fruitless is sex-differentially spliced and is important for the courtship behavior and development of silkmoth Bombyx mori.

Sexual dimorphisms of the brain play essential roles in successful reproduction. Silkmoth Bombyx mori exhibits extensive sexual differences in sexual behavior, as well as their morphology. Although the neural circuits that transmit information about sex pheromone in the male brain are extensively analyzed, the molecular mechanisms that regulate their development are still elusive. In the present study, we focused on the silkmoth ortholog of fruitless (fru) as a candidate gene that regulates sexual dimorphisms of the brain. fru transcripts were expressed from multiple promoters in various tissues, and brain-specific transcripts were sex-specifically spliced, in a manner similar to Drosophila. Interestingly, fru was highly expressed in the adult female brain and the male larval testis. Analysis of CRISPR/Cas9-mediated fru knockout strains revealed that fru plays important roles in survival during late larval and pupal stages, testis development, and adult sexual behavior. fru mutant males exhibited highly reduced levels of courtship and low copulation rate, indicating that fru plays significant roles in the sexual behavior of silkmoths, although it is not absolutely necessary for copulation. In the fru mutant males, sexually dimorphic pattern of the odorant receptor expression was impaired, possibly causing the defects in courtship behavior. These results provide important clues to elucidate the development of sexual dimorphisms of silkmoth brains, as well as the evolution of fruitless gene in insects.

Molecular comparison of concurrent components of high-grade dysplasia, adenocarcinoma, and sarcomatoid carcinoma in a case of sarcomatoid carcinoma of the gallbladder.

Detailed genetic and immunohistochemical features of a sarcomatoid carcinoma of the gallbladder were reported. Studied was a resected gallbladder tumor involving the transverse colon, which was consisted of 3 histopathological neoplastic components, i.e., high-grade dysplasia, adenocarcinoma, and sarcomatoid carcinoma. The targeted amplicon sequencing showed somatic mutations in TP53 (p.S90fs) and ARID1A (c.4993 + 1G > T) in all of the 3 components. Copy numbers of CDKN2A and SMAD4 were decreased in the adenocarcinoma and the sarcomatoid component. Immunohistochemistry showed loss of expression of p53 and ARID1A in all components. p16 expression was lost in the adenocarcinoma and the sarcomatoid component, while SMAD4 expression was lost only in the latter. These results suggest that this sarcomatoid carcinoma may have developed by progression from high-grade dysplasia via adenocarcinoma with sequential accumulation of molecular aberrations involving p53, ARID1A, p16, and SMAD4. This information should serve to understand the molecular mechanism of this very intractable tumor.

Mesentero-axial gastric volvulus treated with laparoscopic gastropexy: a case report.

BACKGROUND: Mesentero-axial gastric volvulus (MAGV) is an uncommon subtype of gastric volvulus (GV). However, reports of such cases in adult patients are very rare. We present an unusual case of idiopathic MAGV in an old woman. CASE PRESENTATION: An 84-year-old woman was referred to the emergency department for vomiting and abdominal pain. An abdominal computed tomography scan revealed a mesentero-axial gastric volvulus, which could be corrected endoscopically, and the symptoms were relieved. Contrast-enhanced examination was performed before the elective surgery to confirm the presence of short-axis dorsal 180-degree volvulus. The patient underwent laparoscopic surgery on a wait-and-watch basis. After releasing the torsion, the stomach returned to normal position. The gastric fornix was sutured to the left diaphragm and the gastric body and antrum were sutured to the abdominal wall using non-absorbable thread. Symptoms did not flare after the surgery. CONCLUSIONS: We experienced a rare case of adult MAGV presenting with incomplete obstruction. Laparoscopic gastropexy is useful when gastric decompression is achieved.

Preoperative CA19-9 is a prognostic factor in pT3N0 gastric cancer patients undergoing curative resection.

