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Abstract Habitat transitions in living organisms are key innovations often coupled with species diversification after their successful adaptation to new environment. The Cyrenidae is among the most well-known heterodont bivalve groups that have successfully invaded freshwater systems from brackish water environments and display diverse lineage-specific developmental modes. Phylogenetic and molecular clock-based divergence time analyses using 12 complete mitochondrial genome sequences suggest that Cyrenidae species independently colonized freshwater habitats during three distinct spatial and geological periods: one from the American continents approximately in the Early Jurassic and the two others from Australasian/East Asian continents in the Early/Middle Cretaceous and the Paleogene-Neogene boundary, respectively. This study provides significant insight into the temporal and spatial patterns of multiple freshwater invasions, aligning with ancient vicariance events inferred from different geological timelines of plate tectonics. Additionally, mitogenome phylogeny confirms the earlier hypothesis of the repeated parallel evolution of parental care system within this bivalve group.

Expression of α‐smooth muscle actin (αSMA) is constitutive in vascular smooth muscle cells, but is induced in nonmuscle cells such as hepatic stellate cells (HSCs). HSCs play important roles in both physiological homeostasis and pathological response. HSC activation is characterized by αSMA expression, which is regulated by the TGFβ‐induced Smad pathway. Recently, protein kinase C (PKC) was identified to regulate αSMA expression. Diacylglycerol kinase (DGK) metabolizes a second‐messenger DG, thereby controlling components of DG‐mediated signaling, such as PKC. In the present study we aimed to investigate the putative role of DGKα in αSMA expression. Use of a cellular model indicated that the DGK inhibitor R59949 promotes αSMA expression and PKCδ phosphorylation. It also facilitates Smad2 phosphorylation after 30 min of TGFβ stimulation. Furthermore, immunocytochemical analysis revealed that DGK inhibitor pretreatment without TGFβ stimulation engenders αSMA expression in a granular pattern, whereas DGK inhibitor pretreatment plus TGFβ stimulation significantly induces αSMA incorporation in stress fibers. Through animal model experiments, we observed that DGKα‐knockout mice exhibit increased expression of αSMA in the liver after 48 h of carbon tetrachloride injection, together with enhanced phosphorylation levels of Smad2 and PKCδ. Together, these findings suggest that DGKα negatively regulates αSMA expression by acting on the Smad and PKCδ signaling pathways, which differentially regulate stress fiber incorporation and protein expression of αSMA, respectively.

Abstract Ellobium chinense is an airbreathing, pulmonate gastropod species that inhabits saltmarshes in estuaries of the northwestern Pacific. Due to a rapid population decline and their unique ecological niche in estuarine ecosystems, this species has attracted special attention regarding their conservation and the genomic basis of adaptation to frequently changing environments. Here we report a draft genome assembly of E. chinense with a total size of 949.470 Mb and a scaffold N50 of 1.465 Mb. Comparative genomic analysis revealed that the GO terms enriched among four gastropod species are related to signal transduction involved in maintaining electrochemical gradients across the cell membrane. Population genomic analysis using the MSMC model for 14 re-sequenced individuals revealed a drastic decline in Korean and Japanese populations during the last glacial period, while the southern Chinese population retained a much larger effective population size (N e ). These contrasting demographic changes might be attributed to multiple environmental factors during the glacial–interglacial cycles. This study provides valuable genomic resources for understanding adaptation and historical demographic responses to climate change.

Brittle stars (class Ophiuroidea) are marine invertebrates comprising approximately 2,100 extant species, and are considered to constitute the most diverse taxon of the phylum Echinodermata. As a non-invasive method for monitoring biodiversity, we developed two new sets of PCR primers for metabarcoding environmental DNA (eDNA) from brittle stars. The new primer sets were designed to amplify 2 short regions of the mitochondrial 16S rRNA gene, comprising a conserved region (111–115 bp, 112 bp on average; named “16SOph1”) and a hyper-variable region (180–195 bp, 185 bp on average; named “16SOph2”) displaying interspecific variation. The performance of the primers was tested using eDNA obtained from two sources: a) rearing water of an 2.5 or 170 L aquarium tanks containing 15 brittle star species and b) from natural seawater collected around Misaki, the Pacific coast of central Japan, at depths ranging from shallow (2 m) to deep (> 200 m) sea. To build a reference library, we obtained 16S rRNA sequences of brittle star specimens collected from around Misaki and from similar depths in Japan, and sequences registered in International Nucleotide Sequence Database Collaboration. As a result of comparison of the obtained eDNA sequences with the reference library 37 (including cryptic species) and 26 brittle star species were detected with certain identities by 16SOph1 and 16SOph2 analyses, respectively. In shallow water, the number of species and reads other than the brittle stars detected with 16SOph1 was less than 10% of the total number. On the other hand, the number of brittle star species and reads detected with 16SOph2 was less than half of the total number, and the number of detected non-brittle star metazoan species ranged from 20 to 46 species across 6 to 8 phyla (only the reads at the “Tank” were less than 0.001%). The number of non-brittle star species and reads at 80 m was less than 10% with both of the primer sets. These findings suggest that 16SOph1 is specific to the brittle star and 16SOph2 is suitable for a variety of marine metazoans. It appears, however, that further optimization of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts. Moreover, a detailed elucidation of the brittle star fauna in the examined area, and the accurate identification of brittle star species in the current DNA databank is required.

