Usefulness of "AcT ratio" in diagnosis of internal carotid artery stenosis: a multicenter, retrospective, observational study.

PURPOSE: The ratio of the internal carotid artery (ICA) to the common carotid artery (CCA), especially the "AcT ratio," which is a modified measurement method of acceleration time, is useful for diagnosing ICA-origin stenosis. However, previous studies were single-center studies. Therefore, this multicenter, retrospective, cross-sectional study aimed to determine whether a method using the AcT ratio is useful for estimating stenosis rates. METHODS: This study included 461 vessels subjected to carotid artery ultrasonography and evaluation for ICA-origin stenosis via NASCET at four hospitals. The duration from the steep rise point to the inflection point or the first peak was defined as AcT on pulsed wave Doppler. The AcT ratio was calculated as AcT of ICA/AcT of ipsilateral CCA. The AcT ratio and rate of ICA-origin stenosis were analyzed using Pearson's correlation coefficient, simple regression analysis, and ROC curve. RESULTS: A significant positive correlation was observed between the AcT ratio and NASCET stenosis. NASCET stenosis of ≥ 50% had a sensitivity, specificity, and negative predictive value (NPV) of 70.2%, 71.6%, and 91.5%, respectively, when the cut-off value of the AcT ratio was 1.17. NASCET stenosis of ≥ 70% had a sensitivity, specificity, and NPV of 70.5%, 72.1%, and 95.9%, respectively, when the cut-off value of the AcT ratio was 1.22. CONCLUSIONS: The findings of this multicenter, retrospective, cross-sectional study suggest that the AcT ratio is useful for diagnosing ICA-origin stenosis, especially for diagnosis by exclusion. NASCET stenosis of ≥ 50% was considered unlikely if the Act ratio was ≤ 1.17, whereas NASCET stenosis of ≥ 70% was considered unlikely if it was ≤ 1.22.

Nanobubbles in vase water inhibit transpiration and prolong the vase life of cut chrysanthemum flowers.

Nanobubble (NB) water has been shown to promote the growth of several types of plants and animals, but the mechanism underlying this promoting effect remains unclear. The present study evaluated the mechanism by which NBs maintain the freshness of cut flowers by keeping cut chrysanthemum (Chrysanthemum morifolium Ramat.) flowers at the bud stage in vase water containing air NBs. The condition of petals and leaves was assessed to determine the vase life of these cut flowers. The NB treatment delayed bud opening and petal senescence of the inflorescences. Water absorption and transpiration by cut flower stems were lower in NB water than in distilled water (DW). Furthermore, when all the leaves were removed from the cut flower stems, no significant difference in vase life was observed between NB water and DW. These findings indicate that the inhibition of transpiration from leaves prolonged the vase life of NB-treated cut chrysanthemum flowers. In the early stage of the treatment, NB treatment significantly reduced transpiration without closing stomata, suggesting that the reduction in transpiration observed in the NB-treated plants might be due to the suppression of cuticular transpiration, defined as water loss through the epidermis. Surface tension, one of the important driving forces of water movement in plants, was not affected by the presence of NBs in water. To our knowledge, this is the first report to show that transpiration from leaves is inhibited by NB treatment.

Amplified Risk of Intracranial Artery Stenosis/Occlusion Associated With RNF213 p.R4810K in Familial Hypercholesterolemia.

Background: The RNF213 p.R4810K variant is associated with moyamoya disease in East Asian individuals and increases the risk of developing intracranial major artery stenosis/occlusion (ICASO) that affects anterior circulation. Meanwhile, 0.5% to 2.5% of asymptomatic East Asian individuals also carry this variant. As such, additional factors are likely required to develop ICASO in variant carriers. Familial hypercholesterolemia (FH) is a common genetic disorder in Japan that has a significant associated risk of developing premature coronary atherosclerosis; however, the relationship between ICASO and FH remains unknown. Objectives: This study aimed to determine if FH facilitates RNF213 p.R4810K carriers to develop ICASO. Methods: We enrolled patients with FH who had undergone brain magnetic resonance angiography at our hospital from May 2005 to March 2020. The RNF213 p.R4810K variant, and LDLR and PCSK9 mutations were genotyped. ICASO lesions in the brain magnetic resonance angiogram were analyzed. Results: Six RNF213 p.R4810K variant carriers were identified among 167 patients with FH (LDLR, n = 104; PCSK9, n = 22). Five of the carriers (83.3%) exhibited ICASO in the anterior circulation; a significant difference in ICASO frequency was observed between the variant carriers and noncarriers (P = 0.025). The median number of stenotic or occluded arteries in the anterior circulation was also significantly larger in the variant carriers (3 vs 1, P = 0.01); however, did not differ between patients with FH with LDLR and PCSK9 mutations. Conclusions: Patients with FH exhibit increased prevalence and severity of ICASO associated with RNF213 p.R4810K. Gene mutations for FH may confer an increased risk of ICASO in RNF213 p.R4810K carriers.

Neovascularization From the Carotid Artery Lumen Into the Carotid Plaque Confirmed by Contrast-Enhanced Ultrasound and Histology.

OBJECTIVE: This study was aimed at assessing intraplaque neovessels, focusing on neovascularization from the vascular luminal side using contrast-enhanced ultrasound (CEUS) and determining that this contrast effect indicates that the neovessel is connected to the vessel lumen histopathologically. Whether plaque vulnerability can be assessed more accurately was also investigated. METHODS: We enrolled consecutive patients with internal carotid artery stenosis who underwent carotid endarterectomy (CEA) and pre-operative CEUS with perflubutane of the carotid arteries. We graded the contrast effect semi-quantitatively from the vascular luminal and adventitial sides. We compared the contrast effect with the pathological findings, especially the neovascularization of the CEA specimens. RESULTS: In total, 68 carotid arterial atheromatous plaques (47 symptomatic) were analyzed. Symptomatic plaques were significantly correlated with stronger contrast effects from the luminal side than from the adventitial side (p = 0.0095). Microbubbles from the luminal side appeared to flow mainly into the plaque shoulder. The contrast effect value for the plaque shoulder and neovessel density were significantly correlated (ρ = 0.35, p = 0.031). Neovessel density was significantly higher in symptomatic than in asymptomatic plaques (56.2 ± 43.7/mm2 and 18.1 ± 15.2/mm2, respectively, p < 0.0001). Serial histological sections of CEA specimens in a symptomatic plaque with a strong contrast effect from the luminal side revealed multiple neovessels fenestrated to the vessel lumen with endothelial cells, consistent with the CEUS findings. CONCLUSION: Contrast-enhanced ultrasound can be used to evaluate neovessels originating from the luminal side, histopathologically confirmed in serial sections. Symptomatic vulnerable plaque is correlated more significantly with intraplaque neovascularization from the luminal side than with neovascularization from the adventitia.

Ultrasound-guided computed tomography angiography for the diagnosis of rotational vertebral artery occlusion: 2 case reports and technical notes.

Rotational vertebral artery occlusion is a rare cause of ischemic stroke in the vertebrobasilar arteries. While computed tomography angiography (CTA) is less invasive for the diagnosis of rational vertebral artery occlusion than digital subtraction angiography and more useful for elucidating the correlation between vertebrobasilar arteries and the surrounding structure, carotid ultrasound is noninvasive and more beneficial for the real-time evaluation of the hemodynamic change with neck rotation compared to CTA. Here, we reported 2 cases of rotational vertebral artery occlusion in patients aged 81 and 38 years and proposed a novel technique for its diagnosis using ultrasound-guided CTA. We suggest that the combination of ultrasound and CTA is useful for diagnosing rotational vertebral artery occlusion, which would compensate for the disadvantages of CTA alone.

Systemic Supplementation of Collagen VI by Neonatal Transplantation of iPSC-Derived MSCs Improves Histological Phenotype and Function of Col6-Deficient Model Mice.

Collagen VI is distributed in the interstitium and is secreted mainly by mesenchymal stromal cells (MSCs) in skeletal muscle. Mutations in COL6A1-3 genes cause a spectrum of COL6-related myopathies. In this study, we performed a systemic transplantation study of human-induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) into neonatal immunodeficient COL6-related myopathy model (Col6a1 KO /NSG) mice to validate the therapeutic potential. Engraftment of the donor cells and the resulting rescued collagen VI were observed at the quadriceps and diaphragm after intraperitoneal iMSC transplantation. Transplanted mice showed improvement in pathophysiological characteristics compared with untreated Col6a1 KO /NSG mice. In detail, higher muscle regeneration in the transplanted mice resulted in increased muscle weight and enlarged myofibers. Eight-week-old mice showed increased muscle force and performed better in the grip and rotarod tests. Overall, these findings support the concept that systemic iMSC transplantation can be a therapeutic option for COL6-related myopathies.

DMRT1-mediated reprogramming drives development of cancer resembling human germ cell tumors with features of totipotency.

In vivo reprogramming provokes a wide range of cell fate conversion. Here, we discover that in vivo induction of higher levels of OSKM in mouse somatic cells leads to increased expression of primordial germ cell (PGC)-related genes and provokes genome-wide erasure of genomic imprinting, which takes place exclusively in PGCs. Moreover, the in vivo OSKM reprogramming results in development of cancer that resembles human germ cell tumors. Like a subgroup of germ cell tumors, propagated tumor cells can differentiate into trophoblasts. Moreover, these tumor cells give rise to induced pluripotent stem cells (iPSCs) with expanded differentiation potential into trophoblasts. Remarkably, the tumor-derived iPSCs are able to contribute to non-neoplastic somatic cells in adult mice. Mechanistically, DMRT1, which is expressed in PGCs, drives the reprogramming and propagation of the tumor cells in vivo. Furthermore, the DMRT1-related epigenetic landscape is associated with trophoblast competence of the reprogrammed cells and provides a therapeutic target for germ cell tumors. These results reveal an unappreciated route for somatic cell reprogramming and underscore the impact of reprogramming in development of germ cell tumors.

AMPK activation reverts mouse epiblast stem cells to naive state.

Despite increasing knowledge on primed and naive pluripotency, the cell signaling that regulates the pluripotency type in stem cells remains not fully understood. Here we show that AMP kinase (AMPK) activators can induce the reversion of primed mouse epiblast stem cells (mEpiSCs) to the naive pluripotent state. The addition of AMPK activators alone or together with leukemia inhibitory factor to primed mEpiSCs induced the appearance of naive-like cells. After passaging in naive culture conditions, the colony morphology, protein expression, and global gene expression profiles indicated the naive state, as did germline transmission ability. Loss-of-function and gain-of-function studies suggested that p38 is a critical downstream target in AMPK activation. Finally, single-cell RNA sequencing analysis revealed that the reversion process through AMPK signaling passes an intermediate naive-like population. In conclusion, the AMPK pathway is a critical driving force in the reversion of primed to naive pluripotency.

Epithelial expression of Gata4 and Sox2 regulates specification of the squamous-columnar junction via MAPK/ERK signaling in mice.

The squamous-columnar junction (SCJ) is a boundary consisting of precisely positioned transitional epithelium between the squamous and columnar epithelium. Transitional epithelium is a hotspot for precancerous lesions, and is therefore clinically important; however, the origins and physiological properties of transitional epithelium have not been fully elucidated. Here, by using mouse genetics, lineage tracing, and organoid culture, we examine the development of the SCJ in the mouse stomach, and thus define the unique features of transitional epithelium. We find that two transcription factors, encoded by Sox2 and Gata4, specify primitive transitional epithelium into squamous and columnar epithelium. The proximal-distal segregation of Sox2 and Gata4 expression establishes the boundary of the unspecified transitional epithelium between committed squamous and columnar epithelium. Mechanistically, Gata4-mediated expression of the morphogen Fgf10 in the distal stomach and Sox2-mediated Fgfr2 expression in the proximal stomach induce the intermediate regional activation of MAPK/ERK, which prevents the differentiation of transitional epithelial cells within the SCJ boundary. Our results have implications for tissue regeneration and tumorigenesis, which are related to the SCJ.

Blood Flow Visualization and Wall Shear Stress Measurement of Carotid Arteries Using Vascular Vector Flow Mapping.

Carotid artery ultrasound is extensively used to assess early- and late-stage atherosclerosis via the intima-media thickness and increased blood flow velocity caused by stenosis, respectively. However, the effect of wall shear stress (WSS) has not been considered to date. This study aimed to visualize the blood flow of carotid arteries and measured WSS using vector flow mapping (VFM) developed specifically for vascular use. Patients with cerebrovascular diseases were prospectively enrolled and examined with carotid ultrasound using VFM Vascular. WSS was calculated in the common carotid artery and internal carotid artery. Blood flow in 82 common carotid arteries was visualized with VFM Vascular. The maximum and mean WSSs were negatively correlated with age and intima-media thickness. The WSS in 16 internal carotid artery plaques was significantly higher upstream of the plaque than downstream. Therefore, VFM Vascular is a promising method that provides a novel indicator of atherosclerosis.

