A major endogenous glycoside hydrolase mediating quercetin uptake in Bombyx mori.

Quercetin is a common plant flavonoid which is involved in herbivore-plant interactions. Mulberry silkworms (domestic silkworm, Bombyx mori, and wild silkworm, Bombyx mandarina) take up quercetin from mulberry leaves and accumulate the metabolites in the cocoon, thereby improving its protective properties. Here we identified a glycoside hydrolase, named glycoside hydrolase family 1 group G 5 (GH1G5), which is expressed in the midgut and is involved in quercetin metabolism in the domestic silkworm. Our results suggest that this enzyme mediates quercetin uptake by deglycosylating the three primary quercetin glycosides present in mulberry leaf: rutin, quercetin-3-O-malonylglucoside, and quercetin-3-O-glucoside. Despite being located in an unstable genomic region that has undergone frequent structural changes in the evolution of Lepidoptera, GH1G5 has retained its hydrolytic activity, suggesting quercetin uptake has adaptive significance for mulberry silkworms. GH1G5 is also important in breeding: defective mutations which result in discoloration of the cocoon and increased silk yield are homozygously conserved in 27 of the 32 Japanese white-cocoon domestic silkworm strains and 12 of the 30 Chinese ones we investigated.

Endothelial cells differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes.

Endothelial cells (ECs) experience two different blood flow patterns: laminar and disturbed flow. Their responses to laminar flow contribute to vascular homeostasis, whereas their responses to disturbed flow result in EC dysfunction and vascular diseases. However, it remains unclear how ECs differentially sense laminar and disturbed flow and trigger signaling that elicits different responses. Here, we showed that ECs differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes in opposite directions. This results in distinct changes in mitochondrial function, namely, increased adenosine triphosphate (ATP) production for laminar flow and increased hydrogen peroxide (H2O2) release for disturbed flow, leading to ATP- and H2O2-mediated signaling, respectively. When cultured human aortic ECs were subjected to laminar or disturbed flow in flow-loading devices, the lipid order of their plasma membranes immediately decreased in response to laminar flow and increased in response to disturbed flow. Laminar flow also decreased the lipid order of mitochondrial membranes and increased mitochondrial ATP production. In contrast, disturbed flow increased the lipid order of mitochondrial membranes and increased the release of H2O2 from the mitochondria. The addition of cholesterol to the cells increased the lipid order of both membranes and abrogated laminar flow-induced ATP production, while treatment of the cells with a cholesterol-depleting reagent, methyl-ß cyclodextrin, decreased the lipid order of both membranes and abolished disturbed flow-induced H2O2 release, indicating that changes in the membrane lipid order and/or cholesterol content are closely linked to flow-induced changes in mitochondrial functions.NEW & NOTEWORTHY How vascular endothelial cells (ECs) differentially sense laminar and disturbed flows and trigger intracellular signaling remains unclear. Here, we show that EC plasma membranes act as mechanosensors to discriminate between laminar and disturbed flows by undergoing opposite changes in their lipid order. Similar lipid order changes occur simultaneously in the mitochondrial membranes, which are linked to changes in mitochondrial function, that is, increased ATP production for laminar flow and increased H2O2 release for disturbed flow.

Fine mapping of Green a, Ga, on chromosome 27 in Bombyx mori.

Some strains of silkworms produce green cocoons of varying intensities. This results from quantitative and qualitative differences in flavonoid pigments, which are influenced by the environment and genetic background. We discovered that the appearance of a faint green cocoon is regulated by a gene (G27) located on chromosome 27. Through mating experiments, we found that G27 is identical to an essential flavonoid cocoon gene, Ga. This locus has not been previously described. Furthermore, we narrowed down the Ga region to 438 kbp using molecular markers. Within this region, several predicted genes for sugar transporters form a cluster structure, suggesting that Ga is among them.

Highly accurate genome assembly of an improved high-yielding silkworm strain, Nichi01.