The subclass Patellogastropoda (called “true limpets”) is one of the most primitive groups of the Gastropoda and contains approximately 350 species worldwide. Within this subclass, internal phylogeny among family members, including relationships of the “Acmaeidae” with other patellogastropod families, remains incompletely clarified. Here, we newly determined two complete mitochondrial genome sequences of “Acmaeidae” (Acmaea mitra and Niveotectura pallida) and one sequence from Lottiidae species (Discurria insessa) and combined them with mitochondrial genome sequences of 20 other published limpet species for phylogenetic analysis of the sequence dataset (nucleotides and amino acids) of 13 protein-coding genes using maximum likelihood and Bayesian inference methods. The resulting phylogenetic trees showed monophyly of Patellogastropoda species that were subsequently subdivided into two clades [clade I (Nacellidae, Pectinodontidae, Acmaeidae, and Patellidae) and clade II (Eoacmaeidae and Lottiidae)]. The sister relationship between the Acmaeidae and Pectinodontidae species revealed by phylogenetic analysis was also supported by sharing their similar gene arrangement patterns, which differ substantially from those of clade II members including the Lottiidae species. The polyphyletic relationship between Acmaeidae (grouped with Pectinodontidae as a sister taxon in clade I) and Lottiidae species (grouped with Eoacmaeidae in clade II) corroborates that they are phylogenetically distinct from each other. This mitochondrial genome phylogeny contradicts previous morphology-based hypotheses, yet highlights that Acmaeidae and Pectinodontidae are the most closely related. Further in-depth analysis of the complete mitochondrial genome sequences based on a broad range of samples including those from relatively unstudied and/or underrepresented taxa is required to fully understand the mitochondrial genome evolution and a more comprehensive phylogeny among the major groups of the Patellogastropoda.

The patellogastropod limpet genus Nipponacmea is widely distributed in Japan and adjacent East Asia. Species identification within Nipponacmea is challenging due to the high variation in shell morphology. In this study, we examined the taxonomy of this genus represented by nine nominal species from 43 localities (including type localities). Results of the molecular phylogenetic analysis revealed that: (1) N. gloriosa, the sole species in this genus inhabiting the subtidal zone, represents the most basal independent branch; (2) the remaining species are divided into two large clades with lower- and higher-apex shell profiles; and (3) the high-apex morphology was derived from the low-apex type. The terminal clades defined using the molecular data were consistent with nine morphospecies and had 100% bootstrap values, strongly supporting the conventional taxonomy of Nipponacmea. Although morphological similarities do not always reflect phylogeny, the set of morphological characters used in the current taxonomy were proven to be adequate for diagnosis. In conclusion, this study provided solid evidence to uphold the monophyly of known species of Nipponacmea in Japan and demonstrated the usefulness of morphological characters for species diagnosis.

Abstract Background Secondary pneumothorax after chemotherapy for a malignant pulmonary tumor is a complication from a large cavity causing refractory pneumothorax. Case presentation A 61-year-old man was referred due to prolonged air leakage from a pulmonary cavity that developed after treatment for pulmonary metastases from renal cell carcinoma. As air leakage continued after thoracic drainage and endobronchial occlusion, we planned thoracoscopy-assisted surgery. Intraoperatively, a large cavity opening to the pulmonary cavity was found in the left upper lobe. As it was difficult to repair the fistula using staplers or direct sutures because the pleura around the cavity was thick and hard, we attempted to plombage the cavity with a pericardial fat pad. After the operation, air leakage immediately disappeared and no recurrence of the pneumothorax was found. Conclusion This novel method can be useful to seal a large bronchopleural fistula that causes refractory pneumothorax.

Diacylglycerol (DG) is unique in lipid metabolism because it serves not only as an intermediate product for triglyceride synthesis, but also as a signaling molecule that activates proteins containing DG-responsive elements, such as protein kinase C. Consequently, DG acts as a hub between energy metabolism and intracellular signaling. Of DG metabolizing pathways, DG kinase (DGK) phosphorylates DG to produce phosphatidic acid, which also serves as a second messenger. Several lines of evidence suggest that DGK is deeply involved in metabolic diseases such as obesity and insulin resistance. Of DGK isozymes, DGKε is simplest in terms of structure, but it is characterized by substrate specificity toward arachidonoyl-DG. Recently, we have reported that DGKε deficiency promotes adipose tissue remodeling in mice during the course of high fat diet (HFD) feeding regimen including obesity, insulin resistance, and beige adipogenesis. DGKε ablation engenders altered expression of other lipid metabolizing enzymes, including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and diacylglycerol acyltransferase (DGAT). Subcellular localization of DGKε in the endoplasmic reticulum suggests involvement of this isozyme in lipid energy homeostasis. This review presents current findings of DGKε in lipid-orchestrated pathophysiology, especially unique phenotypes of DGKε-knockout mice in the early and late stages of obesogenic conditions.

The aim of the present study was to determine which individual or combined CpG sites among O6-methylguanine DNA methyltransferase CpG 74–89 in glioblastoma mainly affects the response to temozolomide resulting from CpG methylation using statistical analyses focused on the tumor volume ratio (TVR). We retrospectively examined 44 patients who had postoperative volumetrically measurable residual tumor tissue and received adjuvant temozolomide therapy for at least 6 months after initial chemoradiotherapy. TVR was defined as the tumor volume 6 months after the initial chemoradiotherapy divided by that before the start of chemoradiotherapy. Predictive values for TVR as a response to adjuvant therapy were compared among the averaged methylation percentages of individual or combined CpGs using the receiver operating characteristic curve. Our data revealed that combined CpG 78 and 79 showed a high area under the curve (AUC) and a positive likelihood ratio and that combined CpG 76–79 showed the highest AUC among all combinations. AUCs of consecutive CpG combinations tended to be higher for CpG 74–82 in exon 1 than for CpG 83–89 in intron 1. In conclusion, the methylation status at CpG sites in exon 1 was strongly associated with TVR reduction in glioblastoma.

Abstract Background Thymomas are typically slow-growing tumors and AB type thymomas are considered no/low risk tumors with a better prognosis. Extra-thoracic metastases are extremely rare. To the best of our knowledge, no patient with an isolated splenic metastasis from a thymoma has been reported. We report a patient who underwent laparoscopic splenectomy for a slow-growing, isolated splenic metastasis, eight years after thymectomy. Case presentation The patient is a 78-year-old man. Eight years previously, the patient underwent extended thymectomy and postoperative radiation therapy for a thymoma. Five years after thymectomy, a nodule appeared in the spleen, and the lesion enlarged gradually for three years thereafter. The patient was referred for further examination and treatment. Computed tomography scan showed a sharply circumscribed 50 mm tumor slightly hypodense and heterogeneous lesion in the spleen. On T2-weighted images on Magnetic Resonance Imaging, the tumor had high intensity, equivalent to or slightly lower than that on T1-weighted images, and no decrease on diffusion-weighted images. The tumor was multinodular and showed a low-signal spoke-wheel sign in the margin, enhanced gradually in the dynamic study. Positron emission tomography-CT scan, showed relatively low accumulation. Surgical resection was undertaken, and pathological examination showed metastatic thymoma. The patient is without recurrence and has no other symptoms three years after splenectomy. Conclusions This is the first report of an isolated splenic metastasis from a thymoma. Further cases are needed to standardize this surgery for such lesions.

Recently developed molecular targeted therapies such as bevacizumab (BEV; Avastin) therapy have therapeutic efficacy for glioblastoma. However, it is difficult to distinguish between a tumor response and nonenhancing tumor progression with conventional magnetic resonance imaging (MRI) after BEV administration. Here we present a recurrent glioblastoma case in which 11C-methionine positron emission tomography (MET-PET) provided useful information for detecting tumor recurrence after complete remission, as assessed by the Response Assessment in Neuro-Oncology criteria. A 47-year-old male with a left frontal lobe glioblastoma experienced recurrence 6 months postoperatively. We administered BEV concomitantly with temozolomide, subsequent to gamma knife surgery. Two months after starting BEV, complete remission was obtained. MET uptake on PET gradually decreased and had nearly disappeared 4 months after initiating BEV. No enhanced area was seen on MRI for 17 months after BEV initiation. Nevertheless, MET-PET revealed recurrence, visualized as nonenhancing tumor progression. MET-PET provides useful information for detecting glioblastoma recurrence, which lacks contrast enhancement on MRI after BEV therapy.

Abstract Background Preoperative evaluation and awareness of anatomical variations in the pulmonary vessel is essential for a secure pulmonary resection. We herein present a patient who underwent complex pulmonary resection for lung cancer with a mediastinal lingular and basal pulmonary artery that had been detected by preoperative three-dimensional computed tomography. Case presentation The patient was an asymptomatic 66-year-old woman who had a 39-pack-year smoking habit. Chest computed tomography (CT) revealed the tumor invading the left upper bronchus and pulmonary artery branches in the left upper lung lobe. Enhanced CT and three-dimensional (3D) images of the pulmonary artery revealed that pulmonary artery branches (A4 + 5, A8, and A9 + 10) were extending into the lingular and basal segment in ventral side of the left upper bronchus. We completed the resection by means of a composite resection of the left upper lobe and the superior segment of the lower lobe, avoiding pulmonary angioplasty to preserve the left lower lobe or pneumonectomy. Conclusions 3D-CT is useful for detecting this rare variation of the left pulmonary artery before operation, allowing for proper resection.

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3-4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.

Oceanic islands lacking connections to other land are extremely isolated from sources of potential colonists and have acquired their biota mainly through dispersal from geographically distant areas. Hence, isolated island biota constitutes interesting models to infer biogeographical mechanisms of dispersal, colonization, differentiation, and speciation. Limpets of the genus Cellana (Nacellidae: Patellogastropoda) show limited dispersal capacity but are broadly distributed across the Indo-Pacific including many endemic species in isolated oceanic islands. Here, we examined main distributional patterns and geographic boundaries among Cellana lineages with special emphasis in the relationships of Southern Hemisphere oceanic islands species. Phylogenetic reconstructions based on mtDNA (COI) recognized three main clades in Cellana including taxa from different provinces of the Indo-Pacific. Clear genetic discontinuities characterize the biogeography of Cellana and several lineages are associated to particular areas of the Indo-Pacific supporting the low dispersal capacity of the genus across recognized biogeographical barriers in the region. However, evolutionary relationships within Cellana suggest that long-distance dispersal processes have been common in the history of the genus and probably associated to the origin of the species in Hawaii and Juan Fernández Archipelago. Therefore, the presence of Cellana species in geographically distant Southern Hemisphere oceanic islands, such as the Juan Fernández Archipelago, suggests that long-distance dispersal mediated by rafting may have played an important role in the biogeography of the genus.

The endoplasmic reticulum (ER) comprises an interconnected membrane network, which is made up of lipid bilayer and associated proteins. This organelle plays a central role in the protein synthesis and sorting. In addition, it represents the synthetic machinery of phospholipids, the major constituents of the biological membrane. In this process, phosphatidic acid (PA) serves as a precursor of all phospholipids, suggesting that PA synthetic activity is closely associated with the ER function. One enzyme responsible for PA synthesis is diacylglycerol kinase (DGK) that phosphorylates diacylglycerol (DG) to PA. DGK is composed of a family of enzymes with distinct features assigned to each isozyme in terms of structure, enzymology and subcellular localization. Of DGKs, DGKε uniquely exhibits substrate specificity toward arachidonate-containing DG and is shown to reside in the ER. Arachidonic acid, a precursor of bioactive eicosanoids, is usually acylated at the sn-2 position of phospholipids, being especially enriched in phosphoinositide. In this review, we focus on arachidonoyl-specific DGKε with respect to the historical context, molecular basis of the substrate specificity and ER-targeting, and functional implications in the ER.

Localization of cyclooxygenase-2 (COX-2) in rat aortae was investigated. In endothelium-intact aortic strips stimulated with interleukin-1 β (IL-1 β ), COX-2 was detected in adventitial fibroblasts and macrophages but not in tunica media. IL-1 β−induced COX-2 expression was also detected in medial smooth muscle cells of endothelium-denuded aortae and cultured rat aortic smooth muscle cells. IL-1β−induced COX-2 expression in all of the above cells was abolished in the presence of U0126, an inhibitor of extracellular signal-regulated kinases (ERK). Thus, IL-1β induces COX-2 through the ERK-mediated pathway in adventitial fibroblasts and macrophages of healthy aortae; and in addition, medial smooth muscle cells are also responsible for COX-2 expression in remodeled aortae. Keywords:: cyclooxygenase-2, remodeling, vascular smooth muscle

The aim of this study was to determine whether the pathway of inducible NO synthase (iNOS) in blood vessels is changed by type 2 diabetes. Lipopolysaccharide (LPS)-induced nitric oxide (NO) production and expression of iNOS and effects of LPS on phenylephrine-induced contractile force were compared in aortae isolated from Goto-Kakizaki (G-K) diabetes rats and aortae isolated from control Wistar rats. Both LPS-stimulated nitrite generation and iNOS expression levels were significantly higher in aortae from G-K rats than in those from control rats. Phenylephrine-induced contractile force in the presence of LPS was significantly lower in aortae from G-K rats than in those from control rats, while contractile force in the absence of LPS was comparable in the diabetic and control groups. On the other hand, incubation of aortae in high glucose-containing medium did not affect the LPS-stimulated nitrite accumulation and iNOS expression and the phenylephrine-induced contractile force, regardless of the presence of LPS. These results suggest that LPS-induced NO production through the iNOS pathway is increased and subsequent attenuation of contractile force by excess NO is enhanced in arteries of rats with type 2 diabetes. Keywords:: artery, glucose, lipopolysaccharide, nitric oxide synthase, type 2 diabetes

The present-day genetic structure of a species reflects both historical demography and patterns of contemporary gene flow among populations. To precisely understand how these factors shape current population structure of the northwestern (NW) Pacific marine gastropod, Thais clavigera, we determined the partial nucleotide sequences of the mitochondrial COI gene for 602 individuals sampled from 29 localities spanning almost the whole distribution of T. clavigera in the NW Pacific Ocean (~3,700 km). Results from population genetic and demographic analyses (AMOVA, ΦST-statistics, haplotype networks, Tajima's D, Fu's FS, mismatch distribution, and Bayesian skyline plots) revealed a lack of genealogical branches or geographical clusters, and a high level of genetic (haplotype) diversity within each of studied population. Nevertheless, low but significant genetic structuring was detected among some geographical populations separated by the Changjiang River, suggesting the presence of geographical barriers to larval dispersal around this region. Several lines of evidence including significant negative Tajima's D and Fu's FS statistics values, the unimodally shaped mismatch distribution, and Bayesian skyline plots suggest a population expansion at marine isotope stage 11 (MIS 11; 400 ka), the longest and warmest interglacial interval during the Pleistocene epoch. The lack of genetic structure among the great majority of the NW Pacific T. clavigera populations may be attributable to high gene flow by current-driven long-distance dispersal of prolonged planktonic larval phase of this species.

Genetic data were used to measure the phylogeographic distribution of the limpet, Cellana toreuma along the China coast in order to acsertain impacts of historic events, ocean currents and especially freshwater discharge from the Yangtze River on the connectivity of intertidal species with limited larval dispersal capability.Genetic variation in 15 populations of C. toreuma (n = 418), ranging from the Yellow Sea (YS), East China Sea (ECS) and South China Sea (SCS), were determined from partial mitochondrial cytochrome c oxidase subunit I gene. Genetic diversity and divergence based on haplotype frequencies were analyzed using CONTRIB, and AMOVA was used to examine genetic population structure. Historic demographic expansions were evaluated from both neutrality tests and mismatch distribution tests. Among the 30 haplotypes identified, a dominant haplotype No. 1 (H1) existed in all the populations, and a relatively abundant private haplotype (H2) in YS. Pairwise F(ST) values between YS and the other two groups were relatively high and the percentage of variation among groups was 10.9%.The high nucleotide and gene diversity in the YS, with large pairwise genetic distances and relatively high percentages of variation among groups, suggests that this group was relatively isolated from ECS and SCS. This is likely driven by historic events, ocean currents, and demographic expansion. We propose that freshwater discharge from the Yangtze River, which may act as physical barrier limiting the southward dispersal of larvae from northern populations, is especially important in determining the separation of the YS group from the rest of the Chinese populations of C. toreuma.

This document is an editorial published in the journal Frontiers in Marine Science. It discusses the biology, ecology, exploitation, and sustainability of true limpets, a group of marine gastropods. Limpets are globally distributed and play a significant role in influencing biological communities on rocky substrata. They are also exploited for various human activities, which has led to sustainability risks for many limpet populations. The editorial highlights recent advances in aquaculture and settlement methodologies that can contribute to the conservation and potential restocking of limpet species. The document also provides an overview of the research articles included in the Research Topic on true limpets, covering aspects such as taxonomy, conservation, and ecology. The collaborative efforts of researchers from different continents contribute to our understanding of these marine gastropods and inform conservation and management strategies. [Extracted from the article]