF-1) is a nuclear receptor that is important in steroid hormone production, and adrenal and gonad development. The SF-1 gene is highly conserved among most vertebrates. However, dog SF-1 registered in public databases, such as CanFam3.1, lacks the 5' end compared to other mammals including mouse, human, bovine, and cat. Whether this defect is due to species differences or database error is unclear. Here, we determined the full-length dog SF-1 cDNA sequence and identified the missing 5' end sequence in the databases. The coding region of the dog SF-1 gene has 1,386 base pairs, and the protein has 461 amino acid residues. Sequence alignment analysis among vertebrates revealed that the 5' end sequence of dog SF-1 cDNA is highly conserved compared to other vertebrates. The genomic position of the first exon was determined, and its promoter region sequence was analyzed. The DNA methylation state at the basal promoter and the expression of dog SF-1 in steroidogenic tissues and non-steroidogenic cells were examined. CpG sites at the basal promoter displayed methylation kinetics inversely correlated with gene expression. The promoter was hypomethylated and hypermethylated in SF-1 expressing and non-SF-1 expressing tissues, respectively. In conclusion, we identified the true full sequence of dog SF-1 cDNA and determined the genome sequence around the first exon. The gene is under the control of epigenetic regulation, such as DNA methylation, at the promoter.

DNA methylation ambiguity in the Fibrillin-1 (FBN1) CpG island shore possibly involved in Marfan syndrome.

Fibrillin-1 (FBN1) is responsible for haploinsufficient and autosomal dominant Marfan syndrome. Even in the same Marfan pedigree, penetrance and expressivity in heterozygous individuals can differ and result in variable disease onset and severity. Thus, other factors in addition to mutations in FBN1 are likely to contribute to the disease. In this study, we examined the regulation of FBN1 in porcine Marfan syndrome model, focusing on DNA methylation patterns distinguishable as wild-type (WT) and FBN1 null (KO) alleles in heterozygous cells. Most importantly, the ratio of the transcriptionally active hypomethylated WT allele was altered during cellular passage and highly correlated with FBN1 mRNA level compared with that in the KO allele. Transcribed FBN1 RNA from the KO allele was abolished after splicing coupled with translational initiation, suggesting that the functional FBN1 mRNA levels were affected by DNA methylation of the WT allele.

Discovery and structure-activity relationships of spiroindolines as novel inducers of oligodendrocyte progenitor cell differentiation.

A novel series of spiroindoline derivatives was discovered for use as inducers of oligodendrocyte progenitor cell (OPC) differentiation, resulting from optimization of screening hit 1. Exploration of structure-activity relationships led to compound 18, which showed improved potency (rOPC EC50 = 0.0032 µM). Furthermore, oral administration of compound 18 significantly decreased clinical severity in an experimental autoimmune encephalomyelitis (EAE) model.

Discovery of novel PTHR1 antagonists: Design, synthesis, and structure activity relationships.

The discovery and optimization of a novel series of PTHR1 antagonists are described. Starting from known PTHR1 antagonists, we identified more potent 1,4-benzodiazepin-2-one derivatives by means of a scaffold-hopping approach. The representative compound 23 (DS08210767) exhibited nanomolar-level PTHR1 antagonist activity and potential oral bioavailability in a pharmacokinetic study.

Deflation of a Rathke cleft cyst triggered rupture of a superior hypophyseal artery aneurysm: a case report.

A 57-year-old woman was diagnosed as a Rathke cleft cyst (RCC). Endoscopic transsphenoidal surgery (TSS) was performed uneventfully. She developed subarachnoid haemorrhage on postoperative day 3. The vessels adhered the cyst had been pulled into the pituitary fossa, causing an aneurysm.

Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors.

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hypermethylated regions compared with embryonic stem cells. These random aberrant hypermethylations might be a cause of the differences in the properties of each of the iPSC lines.

Effectiveness of bioengineered islet cell sheets for the treatment of diabetes mellitus.