The silkworm (Bombyx mori) is an important lepidopteran model insect and an industrial domestic animal traditionally used for silk production. Here, we report the genome assembly of an improved Japanese strain Nichi01, in which the cocoon yield is comparable to that of commercial silkworm strains. The integration of PacBio Sequel II long-read and ddRAD-seq-based high-density genetic linkage map achieved the highest quality genome assembly of silkworms to date; 22 of the 28 pseudomolecules contained telomeric repeats at both ends, and only four gaps were present in the assembly. A total of 452 Mbp of the assembly with an N50 of 16.614 Mbp covered 99.3% of the complete orthologs of the lepidopteran core genes. Although the genome sequence of Nichi01 and that of the previously reported low-yielding tropical strain p50T assured their accuracy in most regions, we corrected several regions, misassembled in p50T, in our assembly. A total of 18,397 proteins were predicted using over 95 Gb of mRNA-seq derived from 10 different organs, covering 96.9% of the complete orthologs of the lepidopteran core genes. The final assembly and annotation files are available in KAIKObase (https://kaikobase.dna.affrc.go.jp/index.html) along with a genome browser and BLAST searching service, which would facilitate further studies and the breeding of silkworms and other insects.

P2X4 receptor stimulation enhances MrgprB2-mediated mast cell activation and pseudoallergic reactions in mice.

Pseudoallergies caused by drugs make disease treatment difficult. Mas-relate G protein-coupled receptor X2 (MRGPRX2), which is specifically expressed in mast cells (MCs), has been implicated in pseudoallergies. High concentrations of therapeutic agents are typically required to stimulate MRGPRX2. Although regulatory mechanisms may enhance this response, the factors involved in this regulation are not well-understood. In this study, the effects of extracellular ATP on MC activation induced by MrgprB2, the mouse ortholog of human MRGPRX2, were examined in mouse peritoneal MCs (PMCs). ATP alone induced minimal PMC degranulation but markedly enhanced degranulation induced by the MrgprB2 agonist compound 48/80 (CP48/80), substance P, PAMP-12, and vancomycin. ATP promoted CP48/80-induced increase in intracellular Ca2+ in PMCs. This enhancement effect of ATP was absent in PMCs prepared from P2X4 receptor (P2X4R)-deficient mice and inhibited by the PI3K inhibitor wortmannin. In addition, P2X4R deficiency reduced the skin-specific and systemic anaphylactic responses to CP48/80 in vivo. In MC-deficient KitW-sh/W-sh mice, reconstitution with MCs obtained from wild-type mice led to a more severe anaphylactic response to CP48/80 compared to that from P2X4R-deficient mice. P2X4R-mediated effect may be involved in MrgprB2-mediated MC activation in vivo and is a potential target for alleviating pseudoallergic reactions.

Endothelial cell malfunction in unruptured intracranial aneurysm lesions revealed using a 3D-casted mold.

Intracranial aneurysms (IA) are major causes of devastating subarachnoid hemorrhages. They are characterized by a chronic inflammatory process in the intracranial arterial walls triggered and modified by hemodynamic force loading. Because IA lesion morphology is complex, the blood flow conditions loaded on endothelial cells in each portion of the lesion in situ vary greatly. We created a 3D-casted mold of the human unruptured IA lesion and cultured endothelial cells on this model; it was then perfused with culture media to model physiological flow conditions. Gene expression profiles of endothelial cells in each part of the IA lesion were then analyzed. Comprehensive gene expression profile analysis revealed similar gene expression patterns in endothelial cells from each part of the IA lesion but gene ontology analysis revealed endothelial cell malfunction within the IA lesion. Histopathological examination, electron microscopy, and immunohistochemical analysis indicated that endothelial cells within IA lesions are damaged and dysfunctional. Thus, our findings reveal endothelial cell malfunction in IA lesions and provided new insights into IA pathogenesis.

Synergistic Cytokine Production by ATP and PGE2 via P2X4 and EP3 Receptors in Mouse Bone-Marrow-Derived Mast Cells.

