A facile access to aliphatic trifluoromethyl ketones via photocatalyzed cross-coupling of bromotrifluoroacetone and alkenes.

Biological molecules incorporating trifluoromethyl ketones (TFMKs) have emerged as reversible covalent inhibitors, aiding in the management and treatment of inflammatory diseases, cancer, and respiratory conditions. TFMKs, renowned for their versatile binding properties and adaptability, are pivotal in the rational design of novel drugs for diverse diseases. The photocatalytic insertion of alkenes, abundant feedstocks, into the α-carbon of trifluoromethylacetone represents a highly effective and atom-economical method for synthesizing valuable TFMKs. However, these processes typically necessitate high-energy photoirradiation (λ > 300 nm, Hg lamp) and stoichiometric oxidants to generate the acetonyl radical from acetone. In our study, we demonstrate the visible-light photocatalytic radical addition into olefins using bromotrifluoroacetone as the trifluoroacetonyl radical precursor under mild conditions. Aliphatic trifluoromethyl ketones or the corresponding bromo-substituted products can be obtained by selecting an appropriate photocatalyst and solvent. Comprehensive experimental investigations, including cyclic voltammetry, Stern-Volmer quenching studies, and kinetic isotope effects, corroborate the synthesis of trifluoroacetonyl radical species from bromotrifluoroacetone under photoredox conditions. Further, we demonstrate the efficient synthesis of an oseltamivir derivative bearing a trifluoromethylketone moiety, which shows promising biological activity. Hence, this methodology will streamline the direct introduction of trifluoromethyl ketone into biological target molecules during drug discovery.

Predicting postpartum hemorrhage in women undergoing planned cesarean section: A multicenter retrospective cohort study in Japan.

Given Japan's unique social background, it is critical to understand the current risk factors for postpartum hemorrhage (PPH) to effectively manage the condition, especially among specific groups. Therefore, this study aimed to identify the current risk factors for PPH during planned cesarean section (CS) in Japan. This multicenter retrospective cohort study was conducted in two tertiary maternal-fetal medicine units in Fukushima, Japan and included 1,069 women who underwent planned CS between January 1, 2013, and December 31, 2022. Risk factors for PPH (of > 1000 g and > 1500 g) were assessed using multivariate logistic regression analysis, considering variables such as maternal age, parity, assisted reproductive technology (ART) pregnancy, pre-pregnancy body mass index (BMI), uterine myoma, placenta previa, gestational age at delivery, birth weight categories, and hypertensive disorders of pregnancy (HDP). Multivariate linear regression analyses were conducted to predict estimated blood loss during planned CS. ART pregnancy, a pre-pregnancy BMI of 25.0-29.9 kg/m2, and uterine myoma increased PPH risk at various levels. Maternal smoking increased the risk of >1500 g PPH (adjusted odds ratio: 3.09, 95% confidence interval [CI]: 1.16-8.20). Multivariate linear analysis showed that advanced maternal age (B: 83 g; 95% CI: 27-139 g), ART pregnancy (B: 239 g; 95% CI: 121-357 g), pre-pregnancy BMI of 25.0-29.9 kg/m2 (B: 74 g; 95% CI: 22-167 g), uterine myoma (B: 151 g; 95% CI: 47-256 g), smoking (B: 107 g; 95% CI: 13-200 g), and birth weight > 3,500 g (B: 203 g; 95% CI: 67-338 g) were associated with blood loss during planned CS. Considering a patient's clinical characteristic may help predict bleeding in planned CSs and help improve patient safety.

Pioneering cord blood transplantation in relapsed/refractory HIV-related lymphoma: a case study with concurrent intramuscular antiretroviral therapy.

