Search

Abstract RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain have been reported in many DCM patients, but the pathogenicity of variants within the RNA-recognition motif remains unknown. Two human patients with the I536T-RBM20 variant without an apparent DCM phenotype were identified in sudden death cohorts. A splicing reporter assay was performed, and an I538T knock-in mouse model (Rbm20I538T) was generated to determine the significance of this variant. The reporter assay demonstrated that the human I536T variant affected the TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice showed different splicing patterns in Ttn, Ldb3, Camk2d, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were upregulated in Rbm20I538T mice, but Rbm20I538T mice showed neither DCM nor cardiac dysfunction on histopathological examination and ultrasound echocardiography. The I536T-RBM20 (I538T-Rbm20) variant changes gene splicing and affects gene expression, but the splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d, and Ryr2 do not cause DCM morphology in the mouse model. Key messages • Two human patients with the I536T-RBM20 variant without a DCM phenotype were identified. • A splicing reporter assay demonstrated that the variant affected the TTN splicing. • Rbm20I538T mice showed neither DCM nor cardiac dysfunction. • Rbm20I538T mice showed different splicing patterns and the gene expressions.

CKLF‐like MARVEL transmembrane domain–containing protein 6 (CMTM6) maintains membrane PD‐L1 expression by controlling its endosomal recycling. However, in patients with hepatocellular carcinoma (HCC), the correlation among CMTM6, B7 family ligands, and CD8‐positive cytotoxic T lymphocytes (CTLs), and the molecular function of CMTM6 in HCC have not been established. We performed immunohistochemistry to evaluate the relationships among CMTM6 expression, clinicopathological factors, B7 family ligands expression, and CTL infiltration in HCC samples. Moreover, we established CMTM6‐knockout human HCC cell lines to evaluate the function of human CMTM6 in immune regulation and tumor viability. CMTM6 expression was positively associated with membrane B7 family ligands expression and CTL infiltration in HCC samples. High CMTM6 expression in HCC tissues was associated with the expression of the proliferation marker Ki‐67 and shorter recurrence‐free survival. In vitro analysis showed the downregulation of membrane B7 family ligands and proliferation potency in the CMTM6‐knockout human HCC cell line. High membrane CMTM6 expression was associated with tumor recurrence and proliferation via the regulation of membranous B7 family ligands expression. Thus, CMTM6 might be a biomarker to predict the risk of HCC recurrence and a therapeutic target to suppress tumor growth and increase CTL activity., CMTM6 expression was positively associated with B7 family ligand expression and cytotoxic T lymphocyte (CTL) infiltration in hepatocellular carcinoma (HCC) samples. The CMTM6‐knockout human HCC cell line showed the downregulation of membrane B7 family ligands expression and proliferation potency. High membrane CMTM6 expression was associated with tumor recurrence and proliferation via the regulation of membranous B7 family expression.

While a KCND3 V392I mutation uniquely displays a mixed electrophysiological phenotype of Kv4.3, only limited clinical information on the mutation carriers is available. We report two teenage siblings exhibiting both cardiac (early repolarization syndrome and paroxysmal atrial fibrillation) and cerebral phenotypes (epilepsy and intellectual disability), in whom we identified the KCND3 V392I mutation. We propose a link between the KCND3 mutation with a mixed electrophysiological phenotype and cardiocerebral phenotypes, which may be defined as a novel cardiocerebral channelopathy.

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. ABO transcription is known to be regulated by a constitutive promoter in a CpG island and regions for regulation of cell-specific expression such as the downstream + 22.6-kb site for epithelial cells and a site in intron 1 for erythroid cells. Here we investigated whether the + 22.6-kb site might play a role in transcriptional regulation of the gene encoding odorant binding protein 2B (OBP2B), which is located on the centromere side 43.4 kb from the + 22.6-kb site. In the gastric cancer cell line KATOIII, quantitative PCR analysis demonstrated significantly reduced amounts of OBP2B and ABO transcripts in mutant cells with biallelic deletions of the site created using the CRISPR/Cas9 system, relative to those in the wild-type cells, and Western blotting demonstrated a corresponding reduction of OBP2B protein in the mutant cells. Moreover, single-molecule fluorescence in situ hybridization assays indicated that the amounts of both transcripts were correlated in individual cells. These findings suggest that OBP2B could be co-regulated by the + 22.6-kb site of ABO.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide as a pandemic throughout 2020. Since the virus uses angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry, increment of ACE2 would lead to an increased risk of SARS-CoV-2 infection. At the same time, an association of the ABO blood group system with COVID-19 has also been highlighted: there is increasing evidence to suggest that non-O individuals are at higher risk of severe COVID-19 than O individuals. These findings imply that simultaneous suppression of ACE2 and ABO would be a promising approach for prevention or treatment of COVID-19. Notably, we have previously clarified that histone deacetylase inhibitors (HDACIs) are able to suppress ABO expression in vitro. Against this background, we further evaluated the effect of HDACIs on cultured epithelial cell lines, and found that HDACIs suppress both ACE2 and ABO expression simultaneously. Furthermore, the amount of ACE2 protein was shown to be decreased by one of the clinically-used HDACIs, panobinostat, which has been reported to reduce B-antigens on cell surfaces. On the basis of these findings, we conclude that panobinostat could have the potential to serve as a preventive drug against COVID-19.

While a KCND3 V392I mutation uniquely displays a mixed electrophysiological phenotype of Kv4.3, only limited clinical information on the mutation carriers is available. We report two teenage siblings exhibiting both cardiac (early repolarization syndrome and paroxysmal atrial fibrillation) and cerebral phenotypes (epilepsy and intellectual disability), in whom we identified the KCND3 V392I mutation. We propose a link between the KCND3 mutation with a mixed electrophysiological phenotype and cardiocerebral phenotypes, which may be defined as a novel cardiocerebral channelopathy.

Immunotherapy targeting immune checkpoint molecules has provided remarkable clinical benefits in cancer patients but no clinically relevant biomarker for predicting treatment outcomes exists. Recently, we demonstrated that glycan structures of serum α1-acid glycoprotein (AGP) changed dramatically in cancer patients and that α1,3fucosylated AGP (fAGP) levels increased along with disease progression and decreased responding to chemotherapy treatments. Here, the fAGP was analyzed in sera prospectively obtained from 39 patients with advanced lung cancer who underwent immunotherapy with anti-PD-1 antibody, nivolumab. Twenty-three patients had significantly high fAGP levels above the cut-off value (H-fAGP) at one month after starting the treatment and 20 patients in this group, whose tumor sizes did not decrease, maintained high fAGP levels continuously and subsequently died. However, the other 16 patients, whose fAGP levels decreased or maintained below the cut-off value (L-fAGP), survived during a 2-year observation even though 5 patients in this group had no tumor shrinkage. Accordingly, the overall survival rate was found to significantly correlate with the fAGP level. Multivariate analyses revealed that the H-fAGP was an independent risk factor for cancer progression. Therefore, the fAGP level appeared to be a reliable biomarker for predicting clinical efficacy of immunotherapy with nivolumab.

Immunotherapy targeting immune checkpoint molecules has provided remarkable clinical benefits in cancer patients but no clinically relevant biomarker for predicting treatment outcomes exists. Recently, we demonstrated that glycan structures of serum α1-acid glycoprotein (AGP) changed dramatically in cancer patients and that α1,3fucosylated AGP (fAGP) levels increased along with disease progression and decreased responding to chemotherapy treatments. Here, the fAGP was analyzed in sera prospectively obtained from 39 patients with advanced lung cancer who underwent immunotherapy with anti-PD-1 antibody, nivolumab. Twenty-three patients had significantly high fAGP levels above the cut-off value (H-fAGP) at one month after starting the treatment and 20 patients in this group, whose tumor sizes did not decrease, maintained high fAGP levels continuously and subsequently died. However, the other 16 patients, whose fAGP levels decreased or maintained below the cut-off value (L-fAGP), survived during a 2-year observation even though 5 patients in this group had no tumor shrinkage. Accordingly, the overall survival rate was found to significantly correlate with the fAGP level. Multivariate analyses revealed that the H-fAGP was an independent risk factor for cancer progression. Therefore, the fAGP level appeared to be a reliable biomarker for predicting clinical efficacy of immunotherapy with nivolumab.

