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The clinical spectrum of Down syndrome (DS) ranges from congenital malformations to premature aging and early-onset senescence. Excessive immunoreactivity and oxidative stress are thought to accelerate the pace of aging in DS patients; however, the immunological profile remains elusive. We investigated whether peripheral blood monocyte-derived dendritic cells (MoDCs) in DS patients respond to lipopolysaccharide (LPS) distinctly from non-DS control MoDCs. Eighteen DS patients (age 2–47 years, 12 males) and 22 controls (age 4–40 years, 15 males) were enrolled. CD14-positive monocytes were immunopurified and cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor and IL-4, yielding MoDCs in vitro. After the LPS-stimulation for 48 hours from days 7 to 9, culture supernatant cytokines were measured by multiplex cytokine bead assays, and bulk-prepared RNA from the cells was used for transcriptomic analyses. MoDCs from DS patients produced cytokines/chemokines (IL-6, IL-8, TNF-α, MCP-1, and IP-10) at significantly higher levels than those from controls in response to LPS. RNA sequencing revealed that DS-derived MoDCs differentially expressed 137 genes (74 upregulated and 63 downregulated) compared with controls. A gene enrichment analysis identified 5 genes associated with Toll-like receptor signaling (KEGG: hsa04620, P = 0.00731) and oxidative phosphorylation (hsa00190, P = 0.0173) pathways. MoDCs obtained from DS patients showed higher cytokine or chemokine responses to LPS than did control MoDCs. Gene expression profiles suggest that hyperactive Toll-like receptor and mitochondrial oxidative phosphorylation pathways configure the immunoreactive signature of MoDCs in DS patients. Excessive immunoreactivity and oxidative stress are associated with aging in patients with Down syndrome; however, their immunological profiles remain elusive. Gene expression profiling in this study illustrates that hyperactive toll-like receptor and mitochondrial oxidative phosphorylation pathways configure the immunoreactive signature in monocyte-derived dendritic cells from patients with Down syndrome. Graphical Abstract [ABSTRACT FROM AUTHOR]

Despite the lack of endogenous synthesis and relevant nuclear receptors, several papers have been published over the decades claiming that the physiology of mollusks is affected by natural and synthetic sex steroids. With scant evidence for the existence of functional steroid nuclear receptors in mollusks, some scientists have speculated that the effects of steroids might be mediated via membrane receptors (i.e. via non-genomic/non-classical actions) - a mechanism that has been well-characterized in vertebrates. However, no study has yet investigated the ligand-binding ability of such receptor candidates in mollusks. The aim of the present study was to further trace the evolution of the endocrine system by investigating the presence of functional membrane sex steroid receptors in a mollusk, the great pond snail (Lymnaea stagnalis). We detected sequences homologous to the known vertebrate membrane sex steroid receptors in the Lymnaea transcriptome and genome data: G protein-coupled estrogen receptor-1 (GPER1); membrane progestin receptors (mPRs); G protein-coupled receptor family C group 6 member A (GPRC6A); and Zrt- and Irt-like protein 9 (ZIP9). Sequence analyses, including conserved domain analysis, phylogenetics, and transmembrane domain prediction, indicated that the mPR and ZIP9 candidates appeared to be homologs, while the GPER1 and GPRC6A candidates seemed to be non-orthologous receptors. All candidates transiently transfected into HEK293MSR cells were found to be localized at the plasma membrane, confirming that they function as membrane receptors. However, the signaling assays revealed that none of the candidates interacted with the main vertebrate steroid ligands. Our findings strongly suggest that functional membrane sex steroid receptors which would be homologous to the vertebrate ones are not present in Lymnaea. Although further experiments are required on other molluscan model species as well, we propose that both classical and non-classical sex steroid signaling for endocrine responses are specific to chordates, confirming that molluscan and vertebrate endocrine systems are fundamentally different. [ABSTRACT FROM AUTHOR]

Background Acute kidney injury (AKI) often occurs in pediatric patients who received allogeneic hematopoietic cell transplantation (HCT). We evaluated the risk and effect of HCT-related AKI in pediatric patients. Methods We retrospectively studied the survival and renal outcome of 69 children 100 days and 1-year posttransplant in our institution in 2004-2016. Stage-3 AKI developed in 34 patients (49%) until 100 days posttransplant. Results The 100-day overall survival (OS) rates of patients with stage-3 AKI were lower than those without it (76.5% vs. 94.3%, P = 0.035). The 1-year OS rates did not differ markedly between 21 post-100-day survivors with stage-3 AKI and 29 without it (80.8% vs. 87.9%, P = 0.444). The causes of 19 deaths included the relapse of underlying disease or graft failure (n = 11), treatment-related events (4), and second HCT-related events (4). Underlying disease of malignancy (crude hazard ratio (HR) 5.7; 95% confidence interval (CI), 2.20 to 14.96), > 1000 ng/mL ferritinemia (crude HR 4.29; 95% CI, 2.11 to 8.71), stem cell source of peripheral (crude HR 2.96; 95% CI, 1.22 to 7.20) or cord blood (crude HR 2.29; 95% CI, 1.03 to 5.06), and myeloablative regimen (crude HR 2.56; 95% CI, 1.24 to 5.26), were identified as risk factors for stage-3 AKI until 100 days posttransplant. Hyperferritinemia alone was significant (adjusted HR 5.52; 95% CI, 2.21 to 13.76) on multivariable analyses. Conclusions Hyperferritinemia was associated with stage-3 AKI and early mortality posttransplant. Pretransplant iron control may protect the kidney of pediatric HCT survivors., Author(s): Mari Kurokawa [sup.1] , Kei Nishiyama [sup.1] , Yuhki Koga [sup.1] , Katsuhide Eguchi [sup.1] , Takashi Imai [sup.1] , Utako Oba [sup.1] , Akira Shiraishi [sup.1] , Hazumu [...]