ATP is an important intercellular messenger in the extracellular space. In mast cells (MCs), ATP stimulates the ionotropic P2X4 receptor (P2X4R), resulting in enhanced degranulation and exacerbation of acute allergic reactions. In this study, we investigate whether ATP regulates inflammatory cytokine production in MCs. Gene expression was analyzed by quantitative RT-PCR, and cytokine production was measured using ELISA. The stimulation of mouse bone-marrow-derived MCs (BMMCs) with ATP alone had little effect on cytokine secretion. However, the co-stimulation with prostaglandin (PG) E2 resulted in a marked increase in the secretion of various cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and IL-13, accompanied by an increase in their mRNA levels. The effects of ATP were inhibited by P2X4R antagonists and diminished in BMMCs derived from P2X4R-deficient mice, suggesting that P2X4R mediated the reaction. The effects of PGE2 were mimicked by an EP3 receptor (EP3R) agonist and blocked by an EP3R antagonist. The synergistic cytokine mRNA elevations induced by ATP and PGE2 were blocked by nuclear factor-κB and Ca2+-calcineurin signaling inhibitors. Altogether, these results suggest that combining P2X4R and EP3R signaling enhances acute degranulation and the subsequent cytokine secretion, exacerbating allergic inflammation.

Hemodynamic Forces, Endothelial Mechanotransduction, and Vascular Diseases.

Cells in the tissues and organs of a living body are subjected to mechanical forces, such as pressure, friction, and tension from their surrounding environment. Cells are equipped with a mechanotransduction mechanism by which they perceive mechanical forces and transmit information into the cell interior, thereby causing physiological or pathogenetic mechano-responses. Endothelial cells (ECs) lining the inner surface of blood vessels are constantly exposed to shear stress caused by blood flow and a cyclic strain caused by intravascular pressure. A number of studies have shown that ECs are sensitive to changes in these hemodynamic forces and alter their morphology and function, sometimes by modifying gene expression. The mechanism of endothelial mechanotransduction has been elucidated, and the plasma membrane has recently been shown to act as a mechanosensor. The lipid order and cholesterol content of plasma membranes change immediately upon the exposure of ECs to hemodynamic forces, resulting in a change in membrane fluidity. These changes in a plasma membrane's physical properties affect the conformation and function of various ion channels, receptors, and microdomains (such as caveolae and primary cilia), thereby activating a wide variety of downstream signaling pathways. Such endothelial mechanotransduction works to maintain circulatory homeostasis; however, errors in endothelial mechanotransduction can cause abnormalities in vascular physiological function, leading to the initiation and progression of various vascular diseases, such as hypertension, thrombosis, aneurysms, and atherosclerosis. Recent advances in detailed imaging technology and computational fluid dynamics analysis have enabled us to evaluate the hemodynamic forces acting on vascular tissue accurately, contributing greatly to our understanding of vascular mechanotransduction and the pathogenesis of vascular diseases, as well as the development of new therapies for vascular diseases.

The ATP-hydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulation of P2X4 receptor-dependent metabolism in myeloid cells.

Extracellular adenosine triphosphate (ATP) released by mucosal immune cells and by microbiota in the intestinal lumen elicits diverse immune responses that mediate the intestinal homeostasis via P2 purinergic receptors, while overactivation of ATP signaling leads to mucosal immune system disruption, which leads to pathogenesis of intestinal inflammation. In the small intestine, hydrolysis of luminal ATP by ectonucleoside triphosphate diphosphohydrolase (E-NTPD)7 in epithelial cells is essential for control of the number of T helper 17 (Th17) cells. However, the molecular mechanism by which microbiota-derived ATP in the colon is regulated remains poorly understood. Here, we show that E-NTPD8 is highly expressed in large-intestinal epithelial cells and hydrolyzes microbiota-derived luminal ATP. Compared with wild-type mice, Entpd8-/- mice develop more severe dextran sodium sulfate-induced colitis, which can be ameliorated by either the depletion of neutrophils and monocytes by injecting with anti-Gr-1 antibody or the introduction of P2rx4 deficiency into hematopoietic cells. An increased level of luminal ATP in the colon of Entpd8-/- mice promotes glycolysis in neutrophils through P2x4 receptor-dependent Ca2+ influx, which is linked to prolonged survival and elevated reactive oxygen species production in these cells. Thus, E-NTPD8 limits intestinal inflammation by controlling metabolic alteration toward glycolysis via the P2X4 receptor in myeloid cells.

Mutations in a ß-group of solute carrier gene are responsible for egg and eye coloration of the brown egg 4 (b-4) mutant in the silkworm, Bombyx mori.

The brown egg 4 (b-4) is a recessive mutant in the silkworm (Bombyx mori), whose egg and adult compound eyes exhibit a reddish-brown color instead of normal purple and black, respectively. By double digest restriction-site associated DNA sequencing (ddRAD-seq) analysis, we narrowed down a region linked to the b-4 phenotype to approximately 1.1 Mb that contains 69 predicted gene models. RNA-seq analysis in a b-4 strain indicated that one of the candidate genes had a different transcription start site, which generates a short open reading frame. We also found that exon skipping was induced in the same gene due to an insertion of a transposable element in other two b-4 mutant strains. This gene encoded a putative amino acid transporter that belongs to the ß-group of solute carrier (SLC) family and is orthologous to Drosophila eye color mutant gene, mahogany (mah). Accordingly, we named this gene Bmmah. We performed CRISPR/Cas9-mediated gene knockout targeting Bmmah. Several adult moths in generation 0 (G0) had totally or partially reddish-brown compound eyes. We also established three Bmmah knockout strains, all of which exhibit reddish-brown eggs and adult compound eyes. Furthermore, eggs from complementation crosses between the b-4 mutants and the Bmmah knockout mutants also exhibited reddish-brown color, which was similar to the b-4 mutant eggs, indicating that Bmmah is responsible for the b-4 phenotypes.

An update of KAIKObase, the silkworm genome database.

KAIKObase was established in 2009 as the genome database of the domesticated silkworm Bombyx mori. It provides several gene sets and genetic maps as well as genome annotation obtained from the sequencing project of the International Silkworm Genome Consortium in 2008. KAIKObase has been used widely for silkworm and insect studies even though there are some erroneous predicted genes due to misassembly and gaps in the genome. In 2019, we released a new silkworm genome assembly, showing improvements in gap closure and covering more and longer gene models. Therefore, there is a need to include new genome and new gene models to KAIKObase. In this article, we present the updated contents of KAIKObase and the methods to generate, integrate and analyze the data sets. Database URL: https://kaikobase.dna.affrc.go.jp.

Functional lipidomics of vascular endothelial cells in response to laminar shear stress.

Laminar shear stress generated by blood flow stimulates endothelial cells and activates signal transduction, which plays an important role in vascular homeostasis. Several lines of evidence indicate that membrane and intracellular lipids are involved in the signal transduction of biomechanical stresses. In this study, we performed global profiling of cellular lipids from human pulmonary artery endothelial cells (HPAEC) exposed to laminar shear stress. A total of 761 species of lipids were successfully annotated, with 198 of these species significantly changed in response to shear stress for 24 hours. Ether-linked lipids containing an alkyl moiety with a medium chain length (C11-C14) were uniquely upregulated, and the administration of their biosynthetic precursor 1-O-dodecyl-rac-glycerol attenuated phorbol 12-myristate 13-acetate (PMA) induced vascular cell adhesion molecule-1 (VCAM-1) expression. Given the pro-inflammatory and atherogenic roles of VCAM-1, our findings suggest that the induction of a specific group of lipids (ie, ether-linked lipids with medium length alkyl side chain) may confer atheroprotective and anti-inflammatory roles to vascular endothelial cells under flow conditions.

Genome-wide assessment and development of molecular diagnostic methods for imidacloprid-resistance in the brown planthopper, Nilaparvata lugens (Hemiptera; Delphacidae).