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. The ABO system is composed of complex carbohydrate structures that are biosynthesized by A- and B-transferases encoded by the ABO gene. However, the mechanisms regulating ABO gene expression in epithelial cells remain obscure. On the basis of DNase I-hypersensitive sites in and around ABO in epithelial cells, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays and histone modifications indicated a novel positive regulatory element, designated the +22.6-kb site, downstream from ABO, and this was shown to enhance ABO promoter activity in an epithelial cell-specific manner. Expression of ABO and B-antigen was reduced in gastric cancer KATOIII cells by biallelic deletion of the +22.6-kb site using the CRISPR/Cas9 system. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the site bound to an epithelial cell-specific transcription factor, Elf5. Mutation of the Ets binding motifs to abrogate binding of this factor reduced the regulatory activity of the +22.6-kb site. Furthermore, ELF5 knockdown with shRNA reduced both endogenous transcription from ABO and B-antigen expression in KATOIII cells. Thus, Elf5 appeared to be involved in the enhancer potential of the +22.6-kb site. These results support the contention that ABO expression is dependent upon a downstream positive regulatory element functioning through a tissue-restricted transcription factor, Elf5, in epithelial cells.

Two series of antifouling microfiltration membranes (MF) with different pure water permeabilities were fabricated using poly(vinyl chloride-co-poly(ethylene glycol)methyl ether methacrylate) (poly(VC-co-PEGMA)) copolymers with different PEGMA segment percentage via nonsolvent-induced phase separation (NIPS) method. Membranes with similar water permeability were obtained for each series by changing the dope solution composition, because the water permeability can affect the membrane fouling property. Bovine serum albumin (BSA) adsorption on copolymer films decreased and membrane surface pore size, hydrophilicity, and antifouling properties increased by increasing the PEGMA segment percentage. BSA filtration results revealed that the hydrophilicity of the membrane and the initial water flux more strongly affected the fouling propensity than the membrane surface pore size. Molecular dynamics simulation was carried out to investigate the state of the PEGMA on the membrane surface for clarifying the fouling mechanism.

Phase separation is one of the major methods to prepare a hollow-fiber membrane in industry. Despite the strong demands for the control of the inner membrane structure and surface geometry, it is still difficult to obtain the desired membrane structure and surface geometry simply by phase separation. In this work, we employed thermally induced phase separation, dip-coating and vapor-induced phase separation to prepare a hollow-fiber membrane with the controlled surface geometry and homogeneous inner structure, which led to high water permeability and high mechanical strength. First a poly(vinylidene fluoride) (PVDF) hollow-fiber membrane was prepared via thermally induced phase separation (TIPS) and then the outer surface of a membrane was dip-coated with another PVDF solution, followed by vapor induced phase separation (VIPS) to reorganize the outer surface of the membrane. The dip-coating and the VIPS treatment produced a highly porous mesh-like layer on the membrane surface. Filtration experiments using nanospheres and protein solutions revealed that the newly-formed layer served as a “separation layer” to determine the separation properties. The membrane pore-size was controlled to some extent by the conditions for the dip-coating and the VIPS treatment. The surface-reorganized membrane kept its intrinsic high water permeability and mechanical strength. In addition, the membrane exhibited improved low-fouling properties in the filtration of humic acid and protein solutions.

The objectives of this study were to evaluate all the non-synonymous single nucleotide polymorphisms (SNPs) in the DNase I and DNase I-like 3 (1L3) genes potentially implicated in autoimmune diseases as a functional SNP in terms of alteration of the activity levels. We examined the genotype distributions of the 32 and 20 non-synonymous SNPs in DNASE1 and DNASE1L3, respectively, in three ethnic groups, and the effect of these SNPs on the DNase activities. Among a total of 44 and 25 SNPs including those characterized in our previous studies [Yasuda et al., Int J Biochem Cell Biol42 (2010) 1216-1225; Ueki et al. Electrophoresis32 (2012) 1465-1472], only four and one, respectively, exhibited genetic heterozygosity in one or all of the ethnic groups examined. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, 11 activity-abolishing and 11 activity-reducing SNPs in DNASE1 and two activity-abolishing and five activity-reducing SNPs in DNASE1L3 were confirmed as a functional SNP. Phylogenetic analysis showed that all of the amino acid residues in activity-abolishing SNPs were completely or well conserved in animal DNase I and 1L3 proteins. Although almost all non-synonymous SNPs in both genes that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of 13 activity-abolishing SNPs producing a loss-of-function variant in both the DNase genes would be a direct genetic risk factor for autoimmune diseases. These findings may have clinical implications in relation to the prevalence of autoimmune diseases.

One standard treatment option for advanced-stage cancer is surgical resection of malignant tumors following by adjuvant chemotherapy and chemoradiotherapy. Additionally, neoadjuvant chemotherapy may be applied if required. During the time course of treatments, patients are generally followed by computed tomography (CT) surveillance, and by tumor marker diagnosis. However, currently, early evidence of recurrence and/or metastasis of tumors with a clinically relevant biomarker remains a major therapeutic challenge. In particular, there has been no validated biomarker for predicting treatment outcomes in therapeutic settings. Recently, we have looked at glycoforms of serum α1-acid glycoprotein (AGP) by using a crossed affinoimmunoelectrophoresis with two lectins and an anti-AGP antibody. The primary glycan structures of AGP were also analyzed by a mass spectrometer and a novel software in a large number of patients with various cancers. Accordingly, the relative abundance of α1,3fucosylated glycans in AGP (FUCAGP) was found to be significantly high in cancer patients as compared with the healthy controls. Further, strikingly elevated levels of FUCAGP were found in patients with poor prognosis but not in patients with good prognosis. In the current study, levels of FUCAGP in serum samples from various cancer patients were analyzed and 17 patients including 13 who had undergone chemotherapy were followed for several years post operation. FUCAGP level determined diligently by using a mass spectrometer was found to change along with disease prognosis as well as with responses to treatments, in particular, to various chemotherapies. Therefore, FUCAGP levels measured during following-up of the patients after operation appeared to be clinically relevant biomarker of treatment intervention.

The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I–hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell–specific manner. Electrophoretic mobility–shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with Bm phenotypes. Therefore, it is plausible that deletion of the erythroid cell–specific regulatory element could down-regulate transcription in the Bm allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells.

A 40-year-old mentally retarded Japanese man was admitted at rehabilitation facility for handicapped persons and found dead in his bed. His neonatal period was complicated by seizures, and he had a medical history of schizophrenia. A postmortem computed tomography scan suggested an intestinal obstruction, but the cause was unknown. To clarify the cause of death, a medicolegal autopsy was carried out. The gastrointestinal tract was found to contain copious amounts of cloth pieces. A diagnosis of intestinal obstruction secondary to pica of clothes was made. Despite still being an essentially neglect condition; mental retardation is cause to significant burden to the patient, his relatives and caregivers and the whole society. Moreover, people with mental retardation may be at increased risk for potentially self-injury due to ingestion of non-eating substance or incongruent intake of eating substances, which may on turn lead to severe or even life-threatening medical and surgical complications as herein reported. Specific attention also to pica in mentally-retarded patients with sudden, severe, gastrointestinal events, should therefore be placed in order to prevent potential death or otherwise severe chronic consequences, ideally aiming at enhancing the early recognition and multi-disciplinary management of those psychological stressors or triggers potentially responsible for pica too.

Case history A 3-month-old infant was found dead in his bed. A postmortem computed tomography (CT) scan suggested fatty attenuation in the liver parenchyma, but no other potentially fatal changes were found. To clarify the cause of death, a medicolegal autopsy was carried out. Autopsy findings Internal examination confirmed the presence of liver steatosis as well as hepatomegaly. There were no other significant findings including encephalitis or brain edema. Mass spectrometry analysis To clarify the mechanism underlying lipid accumulation in the liver, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI–IMS) analysis was conducted. This indicated a significant accumulation of C14:1 acylcarnitine in the liver of the deceased, suggesting very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. Genetic analysis To find the cause of the VLCAD deficiency, genetic analysis of the responsible gene, acyl-CoA dehydrogenase , very long chain ( ACADVL ), was performed. This revealed two novel mutations that may have accounted for the disease. Conclusion A combination of these data revealed that the liver steatosis in this case might have been caused by VLCAD deficiency based on genetic mutations of ACADVL . Thus, the deceased might have been vulnerable to energy crisis and sudden infant death. The present findings show that MALDI–IMS analysis as well as genetic analysis can be useful for elucidating the cause of death.