A wide variety of bioactive peptides have been identified in the central nervous system and several peripheral tissues in the ascidian Ciona intestinalis type A (Ciona robusta). However, hemocyte endocrine peptides have yet to be explored. Here, we report a novel 14-amino-acid peptide, CiEMa, that is predominant in the granular hemocytes and unilocular refractile granulocytes of Ciona. RNA-seq and qRT-PCR revealed the high CiEma expression in the adult pharynx and stomach. Immunohistochemistry further revealed the highly concentrated CiEMa in the hemolymph of the pharynx and epithelial cells of the stomach, suggesting biological roles in the immune response. Notably, bacterial lipopolysaccharide stimulation of isolated hemocytes for 1–4 h resulted in 1.9- to 2.4-fold increased CiEMa secretion. Furthermore, CiEMa-stimulated pharynx exhibited mRNA upregulation of the growth factor (Fgf3/7/10/22), vanadium binding proteins (CiVanabin1 and CiVanabin3), and forkhead and homeobox transcription factors (Foxl2, Hox3, and Dbx) but not antimicrobial peptides (CrPap-a and CrMam-a) or immune-related genes (Tgfbtun3, Tnfa, and Il17-2). Collectively, these results suggest that CiEMa plays roles in signal transduction involving tissue development or repair in the immune response, rather than in the direct regulation of immune response genes. The present study identified a novel Ciona hemocyte peptide, CiEMa, which paves the way for research on the biological roles of hemocyte peptides in chordates. [ABSTRACT FROM AUTHOR]

Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze the cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for plant species other than P. hexandrum , the isolation of the DPS enzyme gene and the type of the enzyme, e.g. whether it is 2-ODD or another type of enzyme such as cytochrome P-450, have not been reported. In this study, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with P. hexandrum deoxypodophyllotoxin synthase (PhDPS). Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using AlphaFold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism. [ABSTRACT FROM AUTHOR]

Schizophyllum commune is a widely distributed basidiomycete fungus that occasionally causes sinusitis or allergic bronchopulmonary mycosis. The invasive infection mostly occurs in immunocompromised adults. The number of reports on S. commune infection have increased in this decade due to the expansion of diagnostic techniques and awareness in clinical practice. However, S.commune infection in patients with primary immunodeficiencies has not been reported yet. Here, we described S. commune -abscesses developed in the brain and lung of a boy with chronic granulomatous disease (CGD) after allogenic hematopoietic cell transplantation (HCT). A 12-year-old CGD patient developed febrile neutropenia from day 4 after HCT, followed by chest pain on day 23. He had no obvious infection before HCT. Diagnostic imaging revealed disseminated lung and brain abscesses. He received administration of voriconazole, and his symptoms improved after engraftment. Chronic administration of voriconazole had also a favorable therapeutic response to brain lesion. A part of the fungus ball exhaled by the patient was cultured to develop a filamentous fungus. S. commune was identified by the analysis of the 28S rRNA gene. The catalase test was positive for S. commune , indicating that S. commune had virulence in this patient with CGD. The assessment of specific-IgG to S. commune suggested peri-transplant infection, although colonization was not excluded. This rare pediatric case of S. commune infection highlights that CGD patients are vulnerable to invasive infection, especially when undergoing HCT. [ABSTRACT FROM AUTHOR]

Backgrounds: Elucidation of peptide-receptor pairs is a prerequisite for many studies in the neuroendocrine, endocrine, and neuroscience fields. Recent omics analyses have provided vast amounts of peptide and G protein-coupled receptor (GPCR) sequence data. GPCRs for homologous peptides are easily characterized based on homology searching, and the relevant peptide-GPCR interactions are also detected by typical signaling assays. In contrast, conventional evaluation or prediction methods, including high-throughput reverse-pharmacological assays and tertiary structure-based computational analyses, are not useful for identifying interactions between novel and omics-derived peptides and GPCRs. Summary: Recently, an approach combining machine learning-based prediction of novel peptide-GPCR pairs and experimental validation of the predicted pairs have been shown to breakthrough this bottleneck. A machine learning method, logistic regression for human class A GPCRs and the multiple subsequent signaling assays led to the deorphanization of human class A orphan GPCRs, namely, the identification of 18 peptide-GPCR pairs. Furthermore, using another machine learning algorithm, the support vector machine (SVM), the peptide descriptor-incorporated SVM was originally developed and employed to predict GPCRs for novel peptides characterized from the closest relative of vertebrates, Ciona intestinalis Type A (Ciona robusta). Experimental validation of the predicted pairs eventually led to the identification of 11 novel peptide-GPCR pairs. Of particular interest is that these newly identified GPCRs displayed neither significant sequence similarity nor molecular phylogenetic relatedness to known GPCRs for peptides. Key Messages: These recent studies highlight the usefulness and versatility of machine learning for enabling the efficient, reliable, and systematic identification of novel peptide-GPCR interactions. [ABSTRACT FROM AUTHOR]