A 44-year-old HIV-positive man diagnosed with diffuse large B-cell lymphoma in 2021 achieved complete remission with six cycles of R-CHOP therapy but had a relapse in November 2022. ESHAP therapy failed to induce remission, leading to complete remission with four cycles of Pola-BR therapy. Post-failure of autologous stem cell harvest, cord blood transplantation (CBT) was performed in June 2023. Notably, this case used recently approved intramuscular antiretroviral therapy (ART) with cabotegravir and rilpivirine, addressing gastrointestinal complications during CBT. This innovative use of intramuscular ART in the treatment of malignancy represents a first in the field, offering a pioneering approach to HIV-related lymphoma.

Hydropic leiomyoma-like ovarian tumor: a case report.

Uterine leiomyomas, benign tumors common in reproductive-aged women, can display rare variants such as hydropic leiomyoma (HL), which exhibit unique histological features like zonal edema and increased vascularity. However, due to its rarity, comprehensive clinical knowledge about HL is limited. We report a case of a 49-year-old Japanese woman who was premenopausal and nulliparous, presenting with a two-year history of abdominal distension. An MRI scan revealed a 20 cm mass in the posterior part of the uterus, exhibiting characteristics suggestive of an ovarian tumor. During laparotomy, a cystic tumor connected with a swollen fibroid was found, and pathology confirmed HL. This case emphasizes that hydropic leiomyomas can mimic malignant tumors on ultrasonography due to their atypical features, necessitating additional evaluations using alternative imaging techniques or histopathological examinations for accurate diagnosis and appropriate management. The patient recovered uneventfully, broadening our understanding of HL's clinical presentation.

Trisomy 12 compromises the mesendodermal differentiation propensity of human pluripotent stem cells.

Trisomy 12 is one of the most frequent chromosomal abnormalities in cultured human pluripotent stem cells (hPSCs). Although potential oncogenic properties and augmented cell cycle caused by trisomy 12 have been reported, the consequences of trisomy 12 in terms of cell differentiation, which is the basis for regenerative medicine, drug development, and developmental biology studies, have not yet been investigated. Here, we report that trisomy 12 compromises the mesendodermal differentiation of hPSCs. We identified sublines of hPSCs carrying trisomy 12 after their prolonged culture. Transcriptome analysis revealed that these hPSC sublines carried abnormal gene expression patterns in specific signaling pathways in addition to cancer-related cell cycle pathways. These hPSC sublines showed a lower propensity for mesendodermal differentiation in embryoid bodies cultured in a serum-free medium. BMP4-induced exit from the self-renewal state was impaired in the trisomy 12 hPSC sublines, with less upregulation of key transcription factor gene expression. As a consequence, the differentiation efficiency of hematopoietic and hepatic lineages was also impaired in the trisomy 12 hPSC sublines. We reveal that trisomy 12 disrupts the genome-wide expression patterns that are required for proper mesendodermal differentiation.

Trophoblast cell surface antigen-2 phosphorylation triggered by binding of galectin-3 drives metastasis through down-regulation of E-cadherin.

The expression of trophoblast cell surface antigen-2 (Trop-2) is enhanced in many tumor tissues and is correlated with increased malignancy and poor survival of patients with cancer. Previously, we demonstrated that the Ser-322 residue of Trop-2 is phosphorylated by protein kinase Cα (PKCα) and PKCδ. Here, we demonstrate that phosphomimetic Trop-2 expressing cells have markedly decreased E-cadherin mRNA and protein levels. Consistently, mRNA and protein of the E-cadherin-repressing transcription factors zinc finger E-Box binding homeobox 1 (ZEB1) were elevated, suggesting transcriptional regulation of E-cadherin expression. The binding of galectin-3 to Trop-2 enhanced the phosphorylation and subsequent cleavage of Trop-2, followed by intracellular signaling by the resultant C-terminal fragment. Binding of ß-catenin/transcription factor 4 (TCF4) along with the C-terminal fragment of Trop-2 to the ZEB1 promoter upregulated ZEB1 expression. Of note, siRNA-mediated knockdown of ß-catenin and TCF4 increased the expression of E-cadherin through ZEB1 downregulation. Knockdown of Trop-2 in MCF-7 cells and DU145 cells resulted in downregulation of ZEB1 and subsequent upregulation of E-cadherin. Furthermore, wild-type and phosphomimetic Trop-2 but not phosphorylation-blocked Trop-2 were detected in the liver and/or lung of some nude mice bearing primary tumors inoculated intraperitoneally or subcutaneously with wild-type or mutated Trop-2 expressing cells, suggesting that Trop-2 phosphorylation, plays an important role in tumor cell mobility in vivo, too. Together with our previous finding of Trop-2 dependent regulation of claudin-7, we suggest that the Trop-2-mediated cascade involves concurrent derangement of both tight and adherence junctions, which may drive metastasis of epithelial tumor cells.

Construction of brain area risk map for decision making using surgical navigation and motor evoked potential monitoring information.

PURPOSE: Surgical devices or systems typically operate in a stand-alone manner, making it difficult to perform integration analysis of both intraoperative anatomical and functional information. To address this issue, the intraoperative information integration system OPeLiNK® was developed. The objective of this study is to generate information for decision making using surgical navigation and intraoperative monitoring information accumulated in the OPeLiNK® database and to analyze its utility. METHODS: We accumulated intraoperative information from 27 brain tumor patients who underwent resection surgery. First, the risk rank for postoperative paralysis was set according to the attenuation rate and amplitude width of the motor evoked potential (MEP). Then, the MEP and navigation log data were combined and plotted on an intraoperative magnetic resonance image of the individual brain. Finally, statistical parametric mapping (SPM) transformation was performed to generate a standard brain risk map of postoperative paralysis. Additionally, we determined the anatomical high-risk areas using atlases and analyzed the relationship with each set risk rank. RESULTS: The average distance between the navigation log corresponding to each MEP risk rank and the anatomical high-risk area differed significantly between the with postoperatively paralyzed and without postoperatively paralyzed groups, except for "safe." Furthermore, no excessive deformation was observed resulting from SPM conversion to create the standard brain risk map. There were cases in which no postoperative paralysis occurred even when MEP decreased intraoperatively, and vice versa. CONCLUSION: The time synchronization reliability of the study data is very high. Therefore, our created risk map can be reported as being functional at indicating the risk areas. Our results suggest that the statistical risks of postoperative complications can be presented for each area where brain surgery is to be performed. In the future, it will be possible to provide surgical navigation with intraoperative support that reflects the risk maps created.

Clinical study of skill assessment based on time sequential measurement changes.

Endoscopic sinus surgery is a common procedure for chronic sinusitis; however, complications have been reported in some cases. Improving surgical outcomes requires an improvement in a surgeon's skills. In this study, we used surgical workflow analysis to automatically extract "errors," indicating whether there was a large difference in the comparative evaluation of procedures performed by experts and residents. First, we quantified surgical features using surgical log data, which contained surgical instrument information (e.g., tip position) and time stamp. Second, we created a surgical process model (SPM), which represents the temporal transition of the surgical features. Finally, we identified technical issues by creating an expert standard SPM and comparing it to the novice SPM. We verified the performance of our methods by using the clinical data of 39 patients. In total, 303 portions were detected as an error, and they were classified into six categories. Three risky operations were overlooked, and there were 11 overdetected errors. We noted that most errors detected by our method involved dangers. The implementation of our methods of automatic improvement points detection may be advantageous. Our methods may help reduce the time for reviewing and improving the surgical technique efficiently.

Transient ETV2 Expression Promotes the Generation of Mature Endothelial Cells from Human Pluripotent Stem Cells.

Differentiation protocols are used for induced pluripotent stem cells (iPSCs) in in vitro disease modeling and clinical applications. Transplantation of endothelial cells (ECs) is an important treatment strategy for ischemic diseases. For example, in vitro generated ECs can be used to provide the vascular plexus to regenerate organs such as the liver. Here, we demonstrate that the E-twenty-six (ETS) transcription factor ETV2 alone can directly convert iPSCs into vascular endothelial cells (iPS-ETV2-ECs) with an efficiency of over 90% within 5 d. Although the stable overexpression of ETV2 induced the expression of multiple key factors for endothelial development, the induced ECs were less mature. Furthermore, doxycycline-inducible transient ETV2 expression could upregulate the expression of von Willebrand factor (vWF) in iPS-ETV2-ECs, leading to a mature phenotype. The findings of this study on generation of mature iPS-ETV2-ECs provide further insights into the exploration of cell reprogramming from iPSCs. Here, we provide a new protocol for differentiation of iPSCs, thus providing a new source of ECs for in vitro disease modeling and clinical applications.

Brain microvascular endothelial cells derived from human induced pluripotent stem cells as in vitro model for assessing blood-brain barrier transferrin receptor-mediated transcytosis.

The blood-brain barrier (BBB), a selective barrier formed by brain microvascular endothelial cells (BMEC), represents a major challenge for the efficient accumulation of pharmaceutical drugs into the brain. The receptor-mediated transcytosis (RMT) has recently gained increasing interest for pharmaceutical industry as it shows a great potential to shuttle large-sized therapeutic cargos across the BBB. Confirming the presence of the RMT pathway by BMEC is therefore important for the screening of peptides or antibody libraries that bind RMT receptors. Herein, a comparative study was performed between a human cell line of BMEC (HBEC) and human induced pluripotent stem cells-derived BMEC-like cells (hiPS-BMEC). The significantly higher gene and protein expressions of transporters and tight junction proteins, excepting CD31 and VE-cadherin were exhibited by hiPS-BMEC than by HBEC, suggesting more biomimetic BBB features of hiPS-BMEC. The presence and functionality of transferrin receptor (TfR), known to use RMT pathway, were confirmed using hiPS-BMEC by competitive binding assays and confocal microscopy observations. Finally, cysteine-modified T7 and cysteine modified-Tfr-T12 peptides, previously reported to be ligands of TfR, were compared regarding their permeability using hiPS-BMEC. The hiPS-BMEC could be useful for the identification of therapeutics that can be transported across the BBB using RMT pathway.

Transport Characteristics of 6-Mercaptopurine in Brain Microvascular Endothelial Cells Derived From Human Induced Pluripotent Stem Cells.

The likelihood of reoccurrence of acute lymphoblastic leukemia is influenced by the cerebral concentration of the therapeutic agent 6-mercaptopurine (6-MP) during treatment. Therefore, it is important to understand the blood-brain barrier (BBB) transport mechanism of 6-MP. The purpose of this study was to characterize this mechanism using human induced pluripotent stem cell-derived microvascular endothelial cells (hiPS-BMECs). The permeability coefficient of 6-MP across hiPS-BMECs monolayer in the basal-to-apical direction (B-to-A) was significantly greater than that in the opposite direction (A-to-B). The inhibition profiles of 6-MP transport in the A-to-B direction were different from those in the B-to-A direction. Transport in the A-to-B direction was mainly inhibited by adenine (an inhibitor of equilibrative nucleobase transporter 1; ENBT1), while transport in the B-to-A direction was significantly reduced by inhibitors of multidrug resistance-associated proteins (MRPs), especially zaprinast (an MRP5 inhibitor). Immunocytochemical analyses demonstrated the expression of ENBT1 and MRP5 proteins in hiPS-BMECs. We confirmed that the cellular uptake of 6-MP is decreased by ENBT1 inhibitors in hiPS-BMECs and by knockdown of ENBT1 in hCMEC/D3 cells. These results suggest that ENBT1 and MRP5 make substantial contributions to the transport of 6-MP in hiPS-BMECs and hCMEC/D3 cells.

Functional Investigation of Solute Carrier Family 35, Member F2, in Three Cellular Models of the Primate Blood-Brain Barrier.

Understanding the mechanisms of drug transport across the blood-brain barrier (BBB) is an important issue for regulating the pharmacokinetics of drugs in the central nervous system. In this study, we focused on solute carrier family 35, member F2 (SLC35F2), whose mRNA is highly expressed in the BBB. SLC35F2 protein was enriched in isolated mouse and monkey brain capillaries relative to brain homogenates and was localized exclusively on the apical membrane of MDCKII cells and brain microvascular endothelial cells (BMECs) differentiated from human induced pluripotent stem cells (hiPS-BMECs). SLC35F2 activity was assessed using its substrate, YM155, and pharmacological experiments revealed SLC35F2 inhibitors, such as famotidine (half-maximal inhibitory concentration, 160 µM). Uptake of YM155 was decreased by famotidine or SLC35F2 knockdown in immortalized human BMECs (human cerebral microvascular endothelial cell/D3 cells). Furthermore, famotidine significantly inhibited the apical (A)-to-basal (B) transport of YM155 in primary cultured monkey BMECs and hiPS-BMECs. Crucially, SLC35F2 knockout diminished the A-to-B transport and intracellular accumulation of YM155 in hiPS-BMECs. By contrast, in studies using an in situ brain perfusion technique, neither deletion of Slc35f2 nor famotidine reduced brain uptake of YM155, even though YM155 is a substrate of mouse SLC35F2. YM155 uptake was decreased significantly by losartan and naringin, inhibitors for the organic anion transporting polypeptide (OATP) 1A4. These findings suggest SLC35F2 is a functional transporter in various cellular models of the primate BBB that delivers its substrates to the brain and that its relative importance in the BBB is modified by differences in the expression of OATPs between primates and rodents. SIGNIFICANCE STATEMENT: This study demonstrated that SLC35F2 is a functional drug influx transporter in three different cellular models of the primate blood-brain barrier (i.e., human cerebral microvascular endothelial cell/D3 cells, primary cultured monkey BMECs, and human induced pluripotent stem-BMECs) but has limited roles in mouse brain. SLC35F2 facilitates apical-to-basal transport across the tight cell monolayer. These findings will contribute to the development of improved strategies for targeting drugs to the central nervous system.

Design and Development of an HBT-Based Ratiometric Fluorescent Probe to Monitor Stress-Induced Premature Senescence.

Stress-induced premature senescence (SIPS) can be induced in tumor cells by reactive oxygen species (ROS) or oncogenes. The antineoplastic drugs cause apoptosis and senescence by damaging the DNA. Although the detection of cellular senescence is important to monitor drug response during anticancer therapy, only a few probes have been studied for imaging SIPS. In this study, we developed 2-(2'-hydroxyphenyl)benzothiazole (HBT)-based fluorescent probes to determine SIPS by monitoring the oxidative stress and ß-galactosidase activity. HBT is a commonly used fluorophore because of its luminescence mechanism via excited-state intramolecular proton transfer, and it has attractive properties, such as a four-level photochemical process and large Stokes shift (151 nm). A novel fluorescent probe, (2-(benzo[d]thiazol-2-yl)phenyl)boronic acid, was prepared for the detection of ROS, including H2O2, via the oxidation reaction of arylboronic acids to form the fluorescent phenol, HBT. In addition, to determine the enzymatic activity of ß-galactosidase, a 2-(4'-chloro-2'-hydroxyphenyl)benzothiazole (CBT)-based enzymatic turn-on probe (CBT-ß-Gal) was designed and synthesized. ß-Galactosidase catalyzed the hydrolysis of ß-galactopyranoside from CBT-ß-Gal to release the fluorescent CBT. These probes were capable of ratiometric imaging the accumulation of H2O2 and the degree of ß-galatosidase activity in contrast to H2O2-untreated and H2O2-treated HeLa cells. Furthermore, these probes were successfully employed for imaging the increased levels of ROS and ß-galactosidase activity in the doxorubicin-treated HeLa cells.

Transcriptomic Dissection of Hepatocyte Heterogeneity: Linking Ploidy, Zonation, and Stem/Progenitor Cell Characteristics.

BACKGROUND & AIMS: There is a long-standing debate regarding the biological significance of polyploidy in hepatocytes. Recent studies have provided increasing evidence that hepatocytes with different ploidy statuses behave differently in a context-dependent manner (eg, susceptibility to oncogenesis, regenerative ability after injury, and in vitro proliferative capacity). However, their overall transcriptomic differences in a physiological context is not known. METHODS: By using microarray transcriptome analysis, we investigated the heterogeneity of hepatocyte populations with different ploidy statuses. Moreover, by using single-cell quantitative reverse-transcription polymerase chain reaction (scPCR) analysis, we investigated the intrapopulational transcriptome heterogeneity of 2c and 4c hepatocytes. RESULTS: Microarray analysis showed that cell cycle-related genes were enriched in 8c hepatocytes, which is in line with the established notion that polyploidy is formed via cell division failure. Surprisingly, in contrast to the general consensus that 2c hepatocytes reside in the periportal region, in our bulk transcriptome and scPCR analyses, the 2c hepatocytes consistently showed pericentral hepatocyte-enriched characteristics. In addition, scPCR analysis identified a subpopulation within the 2c hepatocytes that co-express the liver progenitor cell markers Axin2, Prom1, and Lgr5, implying the potential biological relevance of this subpopulation. CONCLUSIONS: This study provides new insights into hepatocyte heterogeneity, namely 2c hepatocytes are preferentially localized to the pericentral region, and a subpopulation of 2c hepatocytes show liver progenitor cell-like features in terms of liver progenitor cell marker expression (Axin2, Prom1, and Lgr5).

The role of galanin in the differentiation of mucosal mast cells in mice.

Mast cells are generally classified into two phenotypically distinct populations: mucosal-type mast cells (MMCs) and connective tissue-type mast cells (CTMCs). However, the molecular basis determining the different characteristics of the mast cell subclasses still remains unclear. Unfortunately, the number of mast cells that can be obtained from tissues is limited, which makes it difficult to study the function of each mast cell subclass. Here, we report the generation and characterization of MMCs and CTMCs derived from mouse BM mast cells (BMMCs). We found that the expression of galanin receptor 3 was elevated in MMCs when compared to the expression in CTMCs. Moreover, intraperitoneal injection of a galanin antagonist reduced MMCs and inhibited the inflammation of dextran sodium sulfate-induced colitis in mice. Therefore, these results suggest that galanin promotes MMC differentiation in vivo, and provide important insights into the molecular mechanisms underlying the differentiation of mast cell subclasses.

Generation of human hepatic progenitor cells with regenerative and metabolic capacities from primary hepatocytes.

Hepatocytes are regarded as the only effective cell source for cell transplantation to treat liver diseases; however, their availability is limited due to a donor shortage. Thus, a novel cell source must be developed. We recently reported that mature rodent hepatocytes can be reprogrammed into progenitor-like cells with a repopulative capacity using small molecule inhibitors. Here, we demonstrate that hepatic progenitor cells can be obtained from human infant hepatocytes using the same strategy. These cells, named human chemically induced liver progenitors (hCLiPs), had a significant repopulative capacity in injured mouse livers following transplantation. hCLiPs redifferentiated into mature hepatocytes in vitro upon treatment with hepatic maturation-inducing factors. These redifferentiated cells exhibited cytochrome P450 (CYP) enzymatic activities in response to CYP-inducing molecules and these activities were comparable with those in primary human hepatocytes. These findings will facilitate liver cell transplantation therapy and drug discovery studies.

One of the most successful treatments for liver disease is transplanting a donor liver into a patient. But demands for donor livers far outstrips supply. A promising alternative could be, rather than replacing the whole organ, to transplant patients with individual liver cells called hepatocytes. These cells can then move into the liver, replace damaged cells, and help support the organ. However, hepatocytes are also in short supply, as despite the liver's amazing regenerative abilities, these cells struggle to divide outside of the body. Improving how these cells multiply, could therefore help more people receive hepatocyte transplants. In 2017, researchers found a way to convert mouse and rat hepatocytes into cells that could divide more rapidly using a cocktail of three small molecules. These 'chemically induced liver progenitors', or CLiPs for short, were able to mature into working hepatocytes and support injured mouse livers. But, discoveries made in rats and mice are not always applicable to humans. Now, Katsuda et al. ­ including some of the researchers involved in the 2017 work ­ have set out to investigate whether CLiPs can also be made from human cells, and if so, whether these cells can be used for hepatocyte transplantations. Using a similar cocktail of molecules, Katsuda et al. managed to convert infant human hepatocytes into CLiPs. As with the rodent cells, these human CLiPs were able to turn back into mature, working liver cells. When transplanted into mice with genetic liver diseases, the human CLiPs moved into the liver and became part of the organ. These transplanted cells were able to reconstruct the liver tissue of diseased mice, and in some cases, replaced more than 90% of the liver's damaged cells. Developing human CLiP technology could provide a new way to support people on the waiting list for liver transplantation. But there are some obstacles still to overcome. At present the technique only works with hepatocytes from infant donors. The next step is to improve the method so that it works with liver cells donated by adults.

Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption.

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+ /K+ -ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate-mediated excitotoxicity. Under a pyruvate-restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 µM. The OCR monitoring also revealed the contribution of the N-methyl-D-aspartate receptor and Na+ /K+ -ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte-dependent protection of neurons from glutamate-mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.

ETV2-TET1/TET2 Complexes Induce Endothelial Cell-Specific Robo4 Expression via Promoter Demethylation.

Although transcription factors regulating endothelial cell (EC)-specific gene expression have been identified, it is not known how those factors induce EC-specificity. We previously reported that DNA hypomethylation of the proximal promoter elicits EC-specific expression of Roundabout4 (Robo4). However, the mechanisms establishing EC-specific hypomethylation of the Robo4 promoter remain unknown. In this study, we demonstrated that the hypermethylated Robo4 proximal promoter is demethylated as human iPS cells differentiate into endothelial cells. Reporter assays demonstrated that ETV2, an ETS family transcription factor, bound to ETS motifs in the proximal promoter and activated Robo4 expression. Immunoprecipitation demonstrated direct interaction between ETV2 and methylcytosine-converting enzymes TET1 and TET2. Adenoviral expression of ETV2-TET1/TET2 complexes demethylated the Robo4 promoter and induced Robo4 expression in non-ECs. In summary, we propose a novel regulatory model of EC-specific gene expression via promoter demethylation induced by ETV2-TET1/TET2 complexes during endothelial differentiation.

Prenatal diagnosis of premature chromatid separation/mosaic variegated aneuploidy (PCS/MVA) syndrome.

Premature chromatid separation/mosaic variegated aneuploidy (PCS/MVA) syndrome is a rare genetic disorder. In this case report, we describe the prenatal diagnosis of PCS/MVA syndrome in a 24-year-old, gravida 1, para 1, woman who was referred to us in her second trimester due to fetal growth restriction and extreme microcephaly (-5.0 standard deviations). Amniocentesis and chromosomal analysis confirmed PCS in 80% of cultured fetal cells. PCS findings were positive in 9% of paternal cells and 11% of maternal cells, indicative that both were PCS carriers. Genetic analysis confirmed that the fetus carried a combined heterozygote of maternal G > A point mutation of the promoter area of the BUB1B gene and a paternal Alu sequence insertion between intron 8 and exon 9 of the BUB1B gene. As PCS/MVA syndrome is associated with the development of various malignancies in early life, prenatal diagnosis is important for effective planning of post-natal care.

In vitro model of cerebral ischemia by using brain microvascular endothelial cells derived from human induced pluripotent stem cells.

Brain-derived microvascular endothelial cells (BMECs), which play a central role in blood brain barrier (BBB), can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported, they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy, drug screening, and pathological study. In the recent study, an induction method of BMECs from PSCs has been established, making it possible to more precisely study the in vitro human BBB function. Here, using induced pluripotent stem (iPS) cell-derived BMECs, we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function, and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly, TNF-α, which is known to be secreted from astrocytes and microglia in the cerebral ischemia, prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus, we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs.