Theama japonica sp. nov., an Interstitial Polyclad Flatworm Showing a Wide Distribution along Japanese Coasts.

We establish a new interstitial polyclad species, Theama japonica sp. nov., based on specimens collected from coarse-sandy habitats in three Japanese main islands (Hokkaido, Honshu, and Shikoku) along the coasts of the Pacific Ocean and the Sea of Japan. Theama japonica is characterized by i) two pairs of cerebral eyespots and four to six precerebral eyespots; ii) eosinophilic secretion glands distributed in the distal half of the inner ventral part of the prostatic vesicle; iii) a conical penis papilla, bent up dorsally, with a sclerotized inner wall; iv) the prostatic sheath with an inner angular fold on the dorso-distal side; and v) the external cilia longer dorsally than ventrally. Partial sequences of the cytochrome c oxidase subunit I (COI) gene from 20 specimens collected at eight localities along Japanese coasts represented 19 haplotypes. The uncorrected p-distances among these COI haplotypes fell within intraspecific variations observed in other polyclads. A network analysis based on these COI haplotypes suggested a geographically non-cohesive genetic structure of the species, possibly indicating the species' high dispersibility. Molecular phylogenetic analyses based on a concatenated dataset of 18S and 28S rDNA sequences showed T. japonica formed a clade with other Theama species. The resulting tree also indicates that our new species is more closely related to Theama sp. from Colombia than species from Panama and Croatia.

Molecular Phylogenetic Analysis of Acotylea (Platyhelminthes: Polycladida).

Acotylea is a suborder of Polycladida (Rhabditophora, Platyhelminthes) characterized by lack of a cotyl (sucker-like structure) on the ventral surface of the body. We newly determined partial sequences of two mitochondrial (16S ribosomal RNA and cytochrome c oxidase subunit I) and two nuclear (18S and 28S ribosomal RNA) genes from 24 acotylean species (12 families and 14 genera). Based on these sequences in addition to those available in public databases, we inferred the phylogeny of 16 families and 27 genera of Acotylea from molecular phylogenetic analyses (maximum likelihood and Bayesian inference) based on concatenated gene sequences. Our analyses supported three clades corresponding to Discoceloidea, Leptoplanoidea, and Stylochoidea. The phylogenetic position of Callioplanidae remains unclear. Among family- or genus-level taxa, Gnesiocerotidae, Stylochoplanidae, and Comoplana were not monophyletic. We discuss the validities of Notocomplanidae and Koinostylochus, and the family-level assignment of Mirostylochus.

The duodenal tube test is more specific than hepatobiliary scintigraphy for identifying bile excretion in the differential diagnosis of biliary atresia.

PURPOSE: Confirmation of bile excretion into the gastrointestinal tract is important to exclude biliary atresia (BA). We compared the duodenal tube test (DTT) with hepatobiliary scintigraphy (HS) for their efficiency in detecting bile secretion. METHODS: The subjects of this retrospective study were 47 infants who underwent both DTT and HS to diagnose or exclude BA between January 2000 and March 2018. RESULTS: BA was diagnosed in 32 of the 47 patients, and 7 of the remaining 15 non-BA patients underwent intraoperative cholangiography. Among the various DTT parameters, the total bile acid in duodenal fluid (DF-TBA)/serum (S) gamma-glutamyl transferase (γGTP) ratio was found to be the most specific for BA, with sensitivity and specificity of 98.0-100%, respectively. One BA patient in whom cut off values were not met was a premature infant. The sensitivity and specificity of HS were 100-56.3%, respectively. The diagnostic accuracy of the DF-TBA/S-γGTP parameter was higher than that of HS (98.6% vs. 85.1%, respectively). CONCLUSIONS: The DTT could be more a specific method than HS to detect bile excretion. Thus, the DTT should be incorporated into the multidisciplinary diagnostic approach for the differential diagnosis of BA to prevent unnecessary intraoperative cholangiography in patients who do not have BA.

SOCS2 Balances Metabolic and Restorative Requirements during Liver Regeneration.

After significant injury, the liver must maintain homeostasis during the regenerative process. We hypothesized the existence of mechanisms to limit hepatocyte proliferation after injury to maintain metabolic and synthetic function. A screen for candidates revealed suppressor of cytokine signaling 2 (SOCS2), an inhibitor of growth hormone (GH) signaling, was strongly induced after partial hepatectomy. Using genetic deletion and administration of various factors we investigated the role of SOCS2 during liver regeneration. SOCS2 preserves liver function by restraining the first round of hepatocyte proliferation after partial hepatectomy by preventing increases in growth hormone receptor (GHR) via ubiquitination, suppressing GH pathway activity. At later times, SOCS2 enhances hepatocyte proliferation by modulating a decrease in serum insulin-like growth factor 1 (IGF-1) that allows GH release from the pituitary. SOCS2, therefore, plays a dual role in modulating the rate of hepatocyte proliferation. In particular, this is the first demonstration of an endogenous mechanism to limit hepatocyte proliferation after injury.

Dicer-dependent production of microRNA221 in hepatocytes inhibits p27 and is required for liver regeneration in mice.

Dicer processes microRNAs (miRs) into active forms in a wide variety of tissues, including the liver. To determine the role of Dicer in liver regeneration, we performed a series of in vivo and in vitro studies in a murine 2/3 hepatectomy model. Dicer was downregulated after 2/3 hepatectomy, and loss of Dicer inhibited liver regeneration associated with decreased cyclin A2 and miR-221, as well as increased levels of the cell cycle inhibitor p27. In vitro, miR-221 inhibited p27 production in primary hepatocytes and increased hepatocyte proliferation. Specific reconstitution of miR-221 in hepatocyte-specific Dicer-null mice inhibited p27 and restored liver regeneration. In wild type mice, targeted inhibition of miR-221 using a cholesterol-conjugated miR-221 inhibited hepatocyte proliferation after 2/3 hepatectomy. These results identify Dicer production of miR-221 as an essential component of a miRNA-dependent mechanism for suppression of p27 that controls the rate of hepatocyte proliferation after partial hepatectomy.NEW & NOTEWORTHY Our findings demonstrate a direct role for microRNAs in controlling the rate of liver regeneration after injury. By deleting Dicer, an enzyme responsible for processing microRNAs into mature forms, we determined miR-221 is a critical microRNA in the physiological process of restoration of liver mass after injury. miR-221 suppresses p27, releasing its inhibitory effects on hepatocyte proliferation. Pharmaceuticals based on miR-221 may be useful to modulate hepatocyte proliferation in the setting of liver injury.

Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease.

Bioaccumulation and metabolism of polybrominated diphenyl ethers (PBDEs) in coenobitid hermit crabs from marine litter-polluted beaches in remote islands.

Plastic litter containing additives is potentially a major source of chemical contamination in remote areas. We investigated polybrominated diphenyl ethers (PBDEs) and microplastics in crustaceans and sand from beaches with high and low litter volumes on remote islands that were relatively free of other anthropogenic contaminants. Significant numbers of microplastics in the digestive tracts, and sporadically higher concentrations of rare congeners of PBDEs in the hepatopancreases were observed in coenobitid hermit crabs from the polluted beaches than in those from the control beaches. PBDEs and microplastics were detected in high amounts in one contaminated beach sand sample, but not in other beaches. Using BDE209 exposure experiments, similar debrominated products of BDE209 in field samples were detected in the hermit crabs. The results showed that when hermit crabs ingest microplastics containing BDE209, BDE209 leaches out and migrates to other tissues where it is metabolized.

Reversible shifts between interstitial and epibenthic habitats in evolutionary history: Molecular phylogeny of the marine flatworm family Boniniidae (Platyhelminthes: Polycladida: Cotylea) with descriptions of two new species.

Tiny animals in various metazoan phyla inhabit the interstices between sand and/or gravel grains, and adaptive traits in their body plan, such as simplification and size reduction, have attracted research attention. Several possible explanations of how such animals colonized interstitial habitats have been proposed, but their adaptation to this environment has generally been regarded as irreversible. However, the actual evolutionary transitions are not well understood in almost all taxa. In the present study, we show reversible evolutionary shifts from interstitial to epibenthic habitats in the lineage of the polyclad flatworm genus Boninia. In addition, we establish two new species of this genus found from different microhabitats on a single beach in Okinawa Island, Japan: (i) the interstitial species Boninia uru sp. nov. from gravelly sediments and (ii) the epibenthic species Boninia yambarensis sp. nov. from rock undersurfaces. Our observations suggest that rigid microhabitat segregation exists between these two species. Molecular phylogenetic analyses based on the partial 18S and 28S rDNA sequences of the new Boninia species and four other congeners, for which molecular sequences were available in public databases [Boninia antillara (epibenthic), Boninia divae (epibenthic), Boninia neotethydis (interstitial), and an unidentified Boninia sp. (habitat indeterminate)], revealed that the two interstitial species (B. neotethydis and B. uru sp. nov.) were not monophyletic among the three epibenthic species. According to ancestral state reconstruction analysis, the last common ancestor of the analyzed Boninia species inhabited interstitial realms, and a shift to the epibenthic environment occurred at least once. Such an "interstitial to noninterstitial" evolutionary route seems to be rare among Animalia; to date, it has been reported only in acochlidian slugs in the clade Hedylopsacea. Our phylogenetic tree also showed that the sympatric B. uru sp. nov. and B. yambarensis sp. nov. were not in a sister relationship, indicating that they colonized the same beach independently rather than descended in situ from a common ancestor that migrated and settled at the beach.

Descriptions of two new species of Armatoplana (Polycladida: Stylochoplanidae) from the coasts of Japan, with their phylogenetic positions in Leptoplanoidea.

We describe two new species of Armatoplana Faubel, 1983, namely, A. albomaculata sp. nov. and A. kaburakii sp. nov., from Japan. This is the first record of the genus from the West Pacific. Armatoplana albomaculata sp. nov. has the following characteristics: i) no nuchal tentacles; ii) white spots on the dorsal surface of the body; iii) an elongated oval prostatic vesicle directing posteriorly but curving dorsally in the distal part; iv) a long, curved penis stylet; and v) a small, oval Langs vesicle without accessory vesicles. Armatoplana kaburakii sp. nov. is distinguished from other congeners by having i) no nuchal tentacles; ii) a large, elongated Langs vesicle without accessory vesicles; and iii) gonopores opening closely to each other. We propose to re-circumscribe Armatoplana so that it would not become a junior synonym of Candimboides Prudhoe, 1982 and Phylloplana Laidlaw, 1903. We provide partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene as DNA barcodes for the two new species. Our phylogenetic analyses based on concatenated sequences of the 16S, 18S, and 28S ribosomal DNA and COI revealed that A. albomaculata sp. nov. and A. kaburakii sp. nov. were sister taxa; however, they did not form a monophyletic clade with Armatoplana divae (Marcus, 1947) and Armatoplana leptalea (Marcus, 1947).

Characterization of M Cells in Tear Duct-Associated Lymphoid Tissue of Mice: A Potential Role in Immunosurveillance on the Ocular Surface.

The ocular mucosal tissues are exposed to potentially harmful foreign antigens in the air and tear fluid. The tear duct-associated lymphoid tissue (TALT) may contribute to immune surveillance in the eye region. Follicle-associated epithelium (FAE) of TALTs is classified as stratified squamous epithelium and consists of squamous epithelial cells arranged in layers on the basement membrane. In contrast, most mucosa-associated lymphoid tissue is covered by a monolayer of epithelium containing microfold (M) cells. Therefore, antigen uptake and the presence of M cells in TALT are not fully understood. The present study found that a small population of FAE cells in the TALT expressed intestinal M-cell markers, namely Sox8, Tnfaip2, GP2, and OPG. This cell population was identified as functional M cells because of their uptake capacity of luminal nanoparticles. In addition, RANKL, which is essential for M-cell differentiation, was expressed by stroma-like cells at the subepithelial region and its receptor RANK by the FAE in the TALT. The administration of RANKL markedly increased the number of Sox8+ M cells. In contrast, deficiency in OPG, an endogenous inhibitor of RANKL, increased the number of M cells in the TALT. These data demonstrate that the RANKL-RANK axis is essential for M-cell differentiation in the TALT. Furthermore, immunization via eye drops elicited the production of antigen-specific antibodies in tears, which was enhanced by RANKL administration. Thus, TALT M cells play an important role in the immunosurveillance of the eye region.

Assessment of Adenosine Triphosphatase Phospholipid Transporting 8B1 (ATP8B1) Function in Patients With Cholestasis With ATP8B1 Deficiency by Using Peripheral Blood Monocyte-Derived Macrophages.

Adenosine triphosphatase phospholipid transporting 8B1 (ATP8B1) deficiency, an ultrarare autosomal recessive liver disease, includes severe and mild clinical forms, referred to as progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1), respectively. There is currently no practical method for determining PFIC1 or BRIC1 at an early disease course phase. Herein, we assessed the feasibility of developing a diagnostic method for PFIC1 and BRIC1. A nationwide Japanese survey conducted since 2015 identified 25 patients with cholestasis with ATP8B1 mutations, 15 of whom agreed to participate in the study. Patients were divided for analysis into PFIC1 (n = 10) or BRIC1 (n = 5) based on their disease course. An in vitro mutagenesis assay to evaluate pathogenicity of ATP8B1 mutations suggested that residual ATP8B1 function in the patients could be used to identify clinical course. To assess their ATP8B1 function more simply, human peripheral blood monocyte-derived macrophages (HMDMs) were prepared from each patient and elicited into a subset of alternatively activated macrophages (M2c) by interleukin-10 (IL-10). This was based on our previous finding that ATP8B1 contributes to polarization of HMDMs into M2c. Flow cytometric analysis showed that expression of M2c-related surface markers cluster of differentiation (CD)14 and CD163 were 2.3-fold and 2.1-fold lower (95% confidence interval, 2.0-2.5 for CD14 and 1.7-2.4 for CD163), respectively, in patients with IL-10-treated HMDMs from PFIC1 compared with BRIC1. Conclusion: CD14 and CD163 expression levels in IL-10-treated HMDMs may facilitate diagnosis of PFIC1 or BRIC1 in patients with ATP8B1 deficiency.

Description of Pericelis flavomarginata sp. nov. (Polycladida: Cotylea) and its predatory behavior on a scaleworm.

We describe a new species of polyclad flatworm, Pericelis flavomarginata sp. nov., from the intertidal and subtidal zones along localities on the Pacific coast of Japan. Pericelis flavomarginata sp. nov. is characterized by i) the dorsal surface of the body fringed by a lemon-yellow line except for the tip of tentacles, with a narrow brown midline running from the anterior edge of the body to the posterior end of the pharynx, ii) the pair of marginal tentacles with the tips extending and tapering, and iii) the presence of a common gonopore. Our molecular phylogenetic analysis revealed that selected Pericelis species were divided into two clades, each of which may be agreed with a characteristic dorsal color pattern. Additionally, we report an observation on the feeding behavior of P. flavomarginata sp. nov. on the polychaete Iphione muricata (Savigny in Lamarck, 1818).

Description of a new species of Paraplehnia (Polycladida, Stylochoidea) from Japan, with inference on the phylogenetic position of Plehniidae.

We describe a new species of polyclad flatworm, Paraplehniaseisuiae sp. nov., from 298-310 m depths in the Sea of Kumano, West Pacific, Japan. Paraplehniaseisuiae sp. nov. is characterized by i) a developed muscular wall proximally occupying about one-third of the prostatic vesicle, ii) no common duct between the spermiducal bulbs and the prostatic vesicle, and iii) a genital pit between the male and female gonopores. We provide a partial sequence (712 bp) of the mitochondrial cytochrome c oxidase subunit I gene as a DNA barcode for the species. Our phylogenetic analyses based on 603-bp 28S rDNA sequences indicate that P.seisuiae sp. nov. is nested in a clade consisting of stylochoid species along with unidentified species of Stylochus. It suggests that Plehniidae belongs to Stylochoidea, although this should be confirmed by future studies that contain Plehniaarctica (Plehn, 1896), the type species of the type genus of the family. The interfamily relationship among the superfamily Stylochoidea remains poorly resolved.

Single incision laparoscopic surgery (SILS) for Meckel's diverticulum.

A 65-year-old male patient presented with a chief complaint of abdominal pain. Abdominal computed tomography (CT) showed slight intestinal dilation and obstruction of the upper right quadrant of the small intestine, while ectopic gastric mucosal scintigraphy revealed abnormal accumulation in agreement with the CT-identified structure. The cause of bowel obstruction was diagnosed as Meckel's diverticulum; the patient was referred for surgery. A small laparotomy was performed with a 35-mm skin incision to the center of the navel. Once a lap disk was attached, a laparoscope was inserted to visualize the abdominal cavity. The small intestine that includes the structure was pulled out from the umbilicus to the outside of the peritoneal cavity and partially resected. On the pathological tissue findings, the patient was diagnosed with Meckel's diverticulum. We report our experience with single-lap laparoscopic surgery for a case of intestinal obstruction caused by Meckel's diverticulum and review pertinent literature.

Feasibility of Monotherapy by Rituximab Without Additional Desensitization in ABO-incompatible Living-Donor Liver Transplantation.

BACKGROUND: Rituximab is a cornerstone in the regimens of desensitization for ABO-incompatible living-donor liver transplantation (ABO-i LDLT) that makes this modality an acceptable option for liver transplantation. Plasmapheresis (PP) to reduce anti-ABO antibody titer and local infusion (LI) therapy were practiced as the strategies for desensitization before the application of rituximab and were reported as additional treatments. The aim of this study was to clarify the feasibility of monotherapy by rituximab without any additional desensitization treatments in ABO-i LT. METHODS: Forty patients receiving ABO-i LDLT with rituximab were enrolled in this retrospective study. The patients were divided into 2 groups: the rituximab with pretransplant PP and posttransplant LI (RPL) group (n = 20) and the rituximab monotherapy (RM) without any additional treatment group (n = 20). The groups were then compared in terms of the rates of patient survival, antibody-mediated rejection (AMR), and infection. RESULTS: The 1-, 3-, and 5-year patient survival rates were 85%, 85%, and 85% in the RPL group and 89%, 80%, and 80% in the RM group, respectively. There was no significant difference in patient survival between the 2 groups. There were no episodes of AMR in either group. The RM group had a lower rate of fungal and viral infections than the RPL group. CONCLUSIONS: Pretransplant rituximab without additional treatments yielded satisfactory outcomes comparable to that with additional treatments, such as PP and LI.

Ledipasvir and sofosbuvir for recurrent hepatitis C after liver transplantation.

Management of recurrent hepatitis C following liver transplantation still remains a challenge. Here, we report five patients who achieved viral responses following combined treatment with ledipasvir and sofosbuvir. All the patients received tacrolimus for immunosuppression. No dose adjustment was made before the ledipasvir and sofosbuvir therapy. All completed the intended 12-week treatment course with the full dose of ledipasvir and sofosbuvir. There were no significant adverse events greater than grade 2. During the study period, no acute rejection episodes were detected. The trough levels of tacrolimus were maintained stably. Hepatitis C virus RNA was not detected at week 12 in any of the patients. Based on the findings from this pilot study, combined ledipasvir and sofosbuvir therapy for 12 weeks is effective and safe for living - donor liver transplantation recipients with recurrence of hepatitis C virus.