O -Methyltransferases (OMTs) play important roles in antitumor lignan biosynthesis. To date, six OMTs catalyzing the methylation of dibenzylbutyrolactone lignans as biosynthetic precursors of antitumor lignans have been identified. However, there is still no systematic understanding of the diversity and regularity of the biosynthetic mechanisms among various plant lineages. Herein, we report the characterization of two OMTs from Anthriscus sylvestris and Thujopsis dolabrata var. hondae [designated as AsSecoNorYatein (SNY) OMT and TdSNYOMT] together with the six known OMTs to evaluate their diversity and regularity. Although A. sylvestris 5- O -methylthujaplicatin (SecoNorYatein) and 4- O -demethylyatein (NorYatein) OMT (AsSNYOMT) and TdSNYOMT accept 5- O -methylthujaplicatin and 4- O -demethylyatein as substrates, phylogenetic analysis indicated that these two OMTs shared low amino acid sequence identity, 33.8%, indicating a signature of parallel evolution. The OMTs and the six previously identified OMTs were found to be diverse in terms of their substrate specificity, regioselectivity and amino acid sequence identity, indicating independent evolution in each plant species. Meanwhile, two-entropy analysis detected four amino acid residues as being specifically acquired by dibenzylbutyrolactone lignan OMTs. Site-directed mutation of AsSNYOMT indicated that two of them contributed specifically to 5- O -methylthujaplicatin methylation. The results provide a new example of parallel evolution and the diversity and regularity of OMTs in plant secondary (specialized) metabolism. [ABSTRACT FROM AUTHOR]

The Gly-Asp-Ser-Leu (GDSL) motif of esterase/lipase family proteins (GELPs) generally exhibit esterase activity, whereas transferase activity is markedly preferred in several GELPs, including the Tanacetum cinerariifolium GDSL lipase TciGLIP, which is responsible for the biosynthesis of the natural insecticide, pyrethrin I. This transferase activity is due to the substrate affinity regulated by the protein structure and these features are expected to be conserved in transferase activity-exhibiting GELPs (tr-GELPs). In this study, we identified two amino acid residues, [N/R]208 and D484, in GELP sequence alignments as candidate key residues for the transferase activity of tr-GELPs by two-entropy analysis. Molecular phylogenetic analysis demonstrated that each tr-GELP is located in the clusters for non-tr-GELPs, and most GELPs conserve at least one of the two residues. These results suggest that the two conserved residues are required for the acquisition of transferase activity in the GELP family. Furthermore, substrate docking analyses using ColabFold-generated structure models of both natives and each of the two amino acids-mutated TciGLIPs also revealed numerous docking models for the proper access of substrates to the active site, indicating crucial roles of these residues of TciGLIP in its transferase activity. This is the first report on essential residues in tr-GELPs for the transferase activity. [ABSTRACT FROM AUTHOR]

Metamorphosis is the dramatic and irreversible reconstruction of animal bodies transitioning from the larval stage. Because of the significant impact of metamorphosis on animal life, its timing is strictly regulated. Invertebrate chordate ascidians are the closest living relatives of vertebrates. Ascidians exhibit metamorphosis that converts their swimming larvae into sessile adults. Ascidian metamorphosis is triggered by a mechanical stimulus generated when adhesive papillae adhere to a substrate. However, it is not well understood how the mechanical stimulus is generated and how ascidian larvae sense the stimulus. In this study, we addressed these issues by a combination of embryological, molecular, and genetic experiments in the model ascidian Ciona intestinalis Type A, also called Ciona robusta. We here showed that the epidermal neuronal network starting from the sensory neurons at the adhesive papillae is responsible for the sensing of adhesion. We also found that the transient receptor potential (TRP) channel PKD2 is involved in sensing the stimulus of adhesion. Our results provide a better understanding of the mechanisms underlying the regulation of the timing of ascidian metamorphosis. [ABSTRACT FROM AUTHOR]

A mouse testis-specific long noncoding RNA (lncRNA), Start, is localized in the cytosol of Leydig cells and in the nucleus of pachytene spermatocytes. We previously showed that Start regulates steroidogenesis through controlling the expression of Star and Hsd3b1 genes in Leydig cells, but its function in germ cells was not known. Here we verified that a spermatocyte-specific protease gene, Prss43/Tessp-3, was downregulated in Start-knockout testes. To investigate the transcriptional regulatory activity of Start in spermatocytes, we first performed a series of reporter gene assays using a thymidine kinase promoter in spermatocyte-derived GC-2spd(ts) cells. A 5.4-kb genome sequence encompassing Start exhibited enhancer activity for this promoter, and the activity was decreased by knockdown of Start. Deletion of the Start promoter and replacement of the Start sequence abolished the enhancer activity and, consistently, the activity was detected in further experiments only when Start was actively transcribed. We then examined whether the Prss43/Tessp-3 gene could be a target of Start. A reporter gene assay demonstrated that the 5.4-kb sequence exhibited enhancer activity for a Prss43/Tessp-3 promoter in GC-2spd(ts) cells and that the activity was significantly decreased by knockdown of Start. These results suggest that Start functions in transcriptional activation of the Prss43/Tessp-3 gene in spermatocytes. Given that Start is presumed to regulate steroidogenic genes at the posttranscriptional level in Leydig cells, the function in spermatocytes is a novel role of Start. These findings provide an insight into multifunctionality of lncRNAs in the testis. [ABSTRACT FROM AUTHOR]

The plant Tanacetum coccineum (painted daisy) is closely related to Tanacetum cinerariifolium (pyrethrum daisy). However, T. cinerariifolium produces large amounts of pyrethrins, a class of natural insecticides, whereas T. coccineum produces much smaller amounts of these compounds. Thus, comparative genomic analysis is expected to contribute a great deal to investigating the differences in biological defense systems, including pyrethrin biosynthesis. Here, we elucidated the 9.4 Gb draft genome of T. coccineum, consisting of 2,836,647 scaffolds and 103,680 genes. Comparative analyses of the draft genome of T. coccineum and that of T. cinerariifolium, generated in our previous study, revealed distinct features of T. coccineum genes. While the T. coccineum genome contains more numerous ribosome-inactivating protein (RIP)-encoding genes, the number of higher-toxicity type-II RIP-encoding genes is larger in T. cinerariifolium. Furthermore, the number of histidine kinases encoded by the T. coccineum genome is smaller than that of T. cinerariifolium, suggesting a biological correlation with pyrethrin biosynthesis. Moreover, the flanking regions of pyrethrin biosynthesis-related genes are also distinct between these two plants. These results provide clues to the elucidation of species-specific biodefense systems, including the regulatory mechanisms underlying pyrethrin production. [ABSTRACT FROM AUTHOR]

Omics studies contribute to the elucidation of genomes and profiles of gene expression. In the ascidian Ciona intestinalis Type A (Ciona robusta), mass spectrometry (MS)-based peptidomic studies have detected numerous Ciona -specific (nonhomologous) neuropeptides as well as Ciona homologs of typical vertebrate neuropeptides and hypothalamic peptide hormones. Candidates for cognate G protein-coupled receptors (GPCRs) for these peptides have been found in the Ciona transcriptome by two ways. First, Ciona homologous GPCRs of vertebrate counterparts have been detected by sequence homology searches of cognate transcriptomes. Second, the transcriptome-derived GPCR candidates have been used for machine learning-based systematic prediction of interactions not only between Ciona homologous peptides and GPCRs but also between novel Ciona peptides and GPCRs. These data have ultimately led to experimental evidence for various Ciona peptide-GPCR interactions. Comparative transcriptomics between the wildtype and Ciona vasopressin (CiVP) gene-edited Ciona provide clues to the biological functions of CiVP in ovarian follicular development and whole body growth. Furthermore, the transcriptomes of follicles treated with peptides, such as Ciona tachykinin and cionin (a Ciona cholecystokinin homolog), have revealed key regulatory genes for Ciona follicle growth, maturation, and ovulation, eventually leading to the verification of essential and novel molecular mechanisms underlying these biological events. These findings indicate that omics studies, combined with artificial intelligence and single-cell technologies, pave the way for investigating in greater details the nervous, neuroendocrine, and endocrine systems of ascidians and the molecular and functional evolution and diversity of peptidergic regulatory networks throughout chordates. [ABSTRACT FROM AUTHOR]

Mucosa‐associated lymphoid tissue lymphoma‐translocation gene 1 (MALT1)‐deficiency is a rare combined immunodeficiency characterized by recurrent infections, dermatitis and enteropathy. We herein investigate the immunological profiles of our patient and previously reported children with MALT1‐deficiency. A mutation analysis was performed by targeted panel sequencing for primary immunodeficiency. Lymphocyte subset, activation and B cell differentiation were analyzed by flow cytometry and t‐distributed stochastic neighbor embedding. Pneumocystis pneumonia developed in a 6‐month‐old Japanese infant with atopic dermatitis, enteritis and growth restriction. This infant showed agammaglobulinemia without lymphopenia. At 8 years of age, the genetic diagnosis of MALT1‐deficiency was confirmed on a novel homozygous mutation of c.1102G>T, p.E368X. T cell stimulation tests showed impairments in the production of interleukin‐2, phosphorylation of nuclear factor kappa B (NF‐κB) p65 and differentiation of B cells. In combination with the literature data, we found that the number of circulatory B cells, but not T cells, were inversely correlated with the age of patients. The hematopoietic cell transplantation (HCT) successfully reconstituted the differentiation of mature B cells and T cells. These data conceptualize that patients with complete MALT1‐deficiency show aberrant differentiation and depletion of B cells. The early diagnosis and HCT lead to a cure of the disease phenotype associated with the loss‐of‐function mutations in human CARD11. [ABSTRACT FROM AUTHOR]