Identification of distinct loci for de novo DNA methylation by DNMT3A and DNMT3B during mammalian development.

De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.

Identification of HUHS190, a human naftopidil metabolite, as a novel anti-bladder cancer drug.

We carried out structure-activity relationship study on anti-cancer effects of naftopidil (1) and its metabolites, resulted in identification of 1-(4-hydroxy-2-methoxyphenyl)piperazin-1-yl)-3-(naphthalen-1-yloxy) propan-2-ol (2, HUHS190), a major human metabolite of 1, which exhibited the most selective toxicities between against normal and cancer cells (Table 1). 2 was more hydrophilic compared to 1, was enough to be prepared in high concentration solution of more than 100 µM in saline for an intravesical instillation drug. Moreover, serum concentration of 2 was comparable to that of 1, an oral preparation drug, after oral administration at 32 mg/kg (Fig. 3). Both of 1 and 2 showed broad-spectrum anti-cancer activities in vitro, for example, 1 and 2 showed inhibitory activity IC50 = 21.1 µM and 17.2 µM for DU145, human prostate cancer cells, respectively, and IC50 = 18.5 µM and 10.5 µM for T24 cells, human bladder cancer cells. In this study, we estimated anticancer effects of 2 in a bladder cancer model after intravesical administration similar to clinical cases. A single intravesical administration of 2 exhibited the most potent inhibitory activities among the clinical drugs for bladder cancers, BCG and Pirarubicin, without obvious side effects and toxicity (Fig. 4). Thus, HUHS190 (2) can be effective for patients after post-TURBT therapy of bladder cancer without side effects, unlike the currently available clinical drugs.

Smarcb1 maintains the cellular identity and the chromatin landscapes of mouse embryonic stem cells.

ES cell (ESC) identity is stably maintained through the coordinated regulation of transcription factors and chromatin structure. SMARCB1, also known as INI1, SNF5, BAF47, is one of the subunits of SWI/SNF (BAF) complexes that play a crucial role in regulating gene expression by controlling chromatin dynamics. Genetic ablation of Smarcb1 in mice leads to embryonic lethality at the peri-implantation stage, indicating that Smarcb1 is important for the early developmental stages. However, the role of SMARCB1 in the maintenance of the ESC identity remains unknown. Here we established mouse ESCs lacking Smarcb1 and investigated the effect of Smarcb1 ablation on the differentiation propensity of ESCs. We found an increased expression of trophectoderm-related genes including Cdx2 in Smarcb1-deficient ESCs. Consistently, they exhibited an extended differentiation propensity into the trophectoderm lineage cells in teratomas. However, although Smarcb1-deficient cells were infrequently incorporated into the trophectoderm cell layer of blastocysts, they failed to contribute to mature placental tissues in vivo. Furthermore, Smarcb1-deficient cells exhibited a premature differentiation in the neural tissue of E14.5 chimeric embryos. Notably, we found that binding motifs for CTCF, which is involved in the maintenance of genomic DNA architecture was significantly enriched in chromatin regions whose accessibility was augmented in Smarcb1-deficient cells, while those for pluripotency factors were overrepresented in regions which have more closed structure in those cells. Collectively, we propose that SMARCB1-mediated remodeling of chromatin landscapes is important for the maintenance and differentiation of ESCs.

De Novo DNA Methylation at Imprinted Loci during Reprogramming into Naive and Primed Pluripotency.

CpG islands (CGIs) including those at imprinting control regions (ICRs) are protected from de novo methylation in somatic cells. However, many cancers often exhibit CGI hypermethylation, implying that the machinery is impaired in cancer cells. Here, we conducted a comprehensive analysis of CGI methylation during somatic cell reprogramming. Although most CGIs remain hypomethylated, a small subset of CGIs, particularly at several ICRs, was often de novo methylated in reprogrammed pluripotent stem cells (PSCs). Such de novo ICR methylation was linked with the silencing of reprogramming factors, which occurs at a late stage of reprogramming. The ICR-preferred CGI hypermethylation was similarly observed in human PSCs. Mechanistically, ablation of Dnmt3a prevented PSCs from de novo ICR methylation. Notably, the ICR-preferred CGI hypermethylation was observed in pediatric cancers, while adult cancers exhibit genome-wide CGI hypermethylation. These results may have important implications in the pathogenesis of pediatric cancers and the application of PSCs.

In vivo reprogramming drives Kras-induced cancer development.

The faithful shutdown of the somatic program occurs in the early stage of reprogramming. Here, we examined the effect of in vivo reprogramming on Kras-induced cancer development. We show that the transient expression of reprogramming factors (1-3 days) in pancreatic acinar cells results in the transient repression of acinar cell enhancers, which are similarly observed in pancreatitis. We next demonstrate that Kras and p53 mutations are insufficient to induce ERK signaling in the pancreas. Notably, the transient expression of reprogramming factors in Kras mutant mice is sufficient to induce the robust and persistent activation of ERK signaling in acinar cells and rapid formation of pancreatic ductal adenocarcinoma. In contrast, the forced expression of acinar cell-related transcription factors inhibits the pancreatitis-induced activation of ERK signaling and development of precancerous lesions in Kras-mutated acinar cells. These results underscore a crucial role of dedifferentiation-associated epigenetic regulations in the initiation of pancreatic cancers.

Novel Metabolically Stable PCA-1/ALKBH3 Inhibitor Has Potent Antiproliferative Effects on DU145 Cells In Vivo.

Novel potent prostate cancer antigen-1 (PCA-1)/alpha-ketoglutarate-dependent dioxygenase alkB homolog 3 (ALKBH3) inhibitors both in vivo and in vivo were designed and evaluated by a stability assay in an S9 mixture, a mixture of rat liver homogenate and co-factors, and oral absorbability assay in rat, as well as enzyme and cell assays, and resulted in the synthesis of a novel potent PCA-1/ALKBH3 inhibitor in vivo. Among them, compound 7l exhibited potent inhibitory activities in a xenograft model bearing DU145 tumor at 10 mg/kg by subcutaneous administration without negative side-effects. This inhibitory activity in vivo was more potent than that of HUHS015 at 32 mg/kg, a known PCA-1/ALKBH3 inhibitor, or docetaxel at 2.5 mg/kg, the drug clinically used for androgen-independent prostate cancer.

Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation.

Inhibitors of Mek1/2 and Gsk3ß, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.

Synthesis of resveratrol derivatives as new analgesic drugs through desensitization of the TRPA1 receptor.

A series of 31 resveratrol derivatives was designed, synthesized and evaluated for activation and inhibition of the TRPA1 channel. Most acted as activators and desensitizers of TRPA1 channels like resveratrol or allyl isothiocyanate (AITC). Compound 4z (HUHS029) exhibited higher inhibitory activity than resveratrol with an IC50 value of 16.1µM. The activity of 4z on TRPA1 was confirmed in TRPA1-expressing HEK293 cells, as well as in rat dorsal root ganglia neurons by a whole cell patch clamp recording. Furthermore, pretreatment with 4z exhibited an analgesic effect on AITC-evoked TRPA1-related pain behavior in vivo.

The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72 Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.

Hybrid Cellular Metabolism Coordinated by Zic3 and Esrrb Synergistically Enhances Induction of Naive Pluripotency.

Naive pluripotent stem cells (PSCs) utilize both glycolysis and oxidative phosphorylation (OXPHOS) to satisfy their metabolic demands. However, it is unclear how somatic cells acquire this hybrid energy metabolism during reprogramming toward naive pluripotency. Here, we show that when transduced with Oct4, Sox2, and Klf4 (OSK) into murine fibroblasts, Zic3 and Esrrb synergistically enhance the reprogramming efficiency by regulating cellular metabolic pathways. These two transcription factors (TFs) cooperatively activate glycolytic metabolism independently of hypoxia inducible factors (HIFs). In contrast, the regulatory modes of the TFs on OXPHOS are antagonistic: Zic3 represses OXPHOS, whereas Esrrb activates it. Therefore, when introduced with Zic3, Esrrb restores OXPHOS activity, which is essential for efficient reprogramming. In addition, Esrrb-mediated OXPHOS activation is critical for the conversion of primed PSCs into the naive state. Our study suggests that the combinatorial function of TFs achieves an appropriate balance of metabolic pathways to induce naive PSCs.