BACKGROUND: The present study aimed to evaluate whether bioengineered mouse islet cell sheets can be used for the treatment of diabetes mellitus. METHODS: Isolated mouse pancreatic islets were dispersed, and cells were plated on temperature-responsive culture plates coated with iMatrix-551. On day 3 of culture, the sheets were detached from the plates and used for further analysis or transplantation. The following parameters were assessed: (1) morphology, (2) expression of ß-cell-specific transcription factors and other islet-related proteins, (3) methylation level of the pancreatic duodenal homeobox-1 (Pdx-1) promoter, as determined by bisulfite sequencing, and (4) levels of serum glucose after transplantation of one or two islet cell sheets into the abdominal cavity of streptozotocin-induced diabetic severe combined immunodeficiency mice. RESULTS: From each mouse, we recovered approximately 233.3 ± 12.5 islets and 1.4 ± 0.1 × 105 cells after dispersion. We estimate that approximately 68.2% of the cells were lost during dispersion. The viability of recovered single cells was 91.3 ± 0.9%. The engineered islet cell sheets were stable, but the messenger RNA levels of various ß-cell-specific transcription factors were significantly lower than those of primary islets, whereas Pdx-1 promoter methylation and the expression of NeuroD, Pdx-1, and glucagon proteins were similar between sheets and islets. Moreover, transplantation of islet cell sheets did not revert serum hyperglycemia in any of the recipient mice. CONCLUSIONS: Engineering effective islet cell sheets require further research efforts, as the currently produced sheets remain functionally inferior compared with primary islets.

[Significance of Thoraco-laparoscopic Observation in Repairing Diaphragmatic Injury by the Penetrating Trauma].

A 53-year-old man with a penetrating trauma was admitted to our hospital. Thoracoabdominal computed tomography (CT) on admission showed left diaphragmatic injury and peritoneal fat in the left thoracic cavity. Under a diagnosis of the traumatic diaphragmatic injury, an emergency operation was performed, and the left diaphragm was repaired. No other injuries were found in the thoracic and abdominal organs by thoraco-laparoscopic observation. The postoperative course was uneventful, and the patient left hospital on the 14th day after surgery. In case of the diaphragm injury, it is important to confirm the probable injuries of other organs by thoraco-laparoscopic observation.

DNA hypermethylation enhanced telomerase reverse transcriptase expression in human-induced pluripotent stem cells.

During reprogramming into human induced pluripotent stem cells (iPSCs), several stem cell marker genes are induced, such as OCT-4, NANOG, SALL4, and TERT. OCT-4, NANOG, and SALL4 gene expression can be regulated by DNA methylation. Their promoters become hypomethylated in iPSCs during reprogramming, leading to their induced expression. However, epigenetic regulation of the TERT gene remains unclear. In this study, we focused on epigenetic regulation of the human TERT gene and identified a differentially methylated region (DMR) at a distal region in the TERT promoter between human iPSCs and their parental somatic cells. Interestingly, the TERT-DMR was highly methylated in iPSCs, but low-level methylation was observed in their parental somatic cells. Region-specific, methylated-promoter assays showed that the methylated TERT-DMR up-regulated the promoter activity in iPSCs. In addition, Lamin B1 accumulated at the TERT-DMR in iPSCs, but not in their parent somatic cells. These results suggested that the TERT transcription was enhanced by DNA methylation at the TERT-DMR via binding to nuclear lamina during reprogramming. Our findings shed light on a new functional aspect of DNA methylation in gene expression.

Evidence of exposure to chemicals and heavy metals during pregnancy in Japanese women.

Purpose: Prenatal exposure to environmental chemicals is a growing concern, because such exposures have been shown to be associated with various diseases. The levels of chemicals and heavy metals in maternal blood, cord blood, maternal urine and amniotic fluid in Japanese pregnant women were investigated. Methods: A total of 145 women, including 14 fetal growth restriction cases, were included in the present study. The levels of phthalates (di[2-ethylhexyl]phthalate and mono[2-ethylhexyl]phthalate), perfluorinated compounds (perfluorooctane sulfonate, perfluorohexanoic acid, perfluorooctanoic acid, and perfluorononanoic acid), pesticides (dimethylphosphate, dimethylthiophosphate, diethylphosphate, diethylthiophosphate, 3-phenoxybenzoic acid, and octachlorodipropyl ether), bisphenol A, nicotine (nicotine, nornicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine), polybrominated diphenyl ethers, and heavy metals were measured. The relationship between fetal growth and the levels of chemicals and heavy metals were investigated. Results: Phthalates, perfluorinated compounds, pesticides, polybrominated diphenyl ethers, and heavy metals were detected in high frequency, whereas nicotine and bisphenol A were almost negative. Phthalates, perfluorinated compounds, and several heavy metals were transferred to the fetus. High perfluorononanoic acid levels in the maternal blood and cord blood, and low perfluorooctanoic acid level in the cord blood were significantly and negatively associated with fetal growth. Conclusions: The present study showed that pregnant women in Japan and their fetuses are exposed to a variety of chemicals and heavy metals.

Evaluation of obliteration of arteriovenous malformations after stereotactic radiosurgery with arterial spin labeling MR imaging.

PURPOSE: Complete obliteration of treated arteriovenous malformations (AVMs) can be diagnosed only by confirming the disappearance of arterio-venous (A-V) shunts with invasive catheter angiography. The authors evaluated whether non-invasive arterial spin labeling (ASL) magnetic resonance (MR) imaging can be used to diagnose the obliteration of AVMs facilitate the diagnosis of AVM obliteration after treatment with stereotactic radiosurgery (SRS). MATERIAL AND METHODS: Seven patients with a cerebral AVM treated by SRS were followed up with ASL images taken with a 3T-MR unit, and received digital subtraction angiography (DSA) after the AVM had disappeared on ASL images. Three patients among the seven received DSA also after the postradiosurgical AVM had disappeared on conventional MR images but A-V shunt was residual on ASL images. Four patients among the seven received contrast-enhanced (CE) MR imaging around the same period as DSA. RESULTS: ASL images could visualize postradiosurgical residual A-V shunts clearly. In all seven patients, DSA after the disappearance of A-V shunts on ASL images demonstrated no evidence of A-V shunts. In all three patients, DSA after the AVM had disappeared on conventional MR images but not on ASL images demonstrated residual A-V shunt. CE MR findings of AVMs treated by SRS did not correspond with DSA findings in three out of four patients. CONCLUSIONS: Findings of radiosurgically treated AVMs on ASL images corresponded with those on DSA. The results of this study suggest that ASL imaging can be utilized to follow up AVMs after SRS and to decide their obliteration facilitate to decide the precise timing of catheter angiography for the final diagnosis of AVM obliteration after SRS.

Establishment of DNA methylation patterns of the Fibrillin1 (FBN1) gene in porcine embryos and tissues.

DNA methylation in transcriptional regulatory regions is crucial for gene expression. The DNA methylation status of the edges of CpG islands, called CpG island shore, is involved in tissue/cell-type-specific gene expression. Haploinsufficiency diseases are caused by inheritance of one mutated null allele and are classified as autosomal dominant. However, in the same pedigree, phenotypic variances are observed despite the inheritance of the identical mutated null allele, including Fibrillin1 (FBN1), which is responsible for development of the haploinsufficient Marfan disease. In this study, we examined the relationship between gene expression and DNA methylation patterns of the FBN1 CpG island shore focusing on transcriptionally active hypomethylated alleles (Hypo-alleles). No difference in the DNA methylation level of FBN1 CpG island shore was observed in porcine fetal fibroblast (PFF) and the liver, whereas FBN1 expression was higher in PFF than in the liver. However, Hypo-allele ratio of the FBN1 CpG island shore in PFF was higher than that in the liver, indicating that Hypo-allele ratio of the FBN1 CpG island shore likely correlated with FBN1 expression level. In addition, oocyte-derived DNA hypermethylation in preimplantation embryos was erased until the blastocyst stage, and re-methylation of the FBN1 CpG island shore was observed with prolonged in vitro culture of blastocysts. These results suggest that the establishment of the DNA methylation pattern within the FBN1 CpG island shore occurs after the blastocyst stage, likely during organogenesis. In conclusion, Hypo-allele ratios of the FBN1 CpG island shore correlated with FBN1 expression levels in porcine tissues.

Cerebral hyperperfusion syndrome resulting in subarachnoid hemorrhage after carotid artery stenting.

A 64-year-old, right-handed man underwent endovascular treatment for internal carotid artery stenosis after experiencing a left-hemispheric transient ischemic attack. 15O-gas and H 215 O positron emission tomography revealed slightly reduced cerebral blood flow (CBF), elevated cerebral blood volume, and severely reduced cerebral vasoreactivity in the ipsilateral hemisphere as determined by an acetazolamide challenge test. The patient underwent left carotid artery stenting (CAS) via a prefemoral approach under local anesthesia without any complications. Follow-up examinations performed 20 h postoperatively showed subarachnoid hemorrhage (SAH) and cerebral hyperperfusion syndrome (CHS) in the left frontal lobe. Although it is a relatively rare phenomenon, SAH resulting from CHS was determined to be specifically caused by CAS. In this case, the causes of SAH may have been related to multiple factors including increased regional CBF, loss of cerebrovascular autoregulation, contrast agent-mediated disruption of major cerebral vessels, and strong antiplatelet therapy.