Tissue/organ-specific genes (TSGs) are important not only for understanding organ development and function, but also for investigating the evolutionary lineages of organs in animals. Here, we investigate the TSGs of 9 adult tissues of an ascidian, Ciona intestinalis Type A (Ciona robusta), which lies in the important position of being the sister group of vertebrates. RNA-seq and qRT-PCR identified the Ciona TSGs in each tissue, and BLAST searches identified their homologs in zebrafish and mice. Tissue distributions of the vertebrate homologs were analyzed and clustered using public RNA-seq data for 12 zebrafish and 30 mouse tissues. Among the vertebrate homologs of the Ciona TSGs in the neural complex, 48% and 63% showed high expression in the zebrafish and mouse brain, respectively, suggesting that the central nervous system is evolutionarily conserved in chordates. In contrast, vertebrate homologs of Ciona TSGs in the ovary, pharynx, and intestine were not consistently highly expressed in the corresponding tissues of vertebrates, suggesting that these organs have evolved in Ciona-specific lineages. Intriguingly, more TSG homologs of the Ciona stomach were highly expressed in the vertebrate liver (17–29%) and intestine (22–33%) than in the mouse stomach (5%). Expression profiles for these genes suggest that the biological roles of the Ciona stomach are distinct from those of their vertebrate counterparts. Collectively, Ciona tissues were categorized into 3 groups: i) high similarity to the corresponding vertebrate tissues (neural complex and heart), ii) low similarity to the corresponding vertebrate tissues (ovary, pharynx, and intestine), and iii) low similarity to the corresponding vertebrate tissues, but high similarity to other vertebrate tissues (stomach, endostyle, and siphons). The present study provides transcriptomic catalogs of adult ascidian tissues and significant insights into the evolutionary lineages of the brain, heart, and digestive tract of chordates. [ABSTRACT FROM AUTHOR]

Oxytocin (OT) and vasopressin (VP) superfamily neuropeptides are distributed in not only vertebrates but also diverse invertebrates. However, no VPergic innervation of invertebrates has ever been documented. In the ascidian, Ciona intestinalis Type A (Ciona robusta), an OT/VP superfamily peptide was identified, and the Ciona vasopressin (CiVP) induces oocyte maturation and ovulation. In the present study, we characterize the innervation and phenotypes of genetically modified Ciona : CiVP promoter-Venus transgenic and CiVP mutants. CiVP promoter-Venus transgenic Ciona demonstrated that CiVP gene was highly expressed in the cerebral ganglion and several nerves. Fluorescence was also detected in the ovary of young CiVP promoter-Venus transgenic ascidians, suggesting that the CiVP gene is also expressed temporarily in the ovary of young ascidians. Furthermore, a marked decrease of post-vitellogenic (stage III) follicles was observed in the ovary of CiVP mutants, whereas pre-vitellogenic (stage I) and vitellogenic (stage II) follicles were increased in the mutant ovary, compared with that of wildtype Ciona. Gene expression profiles showed that the expression of various genes, including genes related to ovarian follicle growth, was altered in the ovary of CiVP mutants. Altogether, these results indicated that CiVP, mainly as a neuropeptide, plays pivotal roles in diverse biological functions, including growth of early-stage ovarian follicles via regulation of the expression of a wide variety of genes. This is the first report describing a VP gene promoter-transgenic and VP gene-edited invertebrate and also on its gene expression profiles and phenotypes. [ABSTRACT FROM AUTHOR]

A 31-day-old infant was admitted to the pediatric intensive care unit due to shock and anemia. The mother had systemic lupus erythematosus and direct antiglobulin test (DAT)-positive hemolytic anemia. The perinatal course of this infant and the mother was uneventful. Regular health check screenings revealed that activity, growth, and development were unremarkable at birth, 5, and 28 days of life. Passive immune hemolytic anemia due to neonatal lupus erythematosus was diagnosed based on a positive DAT for warm-type IgG antibodies, reticulocytosis, and lupus-specific antibodies at rehospitalization. It was complicated by cytomegalovirus (CMV) antigenemia. Umbilical cord blood and peripheral blood samples obtained from the infant at 5 days after birth were negative for CMV DNA. The infant was curatively treated by intensive care with repeated blood transfusions and antiviral therapy. This is the first report indicating that CMV infection exacerbates hemolytic anemia in patients with maternal red blood cell alloantibodies. [ABSTRACT FROM AUTHOR]

The testis expresses many long noncoding RNAs (lncRNAs), but their functions and overview of lncRNA variety are not well understood. The mouse Prss/Tessp locus contains six serine protease genes and two lncRNAs that have been suggested to play important roles in spermatogenesis. Here, we found a novel testis-specific lncRNA, Start (Steroidogenesis activating lncRNA in testis), in this locus. Start is 1822 nucleotides in length and was found to be localized mostly in the cytosol of germ cells and Leydig cells, although nuclear localization was also observed. Start -knockout (KO) mice generated by the CRISPR/Cas9 system were fertile and showed no morphological abnormality in adults. However, in adult Start -KO testes, RNA-seq and qRT-PCR analyses revealed an increase in the expression of steroidogenic genes such as Star and Hsd3b1 , while ELISA analysis revealed that the testosterone levels in serum and testis were significantly low. Interestingly, at 8 days postpartum, both steroidogenic gene expression and testosterone level were decreased in Start -KO mice. Since overexpression of Start in two Leydig-derived cell lines resulted in elevation of the expression of steroidogenic genes including Star and Hsd3b1 , Start is likely to be involved in their upregulation. The increase in expression of steroidogenic genes in adult Start -KO testes might be caused by a secondary effect via the androgen receptor autocrine pathway or the hypothalamus-pituitary-gonadal axis. Additionally, we observed a reduced number of Leydig cells at 8 days postpartum. Collectively, our results strongly suggest that Start is a regulator of steroidogenesis in Leydig cells. The current study provides an insight into the overall picture of the function of testis lncRNAs. [ABSTRACT FROM AUTHOR]

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP. [ABSTRACT FROM AUTHOR]

Summary: Sesamum spp. (sesame) are known to accumulate a variety of lignans in a lineage‐specific manner. In cultivated sesame (Sesamum indicum), (+)‐sesamin, (+)‐sesamolin and (+)‐sesaminol triglucoside are the three major lignans found richly in the seeds. A recent study demonstrated that SiCYP92B14 is a pivotal enzyme that allocates the substrate (+)‐sesamin to two products, (+)‐sesamolin and (+)‐sesaminol, through multiple reaction schemes including oxidative rearrangement of α‐oxy‐substituted aryl groups (ORA). In contrast, it remains unclear whether (+)‐sesamin in wild sesame undergoes oxidation reactions as in S. indicum and how, if at all, the ratio of the co‐products is tailored at the molecular level. Here, we functionally characterised SrCYP92B14 as a SiCYP92B14 orthologue from a wild sesame, Sesamum radiatum, in which we revealed accumulation of the (+)‐sesaminol derivatives (+)‐sesangolin and its novel structural isomer (+)‐7´‐episesantalin. Intriguingly, SrCYP92B14 predominantly produced (+)‐sesaminol either through ORA or direct oxidation on the aromatic ring, while a relatively low but detectable level of (+)‐sesamolin was produced. Amino acid substitution analysis suggested that residues in the putative distal helix and the neighbouring heme propionate of CYP92B14 affect the ratios of its co‐products. These data collectively show that the bimodal oxidation mechanism of (+)‐sesamin might be widespread across Sesamum spp., and that CYP92B14 is likely to be a key enzyme in shaping the ratio of (+)‐sesaminol‐ and (+)‐sesamolin‐derived lignans from the biochemical and evolutionary perspectives. [ABSTRACT FROM AUTHOR]

Summary: Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid‐related plant specialized metabolites. (+)‐Sesamin and (+)‐sesamolin are major hydrophobic lignans, whereas (+)‐sesaminol primarily accumulates as a water‐soluble sesaminol triglucoside (STG) with a sugar chain branched via β1→2 and β1→6‐O‐glucosidic linkages [i.e. (+)‐sesaminol 2‐O‐β‐d‐glucosyl‐(1→2)‐O‐β‐d‐glucoside‐(1→6)‐O‐β‐d‐glucoside]. We previously reported that the 2‐O‐glucosylation of (+)‐sesaminol aglycon and β1→6‐O‐glucosylation of (+)‐sesaminol 2‐O‐β‐d‐glucoside (SMG) are mediated by UDP‐sugar‐dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the β1→2‐O‐glucosylation of SMG and (+)‐sesaminol 2‐O‐β‐d‐glucosyl‐(1→6)‐O‐β‐d‐glucoside [termed SDG(β1→6)]. UGT94AG1 was phylogenetically related to glycoside‐specific glycosyltransferases (GGTs) and co‐ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG‐deficient sesame mutant that predominantly accumulates SDG(β1→6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar–sugar β1→6‐O‐glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two‐hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane‐associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane‐bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main β‐O‐glucosylation sequence of STG biosynthesis is β1→2‐O‐glucosylation of SMG by UGT94AG1 followed by UGT94AA2‐mediated β1→6‐O‐glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio‐selectivity of sequential glucosylations catalyzed by GGTs. Significance Statement: Sesaminol triglucoside (STG) of sesame seeds has unique bioactivities with potential health benefits, yet the STG biosynthesis pathway remains to be fully established. This study has identified the missing enzyme in STG biosynthesis that catalyzes β1→2‐O‐glucosylation, a regioselective sequential glycosylation of sesame lignan biosynthesis. [ABSTRACT FROM AUTHOR]

Theca/interstitial cells are responsible for the growth and maturation of ovarian follicles. However, little is known about the theca/interstitial cell-specific genes and their functions. In this study, we explored transcriptomes of theca/interstitial cells by RNA-seq, and the novel biological roles of a theca cell marker, asporin (Aspn)/periodontal ligament-associated protein 1 (PLAP-1). RNA-seq detected 432 and 62 genes expressed specifically in theca/interstitial cells and granulosa cells isolated from 3-weeks old mouse ovaries. Gene ontology analysis demonstrated that these genes were largely categorized into four major groups: extracellular matrix organization-related terms, chemotaxis-related terms, the angiogenesis-related terms, and morphogenesis-related terms. In situ hybridization demonstrated that the newly detected representative gene, Aspn/PLAP-1 , was detected specifically in the outer layer of theca cells in contrast with the expression of the basal lamina-specific gene, Nidgen-1. Intriguingly, an Aspn/PLAP-1 antibody completely arrested the growth of secondary follicles that is the gonadotropin-independent follicle developmental stage. Furthermore, transforming growth factor-β (TGF-β)-triggered signaling was induced by the Aspn/PLAP-1 antibody treatment, which is consistent with the inhibitory effect of Aspn/PLAP-1 on TGF-β. Altogether, these results suggest that theca cells are classified into subpopulations on the basis of new marker genes and their biological functions, and provide evidence that Aspn/PLAP-1 is expressed exclusively in the outer layer of theca cells and plays a pivotal role in the growth of secondary follicles via downregulation of the canonical TGF-β signaling cascade. [ABSTRACT FROM AUTHOR]

This paper reports the development of a newly modified, noncontact spectrophotometer for clinical use. The instrument is capable of accurately measuring the color within small areas (1 × 2 mm) of a tooth. This spectrophotometer used a 45°/0° geometry, a 150W halogen lamp, and fiberoptics to focus the light, together with a lens having a focal point distance of 85 mm. A movable platform was added to the apparatus to automatically scan and measure the color of specific areas. Short-term repeatability indicated the color difference ΔE to be approximately 0.15. [ABSTRACT FROM AUTHOR]

Cholinergic signaling, which modulates cell activities via nicotinic and muscarinic acetylcholine receptors (n- and mAChRs) in response to internal or external stimuli, has been demonstrated in mammalian non-neuronal cells that synthesize acetylcholine (ACh). One of the major pathways of excitatory transmission in the enteric nervous system (ENS) is mediated by cholinergic transmission, with the transmitter ACh producing excitatory potentials in postsynaptic effector cells. In addition to ACh-synthesizing and ACh-metabolizing elements in the ENS, the presence of non-neuronal ACh machinery has been reported in epithelial cells of the small and large intestines of rats and humans. However, little is known about how non-neuronal ACh controls physiological function in the intestine. Here, experiments using crypt-villus organoids that lack nerve and immune cells in culture suggest that endogenous ACh is synthesized in the intestinal epithelium to drive organoid growth and differentiation through activation of nAChRs. Treatment of organoids with nicotine enhanced cell growth and the expression of marker genes for stem and epithelial cells. On the other hand, the nAChR antagonist mecamylamine strongly inhibited the growth and differentiation of organoids, suggesting the involvement of nAChRs in the regulation of proliferation and differentiation of Lgr5-positive stem cells. More specifically, RNA sequencing analysis revealed that Wnt5a expression was dramatically upregulated after nicotine treatment, and Wnt5a rescued organoid growth and differentiation in response to mecamylamine. Taken together, our results indicate that coordinated activities of nAChR and Wnt signaling maintain Lgr5-positive stem cell activity and balanced differentiation. Furthermore, we could clearly separate the two groups, neuronal ACh in the ENS and non-neuronal ACh in the intestinal epithelium. Dysfunction of the non-neuronal cholinergic system is involved in the pathogenesis of disease. The data will increase our understanding of the cholinergic properties of non-neuronal cells and lead to optimization of drug therapy. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) have been found to form heterodimers and modulate or fine-tune the functions of GPCRs. However, the involvement of GPCR heterodimerization and its functional consequences in gonadal tissues, including granulosa cells, have been poorly investigated, mainly due to the lack of efficient method for identification of novel GPCR heterodimers. In this paper, we identified a novel GPCR heterodimer between prostaglandin E2 (PGE2) receptor 2 (EP2) and calcitonin (CT) receptor (CTR). High-resolution liquid chromatography (LC)-tandem mass spectrometry (MS/MS) of protease-digested EP2-coimmunoprecipitates detected protein fragments of CTR in an ovarian granulosa cell line, OV3121. Western blotting of EP2- and CTR-coimmunoprecipitates detected a specific band for EP2-CTR heterodimer. Specific heterodimerization between EP2 and CTR was also observed by fluorescence resonance energy transfer analysis in HEK293MSR cells expressing cyan- and yellow-fluorescent protein-fused EP2 and CTR, respectively. Collectively, these results provided evidence for heterodimerization between EP2 and CTR. Moreover, Ca2+ mobilization by CT was approximately 40% less potent in HEK293MSR cells expressing an EP2-CTR heterodimer, whereas cAMP production by EP2 or CT was not significantly altered compared with cells expressing EP2- or CTR alone. These functional analyses verified that CTR-mediated Ca2+ mobilization is specifically decreased via heterodimerization with EP2. Altogether, the present study suggests that a novel GPCR heterodimer, EP2-CTR, is involved in some functional regulation, and paves the way for investigation of novel biological roles of CTR and EP2 in various tissues. [ABSTRACT FROM AUTHOR]

Calcitonin (CT) is a hormone that decreases serum calcium level by suppressing osteoclastic activity in the vertebrate bone. In vertebrates, the structure-function relationship of CTs has been studied extensively. We recently identified three CT superfamily peptides, Bf-CTFP1 to 3, and clarified the molecular and functional characteristics of their receptor and receptor activity-modifying protein in amphioxus, Branchiostoma floridae . However, the CT activity of Bf-CTFPs has yet to be investigated. In the present study, a functional analysis of Bf-CTFPs was performed using goldfish scales having both osteoclasts and osteoblasts. All Bf-CTFPs suppressed osteoclastic activity via a goldfish CT receptor. Although the primary amino acid sequences of the Bf-CTFPs showed low sequence similarity to vertebrate CTs, Bf-CTFP1 to 3 share three amino acids, Thr 25 , Thr 27 , and Pro 32 -NH 2 , that are required for receptor binding, with salmon CT. Moreover, homology model analysis revealed that the Bf-CTFPs form alpha-helical structures. The alpha-helical position and length of Bf-CTFP1 and 2 were conserved with those of a highly potent ligand, teleost CT. Interestingly, the composition of the alpha-helix of Bf-CTFP3 differed from those of teleost CT, despite that the action of Bf-CTFP3 on goldfish scales was the same as that of Bf-CTFP1 and 2. Collectively, the present study provides new insights into the structure-function relationship of CT and its functional evolution in chordates. [ABSTRACT FROM AUTHOR]

Stable breast cancer cell ( BCC) lines are valuable tools for the identification of breast cancer stem cell ( BCSC) phenotypes that develop in response to several stimuli as well as for studying the basic mechanisms associated with the initiation and maintenance of BCSCs. However, the characteristics of individual, BCC-derived BCSCs varies and these cells show distinct phenotypes depending on the different BCSC markers used for their isolation. Aldehyde dehydrogenase ( ALDH) activity is just such a recognized biomarker of BCSCs with a CD44+/ CD24− phenotype. We isolated BCSCs with high ALDH activity ( CD44+/ CD24−/Aldefluorpos) from a primary culture of human breast cancer tissue and observed that the cells had stem cell properties compared to BCSCs with no ALDH activity ( CD44+/ CD24−/Aldefluorneg). Moreover, we found Aldefluorpos BCSCs had a greater hypoxic response and subsequent induction of HIF-1α expression compared to the Aldefluorneg BCSCs. We also found that knocking down HIF-1α, but not HIF-2α, in Aldefluorpos BCSCs led to a significant reduction of the stem cell properties through a decrease in the mRNA levels of genes associated with the epithelial-mesenchymal transition. Indeed, HIF-1α overexpression in Aldefluorneg BCSCs led to Slug and Snail mRNA increase and the associated repression of E-cadherin and increase in Vimentin. Of note, prolonged hypoxic stimulation promoted the phenotypic changes of Aldefluorneg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes. [ABSTRACT FROM AUTHOR]

Forsythia spp. are perennial woody plants which are one of the most extensively used medicinal sources of Chinese medicines and functional diets owing to their lignan contents. Lignans have received widespread attention as leading compounds in the development of antitumor drugs and healthy diets for reducing the risks of lifestyle-related diseases. However, the molecular basis of Forsythia has yet to be established. In this study, we have verified de novo deep transcriptome of Forsythia koreana leaf and callus using the Illumina HiSeq 1500 platform. A total of 89 million reads were assembled into 116,824 contigs using Trinity, and 1,576 of the contigs displayed the sequence similarity to the enzymes responsible for plant specialized metabolism including lignan biosynthesis. Notably, gene ontology (GO) analysis indicated the remarkable enrichment of lignan-biosynthetic enzyme genes in the callus transcriptome. Nevertheless, precise annotation and molecular phylogenetic analyses were hindered by partial sequences of open reading frames (ORFs) of the Trinity-based contigs. To obtain more numerous contigs harboring a full-length ORF, we developed a novel overlapping layout consensus-based procedure, virtual primer-based sequence reassembly (VP-seq). VP-seq elucidated 709 full-length ORFs, whereas only 146 full-length ORFs were assembled by Trinity. The comparison of expression profiles of leaf and callus using VP-seq-based full-length ORFs revealed 50-fold upregulation of secoisolariciresinol dehydrogenase (SIRD) in callus. Expression and phylogenetic cluster analyses predicted candidates for matairesinol-glucosylating enzymes. We also performed VP-seq analysis of lignan-biosynthetic enzyme genes in the transcriptome data of other lignan-rich plants, Linum flavum, Linum usitatissimum and Podophyllum hexandrum. The comparative analysis indicated both common gene clusters involved in biosynthesis upstream of matairesinol such as SIRD and plant lineage-specific gene clusters, in particular, genes responsible for biosynthetic pathways for production of podophyllotoxin; CYP71BE54, a key enzyme gene for podophyllotoxin biosynthesis in P. hexandrum, was not found in L. flavum, although both P. hexandrum. and L. flavum yield podophyllotoxin. Altogether, these data have established the fruitful molecular basis of Forsythia and provided insight into the molecular evolution and diversity of lignan biosynthetic pathways. [ABSTRACT FROM AUTHOR]

The expression of estrogen receptor is the key in most breast cancers (BC) and binding of estrogen receptor to the genome correlates to Forkhead protein (FOXA1) expression. We herein assessed the correlation between the cancer stem cell (CSC) population and FOXA1 expression in luminal BC. We established luminal BC cells derived from metastatic pleural effusion and analyzed the potency of CSC and related factors with established luminal BC cell lines. We also confirmed that mammosphere cultures have an increased aldehyde dehydrogenase-positive population, which is one of the CSC markers, compared with adherent culture cells. Using a quantitative PCR analysis, we found that mammosphere forming cells showed a higher expression of FOXA1 and stemness-related genes compared with adherent culture cells. Furthermore, the growth activity and colony-forming activity of 4-hydroxytamoxifen-treated BC cells were inhibited in a mammosphere assay. Interestingly, 4-hydroxytamoxifen-resistant cells had significantly increased FOXA1 gene expression levels. Finally, we established short hairpin RNA of FOXA1 (shFOXA1) MCF-7 cells and investigated the relationship between self-renewal potential and FOXA1 expression. As a result, we found no significant difference in the number of mammospheres but decreased colony formation in shFOXA1 MCF-7 cells compared with control. These results suggest that the expression of FOXA1 appears to be involved in the proliferation of immature BC cells rather than the induction of stemness-related genes and self-renewal potency of CSCs. [ABSTRACT FROM AUTHOR]