BACKGROUND: The brown planthopper, Nilaparvata lugens (Stål), is one of the most notorious pests of rice throughout Asia. The brown planthopper has developed high resistance to imidacloprid, a member of neonicotinoid insecticides. Several genes and mutations conferring imidacloprid resistance in N. lugens, especially in eastern and southeastern Asia populations, have been reported. Thus, the key mechanisms of imidacloprid resistance need to be examined. RESULTS: RNA-seq analyses revealed that only one cytochrome P450 monooxygenase gene, CYP6ER1, was commonly upregulated in the five resistant strains tested. Sequences of CYP6ER1, which were highly expressed in the imidacloprid-resistant strains, contained a three-nucleotide deletion in the coding region, and amino acid substitutions and deletion, compared to that in an imidacloprid-susceptible strain. RNAi-mediated gene knockdown of CYP6ER1 increased imidacloprid susceptibility in a resistant strain. Further, we established two simple and convenient PCR-based molecular diagnostic methods to detect the CYP6ER1 locus with the three-nucleotide deletion. Using these methods, the resistance of F2 progenies derived from the crosses of F1 siblings from susceptible and resistant parents was analyzed, showing that the imidacloprid resistance had a relationship to the CYP6ER1 locus with the three-nucleotide deletion. CONCLUSION: The overexpression of a variant CYP6ER1 with amino acid substitutions and deletion was involved in imidacloprid resistance in N. lugens. Based on these findings, molecular diagnostic methods have been developed and are promising tools for monitoring imidacloprid resistance in paddy fields. © 2020 Society of Chemical Industry.

Shear stress activates mitochondrial oxidative phosphorylation by reducing plasma membrane cholesterol in vascular endothelial cells.

Vascular endothelial cells (ECs) sense and respond to hemodynamic shear stress, which is critical for circulatory homeostasis and the pathophysiology of vascular diseases. The mechanisms of shear stress mechanotransduction, however, remain elusive. We previously demonstrated a direct role of mitochondria in the purinergic signaling of shear stress: shear stress increases mitochondrial adenosine triphosphate (ATP) production, triggering ATP release and Ca2+ signaling via EC purinoceptors. Here, we showed that shear stress rapidly decreases cholesterol in the plasma membrane, thereby activating mitochondrial ATP production. Imaging using domain 4 mutant-derived cholesterol biosensors showed that the application of shear stress to cultured ECs markedly decreased cholesterol levels in both the outer and inner plasma membrane bilayers. Flow cytometry showed that the cholesterol levels in the outer bilayer decreased rapidly after the onset of shear stress, reached a minimum (around 60% of the control level) at 10 min, and plateaued thereafter. After the shear stress ceased, the decreased cholesterol levels returned to those seen in the control. A biochemical analysis showed that shear stress caused both the efflux and the internalization of plasma membrane cholesterol. ATP biosensor imaging demonstrated that shear stress significantly increased mitochondrial ATP production. Similarly, the treatment of cells with methyl-ß-cyclodextrin (MßCD), a membrane cholesterol-depleting agent, increased mitochondrial ATP production. The addition of cholesterol to cells inhibited the increasing effects of both shear stress and MßCD on mitochondrial ATP production in a dose-dependent manner. These findings indicate that plasma membrane cholesterol dynamics are closely coupled to mitochondrial oxidative phosphorylation in ECs.

Extracellular ATP Augments Antigen-Induced Murine Mast Cell Degranulation and Allergic Responses via P2X4 Receptor Activation.

Extracellular ATP released from stimulated and/or damaged cells modulates physiological responses via stimulation of various purinoceptors. We previously showed that ATP potentiated the Ag-induced mast cell (MC) degranulation via purinoceptors pharmacologically similar to the ionotropic P2X4 receptor. In this study, we investigated the role of P2X4 receptor in MC degranulation induced by stimulation of IgE-FcεRI complex with Ag, using bone marrow-derived MCs (BMMCs) prepared from wild type and P2X4 receptor-deficient (P2rx4-/- ) mice. ATP significantly increased Ag-induced degranulation in BMMCs prepared from wild type mice. This effect of ATP was reduced in BMMCs prepared from P2rx4-/- mice. The potentiating effect of ATP was restored by expressing P2X4 receptor in P2rx4-/- BMMCs. The P2X4 receptor-mediated effects were maintained even after differentiating into the connective tissue-type MCs. P2X4 receptor stimulation did not affect the Ag-induced Ca2+ response but enhanced Ag-induced early signals, such as tyrosine phosphorylation of Syk and phospholipase C-γ. Interestingly, these effects of ATP on Syk phosphorylation were not impaired by pretreatment with Cu2+, an inhibitor of the P2X4 receptor channel, or removal of external Ca2+, suggesting that a mechanisms other than Ca2+ influx through ion channel activity may be involved. In vivo experiments revealed that systemic and intradermal passive anaphylaxis responses were significantly alleviated in P2rx4-/- mice. Taken together, the present data suggest that the P2X4 receptor plays an essential role in ATP-induced upregulation of MC degranulation in response to Ag, and also contributes to the Ag-induced allergic response in vivo.

Two Diverse Hemodynamic Forces, a Mechanical Stretch and a High Wall Shear Stress, Determine Intracranial Aneurysm Formation.

Intracranial aneurysm (IA) usually induced at a bifurcation site of intracranial arteries causes a lethal subarachnoid hemorrhage. Currently, IA is considered as a macrophage-mediated inflammatory disease triggered by a high wall shear stress (WSS) on endothelial cells. However, considered the fact that a high WSS can be observed at every bifurcation site, some other factors are required to develop IAs. We therefore aimed to clarify mechanisms underlying the initiation of IAs using a rat model. We found the transient outward bulging and excessive mechanical stretch at a prospective site of IA formation. Fibroblasts at the adventitia of IA walls were activated and produced (C-C motif) ligand 2 (CCL2) as well in endothelial cells loaded on high WSS at the earliest stage. Consistently, the mechanical stretch induced production of CCL2 in primary culture of fibroblasts and promoted migration of macrophages in a Transwell system. Our results suggest that distinct hemodynamic forces, mechanical stretch on fibroblasts and high WSS on endothelial cells, regulate macrophage-mediated IA formation.

Disruption of P2X4 purinoceptor and suppression of the inflammation associated with cerebral aneurysm formation.

OBJECTIVE: There are no effective therapeutic drugs for cerebral aneurysms, partly because the pathogenesis remains unresolved. Chronic inflammation of the cerebral arterial wall plays an important role in aneurysm formation, but it is not clear what triggers the inflammation. The authors have observed that vascular endothelial P2X4 purinoceptor is involved in flow-sensitive mechanisms that regulate vascular remodeling. They have thus hypothesized that shear stress-associated hemodynamic stress on the endothelium causes the inflammatory process in the cerebral aneurysm development. METHODS: To test their hypothesis, the authors examined the role of P2X4 in cerebral aneurysm development by using P2X4-/- mice and rats that were treated with a P2X4 inhibitor, paroxetine, and subjected to aneurysm-inducing surgery. Cerebral aneurysms were induced by unilateral carotid artery ligation and renovascular hypertension. RESULTS: The frequency of aneurysm induction evaluated by light microscopy was significantly lower in the P2X4-/- mice (p = 0.0488) and in the paroxetine-treated male (p = 0.0253) and female (p = 0.0204) rats compared to control mice and rats, respectively. In addition, application of paroxetine from 2 weeks after surgery led to a significant reduction in aneurysm size in the rats euthanized 3 weeks after aneurysm-inducing surgery (p = 0.0145), indicating that paroxetine suppressed enlargement of formed aneurysms. The mRNA and protein expression levels of known inflammatory contributors to aneurysm formation (monocyte chemoattractant protein-1 [MCP-1], interleukin-1ß [IL-1ß], tumor necrosis factor-α [TNFα], inducible nitric oxide synthase [iNOS], and cyclooxygenase-2 [COX-2]) were all significantly elevated in the rats that underwent the aneurysm-inducing surgery compared to the nonsurgical group, and the values in the surgical group were all significantly decreased by paroxetine administration according to quantitative polymerase chain reaction techniques and Western blotting. Although immunolabeling densities for COX-2, iNOS, and MCP-1 were not readily observed in the nonsurgical mouse groups, such densities were clearly seen in the arterial wall of P2X4+/+ mice after aneurysm-inducing surgery. In contrast, in the P2X4-/- mice after the surgery, immunolabeling of COX-2 and iNOS was not observed in the arterial wall, whereas that of MCP-1 was readily observed in the adventitia, but not the intima. CONCLUSIONS: These data suggest that P2X4 is required for the inflammation that contributes to both cerebral aneurysm formation and growth. Enhanced shear stress-associated hemodynamic stress on the vascular endothelium may trigger cerebral aneurysm development. Paroxetine may have potential for the clinical treatment of cerebral aneurysms, given that this agent exhibits efficacy as a clinical antidepressant.

Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (FcεRI) and trigger type I allergic reactions. FcεRI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced FcεRI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E2 (PGE2) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE2-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE2 on MCs may be involved in Ag-independent hypersensitivity in vivo.

A biomechanically derived minimum work model of the fish gill lamellar system exhibits its exquisite morphological arrangement and perfusate regulation for oxygen uptake from water.

To evaluate the efficiency of oxygen (O2) uptake from water through the fish gill lamellar system, a cost function (CF) representing mechanical power expenditure for water ventilation and blood circulation through the gill was formulated, by applying steady-state fluid mechanics to a homogeneous lamellar-channel model. This approach allowed us to express CF as the function of inter-lamellar water channel width (w) and to derive an analytical solution of the width (wmin) at the minimum CF. Morphometric and physiological data for rainbow trout in the literature were referred to calculate CF(w) curves and their wmin values at five intensity stages of swimming exercise. Obtained wmin values were evenly distributed around the standard measure of the width (ws = 24 µm) in this fish. Individual levels of CF(wmin) were also fairly close to the corresponding CF(ws) values within a 10% deviation, suggesting the reliability of approximating [CF(wmin) = CF(ws)]. The cost-performance of O2 uptake through the gill (ηg) was then assessed from reported data of total O2 uptake/CF(ws) at each intensity stage. The ηg levels at any swimming stage exceeded 95% of the theoretical maximum value, implying that O2 uptake is nearly optimally performed in the lamellar-channel system at all swimming speeds. Further analyses of O2 transport in this fresh water fish revealed that the water ventilation by the buccal/opercular pumping evokes a critical limit of swimming velocity, due to confined O2 supply to the peripheral skeletal muscles, which is avoided in ram ventilators such as tuna.

High-quality genome assembly of the silkworm, Bombyx mori.

In 2008, the genome assembly and gene models for the domestic silkworm, Bombyx mori, were published by a Japanese and Chinese collaboration group. However, the genome assembly contains a non-negligible number of misassembled and gap regions due to the presence of many repetitive sequences within the silkworm genome. The erroneous genome assembly occasionally causes incorrect gene prediction. Here we performed hybrid assembly based on 140 × deep sequencing of long (PacBio) and short (Illumina) reads. The remaining gaps in the initial genome assembly were closed using BAC and Fosmid sequences, giving a new total length of 460.3 Mb, with 30 gap regions and an N50 comprising 16.8 Mb in scaffolds and 12.2 Mb in contigs. More RNA-seq and piRNA-seq reads were mapped on the new genome assembly compared with the previous version, indicating that the new genome assembly covers more transcribed regions, including repetitive elements. We performed gene prediction based on the new genome assembly using available mRNA and protein sequence data. The number of gene models was 16,880 with an N50 of 2154 bp. The new gene models reflected more accurate coding sequences and gene sets than old ones. The proportion of repetitive elements was also reestimated using the new genome assembly, and was calculated to be 46.8% in the silkworm genome. The new genome assembly and gene models are provided in SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp).