Bi-allelic variants in the ESAM tight-junction gene cause a neurodevelopmental disorder associated with fetal intracranial hemorrhage.

The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies."

The CAR group of Ig cell adhesion proteins-Regulators of gap junctions?

Members of the CAR group of Ig-like type I transmembrane proteins mediate homotypic cell adhesion, share a common overall extracellular domain structure and are closely related at the amino acid sequence level. CAR proteins are often found at tight junctions and interact with intracellular scaffolding proteins, suggesting that they might modulate tight junction assembly or function. However, impairment of tight junction integrity has not been reported in mouse knockout models or zebrafish mutants of CAR members. In contrast, in the same knockout models deficits in gap junction communication were detected in several organ systems, including the atrioventricular node of the heart, smooth muscle cells of the intestine and the ureter and in Sertoli cells of the testes. Possible interactions between BT-IgSF and connexin41.8 on the disturbed pattern of pigment stripes found in zebrafish mutants and between ESAM and connexin43 during hematopoiesis in the mouse are also discussed. On the basis of the combined data and phenotypic similarities between CAR member mutants and connexin mutants I hypothesize that they primarily play a role in the organization of gap junction communication. Also see the video abstract here: https://youtu.be/i0yq2KhuDAE.

3D Visualization of Human Blood Vascular Networks Using Single-Domain Antibodies Directed against Endothelial Cell-Selective Adhesion Molecule (ESAM).

High-quality three-dimensional (3D) microscopy allows detailed, unrestricted and non-destructive imaging of entire volumetric tissue specimens and can therefore increase the diagnostic accuracy of histopathological tissue analysis. However, commonly used IgG antibodies are oftentimes not applicable to 3D imaging, due to their relatively large size and consequently inadequate tissue penetration and penetration speed. The lack of suitable reagents for 3D histopathology can be overcome by an emerging class of single-domain antibodies, referred to as nanobodies (Nbs), which can facilitate rapid and superior 2D and 3D histological stainings. Here, we report the generation and experimental validation of Nbs directed against the human endothelial cell-selective adhesion molecule (hESAM), which enables spatial visualization of blood vascular networks in whole-mount 3D imaging. After analysis of Nb binding properties and quality, selected Nb clones were validated in 2D and 3D imaging approaches, demonstrating comparable staining qualities to commercially available hESAM antibodies in 2D, as well as rapid and complete staining of entire specimens in 3D. We propose that the presented hESAM-Nbs can serve as novel blood vessel markers in academic research and can potentially improve 3D histopathological diagnostics of entire human tissue specimens, leading to improved treatment and superior patient outcomes.

Sox17-mediated expression of adherent molecules is required for the maintenance of undifferentiated hematopoietic cluster formation in midgestation mouse embryos.

Hematopoietic stem cell-containing intra-aortic hematopoietic cell clusters (IAHCs) emerge in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation mouse embryos. We previously showed that transduction of Sox17 in CD45lowc-Kithigh cells, which are one component of IAHCs, maintained the cluster formation and the undifferentiated state, but the mechanism of the cluster formation by Sox17 has not been clarified. By microarray gene expression analysis, we found that genes for vascular endothelial-cadherin (VE-cad) and endothelial cell-selective adhesion molecule (ESAM) were expressed at high levels in Sox17-transduced c-Kit+ cells. Here we show the functional role of these adhesion molecules in the formation of IAHCs and the maintenance of the undifferentiated state by in vitro experiments. We detected VE-cad and ESAM expression in endothelial cells of dorsal aorta and IAHCs in E10.5 embryos by whole mount immunohistochemistry. Cells with the middle expression level of VE-cad and the low expression level of ESAM had the highest colony-forming ability. Tamoxifen-dependent nuclear translocation of Sox17-ERT fusion protein induced the formation of cell clusters and the expression of Cdh5 (VE-cad) and ESAM genes. We showed the induction of the Cdh5 (VE-cad) and ESAM expression and the direct interaction of Sox17 with their promoter by luciferase assay and chromatin immunoprecipitation assay, respectively. Moreover, shRNA-mediated knockdown of either Cdh5 (VE-cad) or ESAM gene in Sox17-transduced cells decreased the multilineage-colony forming potential. These findings suggest that VE-cad and ESAM play an important role in the high hematopoietic activity of IAHCs and cluster formation.

Distinct and redundant functions of Esama and VE-cadherin during vascular morphogenesis.

The cardiovascular system forms during early embryogenesis and adapts to embryonic growth by sprouting angiogenesis and vascular remodeling. These processes require fine-tuning of cell-cell adhesion to maintain and re-establish endothelial contacts, while allowing cell motility. We have compared the contribution of two endothelial cell-specific adhesion proteins, VE-cadherin (VE-cad/Cdh5) and Esama (endothelial cell-selective adhesion molecule a), during angiogenic sprouting and blood vessel fusion (anastomosis) in the zebrafish embryo by genetic analyses. Different combinations of mutant alleles can be placed into a phenotypic series with increasing defects in filopodial contact formation. Contact formation in esama mutants appears similar to wild type, whereas esama-/-; ve-cad+/- and ve-cad single mutants exhibit intermediate phenotypes. The lack of both proteins interrupts filopodial interaction completely. Furthermore, double mutants do not form a stable endothelial monolayer, and display intrajunctional gaps, dislocalization of Zo-1 and defects in apical-basal polarization. In summary, VE-cadherin and Esama have distinct and redundant functions during blood vessel morphogenesis, and both adhesion proteins are central to endothelial cell recognition during anastomosis.

Regulation of Type III Secretion of Translocon and Effector Proteins by the EsaB/EsaL/EsaM Complex in Edwardsiella tarda.

The type III secretion system (T3SS) plays a crucial role in the pathogenesis of many Gram-negative bacteria, including Edwardsiella tarda, an important fish pathogen. Within the E. tarda T3SS, there are three proteins (EsaB/EsaL/EsaM) that are homologous to proteins present in many other bacteria, including SpiC/SsaL/SsaM in Salmonella, SepD/SepL/CesL in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and YscB/YopN/SycN in Yersinia EsaL was found to interact with both EsaB and EsaM within the bacterial cell, as revealed by a coimmunoprecipitation assay. Moreover, EsaM is required for EsaB stability, and the two proteins interact with each other. EsaB, EsaL, and EsaM are all indispensable for the secretion of the T3SS translocon protein EseC into supernatants under pH 5.5 and pH 7.2 conditions. Unlike EseC, EseG is a T3SS effector whose secretion is suppressed by EsaL at pH 7.2 while it is promoted at pH 5.5 condition. Despite this finding, mutant strains lacking EsaB, EsaL, or EsaM (i.e., the ΔesaB, ΔesaL, or ΔesaM strain, respectively) were all outcompeted by wild-type E. tarda during a coinfection model. These results demonstrate that EsaB/EsaL/EsaM form a ternary complex controlling the secretion of T3SS translocon and effector proteins and contributing to E. tarda pathogenesis.

Identification of the Porcine Vascular Endothelial Cell-Specific Promoter ESAM1.0 Using Transcriptome Analysis.

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection.

Extraretinal Fibrovascular Proliferation in a Neonate Possibly Associated with an ESAM Gene Variant

A female infant born with a gestational age of 35 weeks and birth weight of 2500 g was referred for ophthalmic examination on the second postnatal day. Bilateral venous dilatation and arterial tortuosity, severe extraretinal fibrovascular proliferation, and peripheral ischemia were detected. Fluorescein angiography showed profoundly delayed arteriovenous transit and peripheral avascularity. Both eyes were treated with diode laser photocoagulation and bevacizumab injection. Cranial magnetic resonance imaging (MRI) revealed hydrocephalus, ventricular dilatation, and cerebral atrophy. Her family history revealed that the patient's brother presented to the ophthalmology outpatient clinic at postnatal 3 months with inoperable total retinal detachment and similar cranial MRI findings. No systemic or ocular findings were detected in the parents. A recent study showed that in 13 cases, including our patients, bi-allelic variants in the ESAM gene lead to a new neurodevelopmental disease whose main clinical features include impaired speech and language development, seizures, varying degrees of spasticity, ventriculomegaly, intracranial hemorrhage, and developmental delay/mental disability. Newborn siblings of children with serious pathological retinal findings should undergo a detailed ophthalmic examination as soon as possible after birth to prevent total retinal detachment, even without a diagnosis of specific inherited retinal vascular diseases. Further investigations performed in collaboration with an international network may reveal more candidate gene variants possibly related to retinopathy of prematurity-like ophthalmological findings such as extraretinal fibrovascular proliferation.

miR-7 Reduces Breast Cancer Stem Cell Metastasis via Inhibiting RELA to Decrease ESAM Expression.

This study aimed to present evidence that miR-7 inhibited the metastasis of breast cancer stem cells (BCSCs) and elucidated the mechanisms that have remained unknown. The samples collected from miR-7 agomir-treated, BCSC-driven tumors were subjected to a protein array to analyze the protein expression profiles. A dual-luciferase reporter and chromatin immunoprecipitation-PCR were used to validate and evaluate the molecular expressions of interest in the collected breast cancer tissues and cell lines. miR-7 overexpression affecting metastasis of BCSCs was further evaluated in mice. The endothelial cell-selective adhesion molecule (ESAM) was highly expressed in breast cancer tissues and in BCSC-driven xenografts. Results of the dual-luciferase reporter and chromatin immunoprecipitation-PCR indicated that the miR-7 mimic reduced RELA expression by directly targeting the 3' UTR of RELA to inhibit ESAM expression in MDA-MB-231 cells. Moreover, the expression levels of RELA, CD44, and ESAM were significantly decreased in lentivirus (Lenti)-miR-7-BCSC-driven xenografts compared with the control xenografts, accompanied with an increase in E-cadherin and a decrease in vimentin expression, as well as reduction in tumor growth and metastasis to lungs. Our data demonstrated that miR-7 overexpression reduced the metastasis of BCSCs via inhibiting ESAM, suggesting that ESAM could be a potential target for breast cancer therapy.

The Tradeoff Analysis for Remote Sensing Image Fusion Using Expanded Spectral Angle Mapper.

Image fusion is a useful tool in integrating a high-resolution panchromaticimage (HRPI) with a low-resolution multispectral image (LRMI) to produce a highresolutionmultispectral image (HRMI). To date, many image fusion techniques have beendeveloped to try to improve the spatial resolution of the LRMI to that of the HRPI with itsspectral property reliably preserved. However, many studies have indicated that thereexists a trade- off between the spatial resolution improvement and the spectral propertypreservation of the LRMI, and it is difficult for the existing methods to do the best in bothaspects. Based on one minimization problem, this paper mathematically analyzes thetradeoff in fusing remote sensing images. In experiment, four fusion methods are evaluatedthrough expanded spectral angle mapper (ESAM). Results clearly prove that all the testedmethods have this property.

Adiponectin is related to markers of endothelial dysfunction and neoangiogenesis in diabetic patients.

PURPOSE: Adiponectin an adipokine, produced by mature adipocyte, has an important effect on several aspects of endothelial function, including leukocyte adhesion (mediated by adhesion molecules like intercellular adhesion molecule 1 (ICAM1) endothelial cell selective adhesion molecule ESAM). Recently, it has been linked to vascular endothelial growth factor (VEGF)-modulated angiogenesis. ESAM might also be involved in modulating VEGF-dependent actions. We studied relationship of adiponectin to ESAM, ICAM1, and VEGF in type 2 diabetic patients (T2DP) with or without microvascular complications. METHODS: Incident T2DP referred for nephrologic evaluation were included (patients with no nephropathy or stage 1-4 nephropathy). T2DP with stage 5 chronic kidney disease (CKD) were selected from a dialysis center. Clinical, standard laboratory assessment and adiponectin, ESAM, ICAM1, and VEGF (ELISA) were recorded. RESULTS: Eighty-seven patients were included, 15 had no CKD, 30 with stage 1 or 2 CKD, 20 with stage 3 or 4 CKD and 22 patients on dialysis. ESAM was higher in patients with CKD than in those without CKD (p = 0.02), adiponectin, ICAM1, and VEGF were similar. Adiponectin correlated in univariate analysis to ESAM (r = 0.32, p = 0.002), ICAM1 (r = 0.23, p = 0.038), and CRP (r = 0.31, p = 0.012), and inversely to serum albumin (r = - 0.57, < 0.0001). In predialysis patients, adiponectin also correlated to albuminuria (r = 0.54, p < 0.0001) and glomerular filtration rate (r = - 0.46, p = 0.0001). In multivariate regression ESAM (p = 0.04), VEGF (p = 0.03), and albumin (p < 0.0001) are significant predictors of adiponectin. None of these cytokines were different when comparing patients with and without retinopathy. CONCLUSION: Adiponectin is directly linked to adhesion molecules and VEGF in T2DP.

A verification strategy for web services composition using enhanced stacked automata model.

Currently, Service-Oriented Architecture (SOA) is becoming the most popular software architecture of contemporary enterprise applications, and one crucial technique of its implementation is web services. Individual service offered by some service providers may symbolize limited business functionality; however, by composing individual services from different service providers, a composite service describing the intact business process of an enterprise can be made. Many new standards have been defined to decipher web service composition problem namely Business Process Execution Language (BPEL). BPEL provides an initial work for forming an Extended Markup Language (XML) specification language for defining and implementing business practice workflows for web services. The problems with most realistic approaches to service composition are the verification of composed web services. It has to depend on formal verification method to ensure the correctness of composed services. A few research works has been carried out in the literature survey for verification of web services for deterministic system. Moreover the existing models did not address the verification properties like dead transition, deadlock, reachability and safetyness. In this paper, a new model to verify the composed web services using Enhanced Stacked Automata Model (ESAM) has been proposed. The correctness properties of the non-deterministic system have been evaluated based on the properties like dead transition, deadlock, safetyness, liveness and reachability. Initially web services are composed using Business Process Execution Language for Web Service (BPEL4WS) and it is converted into ESAM (combination of Muller Automata (MA) and Push Down Automata (PDA)) and it is transformed into Promela language, an input language for Simple ProMeLa Interpreter (SPIN) tool. The model is verified using SPIN tool and the results revealed better recital in terms of finding dead transition and deadlock in contrast to the existing models.

Emotional Effect of Excessive Negative Air Ions to Human Emotion Spectrum / 日本温泉気候物理医学会雑誌

It is believed that one of the principal effects of negative air ion is refreshing human psychological condition. Negative air ion can be observed in natural environments such as forest, fountain, and hot spring. In order to elucidate scientific reason of psychological effects by negative air ions, quantifying psychological effects will be important.<br>Healthy sixteen examinees (8 young candidates around 20 years, 8 old candidates around 60 years) are tested by SRS-18, blood pressure, questionnaire, and electroencephalogram. SRS-18, blood pressure, questionnaire was performed before and after experiment. Electroencephalogram was continuously measured during whole experiment and used for emotional spectrum analysis method (ESAM) calculation. Experiment was divided into three conditions (initial normal condition, negative air ion boosted condition at middle concentration, after ventilated condition) and three phases (accommodation, rest, calculation) at each condition.<br>At young group, excessive negative air ions increased human psychological concentration which is calculated by ESAM. At calculation phase, psychological concentration has a positive correlation with psychological stress (correlation factor 0.505). Otherwise at old aged group, no significant change was observed. It shows that sensitivity of negative air ions at old persons will be reduced. It also suggests that human sensitivity for negative air ion decreases with aging.<br>At rest phase of young group in negative air ion boosted condition, elementary emotions are balanced at ESAM. This shows that balancing among four elementary emotions is related with psychological effects of negative air ions.