BACKGROUND: The standard treatment for pT3N0 gastric cancer (GC) in Japanese guidelines is radical surgery without adjuvant chemotherapy. However, certain percentages of these patients develop recurrences; therefore, detecting the high-risk subgroup of recurrence may contribute to improve patient's outcome by adjuvant chemotherapy. In this study, we aimed to identify a predictive indicator of poor prognosis in pT3N0 GC. METHODS: Eighty-one patients who were diagnosed as pT3N0 GC after curative surgical resection and had not received adjuvant chemotherapy were included. The clinicopathological factors and laboratory parameters were evaluated by univariate and multivariate analyses to identify prognostic factors of tumor recurrence. Survival analysis was performed by Kaplan-Meier method. RESULTS: Male (P = 0.027), a high body mass index (BMI) (P = 0.031), a high CA19-9 value (P = 0.025), and a lower number of retrieved lymph nodes (P = 0.018) were found to be significantly associated with a shorter recurrence free survival (RFS). In a multivariate analysis, high CA19-9 value (> 37 U/ml) [(hazard ratio (HR): 3.326; 95% confidence interval (CI): 1.044 to 10.596; P = 0.042] was found to be an independent predictor of RFS. CONCLUSION: The preoperative high CA19-9 value is considered a useful prognostic marker for predicting cancer recurrence after curative surgery in pT3N0 GC patients. For those patients, adjuvant chemotherapy might be considered to improve the survival outcome.

Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail.

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

Long-term survival after esophagectomy with distal pancreatectomy for locally advanced esophageal cancer with pancreatic invasion: a case report.

BACKGROUND: The treatment for the locally advanced esophageal cancer invading adjacent organs is controversial. We performed a radical surgery for a patient suffering from lower esophageal cancer with pancreatic invasion, and led to long-term survival. CASE PRESENTATION: A 62-year-old man with dysphagia, was endoscopically diagnosed lower esophageal cancer. Abdominal computed tomography shows that the tumor formed a mass with the solitary metastatic abdominal lymph node, which invaded pancreas body and gastric body. He was diagnosed locally advanced esophageal cancer cStage IIIC. As chemoradiotherapy was difficult because of the high risk of gastric mucosal damage, radical esophagectomy with distal pancreatectomy and reconstruction of gastric conduit were performed. The postoperative course was uneventful and the patient was discharged 16 days after operation. At present, 7 years after surgery, he is still alive with disease-free condition. CONCLUSION: Esophagectomy with distal pancreatectomy may be feasible for locally advanced esophageal cancer with pancreatic invasion in terms of curability and long-term survival.

The role of toxic planocerid flatworm larvae on tetrodotoxin accumulation in marine bivalves.

Tetrodotoxin (TTX), also known as pufferfish toxin, has been detected in marine edible bivalves worldwide. In this study, several bivalve species, Azumapecten farreri subsp. akazara, Patinopecten yessoensis and Mytilus galloprovincialis, collected from the Pacific side of the northern Japanese Islands, were studied for the accumulation of TTX in the presence of toxic planocerid larvae. LC-MS/MS analysis demonstrated that TTX was detected only in the midgut gland of A. farreri subsp. akazara. Toxic flatworm-specific PCR and direct sequencing of the amplicons showed that the DNA fragments of the Planocera multitentaculata COI gene were detected in the gut contents of the toxified bivalves. The planocerid larvae were also detected in the environmental seawaters. Toxification experiments in the aquarium demonstrated that the mussel M. galloprovincialis was also toxified by feeding on the toxic flatworm larvae. These results suggest that the source of TTX accumulation in edible bivalves is toxic flatworm larvae.

Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

PPP3CB contributes to poor prognosis through activating nuclear factor of activated T-cells signaling in neuroblastoma.

We previously identified a gain-of-function mutation in PPP3CB in a neuroblastoma (NB) with MYCN amplification. Here we investigated the functional and clinical role of PPP3CB in NB. High PPP3CB expression was an independent indicator predicting poor prognosis of NB. Overexpression of wildtype or mutated PPP3CB (PPP3CBmut) promoted cell growth, but PPP3CB knockdown decreased cell growth in NB cells. Forced expressions of PPP3CB and PPP3CBmut activated NFAT2 and NFAT4 transcription factors and inhibited GSK3ß activity, resulting in the increase in the expressions of c-Myc, MYCN, and ß-catenin, which were downregulated in response to PPP3CB knockdown. Treatment with calcineurin inhibitor cyclosporin A (CsA) or FK506 suppressed cell proliferation and induced apoptotic cell death in both MYCN-amplified and MYCN-non-amplified NB cell lines. Expression of PPP3CB protein was decreased in response to two calcineurin inhibitors. c-Myc, MYCN, and ß-catenin were downregulated at the mRNA and protein levels in CsA or FK506-treated NB cells. Our data indicate that elevated expression of PPP3CB and the expression of its constitutively active mutant contribute to the aggressive behavior of NB tumors and therefore suggest that inhibition of calcineurin activity might have therapeutic potential for high-risk NB.

Sendai virus-mediated expression of reprogramming factors promotes plasticity of human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial solid tumor that originates from multipotent neural crest cells. NB cell populations that express embryonic stem cell-associated genes have been identified and shown to retain a multipotent phenotype. However, whether somatic reprogramming of NB cells can produce similar stem-cell like populations is unknown. Here, we sought to reprogram NB cell lines using an integration-free Sendai virus vector system. Of four NB cell lines examined, only SH-IN cells formed induced pluripotent stem cell-like colonies (SH-IN 4F colonies) at approximately 6 weeks following transduction. These SH-IN 4F colonies were alkaline phosphatase-positive. Array comparative genomic hybridization analysis indicated identical genomic aberrations in the SH-IN 4F cells as in the parental cells. SH-IN 4F cells had the ability to differentiate into the three embryonic germ layers in vitro, but rather formed NBs in vivo. Furthermore, SH-IN 4F cells exhibited resistance to cisplatin treatment and differentiated into endothelial-like cells expressing CD31 in the presence of vascular endothelial growth factor. These results suggest that SH-IN 4F cells are partially reprogrammed NB cells, and could be a suitable model for investigating the plasticity of aggressive tumors.

Functional interplay between MYCN, NCYM, and OCT4 promotes aggressiveness of human neuroblastomas.

Neuroblastoma is a pediatric solid tumor that originates from embryonic neural crest cells. The MYCN gene locus is frequently amplified in unfavorable neuroblastomas, and the gene product promotes the progression of neuroblastomas. However, the molecular mechanisms by which MYCN amplification contributes to stem cell-like states of neuroblastoma remain elusive. In this study, we show that MYCN and its cis-antisense gene, NCYM, form a positive feedback loop with OCT4, a core regulatory gene maintaining a multipotent state of neural stem cells. We previously reported that NCYM is co-amplified with the MYCN gene in primary human neuroblastomas and that the gene product promotes aggressiveness of neuroblastoma by stabilization of MYCN. In 36 MYCN-amplified primary human neuroblastomas, OCT4 mRNA expression was associated with unfavorable prognosis and was correlated with that of NCYM. The OCT4 protein induced both NCYM and MYCN in human neuroblastoma cells, whereas NCYM stabilized MYCN to induce OCT4 and stem cell-related genes, including NANOG, SOX2, and LIN28. In sharp contrast to MYCN, enforced expression of c-MYC did not enhance OCT4 expression in human neuroblastoma cells. All-trans retinoic acid treatment reduced MYCN, NCYM, and OCT4 expression, accompanied by the decreased amount of OCT4 recruited onto the intron 1 region of MYCN. Knockdown of NCYM or OCT4 inhibited formation of spheres of neuroblastoma cells and promoted asymmetric cell division in MYCN-amplified human neuroblastoma cells. These results suggest that the functional interplay between MYCN, NCYM, and OCT4 contributes to aggressiveness of MYCN-amplified human neuroblastomas.

NCYM promotes calpain-mediated Myc-nick production in human MYCN-amplified neuroblastoma cells.

NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas.

NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3ß resulting in the stabilization of MYCN in human neuroblastomas.

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3ß, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3ß, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3ß inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3ß activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

Positive auto-regulation of MYCN in human neuroblastoma.

MYCN oncogene is one of the most important regulators affecting the prognosis of neuroblastoma and is frequently amplified in the high-risk subsets. Despite its clinical significance, it remains unclear how the MYCN expression is regulated in human neuroblastomas. Here, we found the presence of a positive auto-regulatory mechanism of MYCN. Enforced expression of MYCN induced endogenous MYCN mRNA expression in SK-N-AS neuroblastoma cells with a single copy of MYCN gene. Luciferase reporter assay revealed that MYCN protein activates its own promoter activity in a dose-dependent manner and the downstream region relative to the transcription start sites is responsible for the activation. Furthermore, ChIP analysis showed that MYCN is directly recruited onto the intron 1 region of MYCN gene which contains two putative E-box sites. Intriguingly, in response to all-trans-retinoic acid (ATRA), MYCN was down-regulated in MYCN-amplified SK-N-BE neuroblastoma cells, and the recruitment of MYCN protein onto its own intron 1 region was reduced in association with an induction of neuronal differentiation. Collectively, our present results suggest that MYCN contributes to its own expression by forming a positive auto-regulatory loop in neuroblastoma cells.