Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia.

Wolbachia, a maternally transmitted bacterium, shows male-killing, an adaptive phenotype for cytoplasmic elements, in various arthropod species during the early developmental stages. In lepidopteran insects, lethality of males is accounted for by improper dosage compensation in sex-linked genes owing to Wolbachia-induced feminization. Herein, we established Ostrinia scapulalis cell lines that retained sex specificity per the splicing pattern of the sex-determining gene doublesex (Osdsx). We found that Wolbachia transinfection in male cell lines enhanced the female-specific splice variant of Osdsx (OsdsxF ) while suppressing the male-specific variant (OsdsxM ), indicating that Wolbachia affects sex-determining gene signals even in vitro. Comparative transcriptome analysis isolated only two genes that behave differently upon Wolbachia infection. The two genes were respectively homologous to Masculinizer (BmMasc) and zinc finger-2 (Bmznf-2), male-specifically expressed sex-determining genes of the silkworm Bombyx mori that encode CCCH-type zinc finger motif proteins. By using cultured cells and organismal samples, OsMasc and Osznf-2 were found to be sex-determining genes of O. scapulalis that are subjected to sex-specific alternative splicing depending upon the chromosomal sex, developmental stage, and infection status. Overall, our findings expound the cellular autonomy in insect sex determination and the mechanism through which sex is manipulated by intracellular selfish microbes.

Primary angiosarcoma of the ascending colon diagnosed after the discovery of intraoral tumor.

Here, we report a case of primary angiosarcoma of the ascending colon diagnosed after the discovery of an oral tumor. An 86-year-old woman presented to our hospital with severe anemia. Although she did not report any oral symptoms, an intraoral mass 15 mm in size with ulceration was observed. Since colonic tumors and osteolytic tumors in the maxilla and sacra were also identified by fluorodeoxyglucose (FDG)-positron emission tomography (PET), colonoscopy was performed. A semi-peripheral tumor with ulceration from the cecum to the ascending colon was detected. Biopsies of the oral cavity and colon revealed a poorly differentiated tumor, and multiple additional immunohistochemical stains were performed to confirm the diagnosis of angiosarcoma. Angiosarcoma progresses rapidly and has a very poor prognosis. Hence, although it is rare, angiosarcoma should always be considered in the differential diagnoses of malignancy of the gastrointestinal tract.

Large submucosal hematoma after cold snare polypectomy for colorectal adenoma.

Cold snare polypectomy (CSP), for the treatment of colorectal polyps, has become widespread due to its low incidence of adverse events compared to that of endoscopic procedures such as endoscopic mucosal resection. However, we experienced a case of large hematoma development shortly after CSP for a colorectal adenoma despite no bleeding during the procedure. The patient underwent CSP for a 7-mm type Isp lesion in the ascending colon. She returned the following day because of hematochezia. Computed tomography showed a 70-mm, high-intensity mass in the ascending colon, consistent with the large hematoma that was detected by colonoscopy. Although the patient initially had right-sided abdominal pain, it gradually improved with conservative treatment. The hematoma decreased in size, and she was discharged 20 days after emergency admission. Although CSP can be a favorable alternative to more invasive procedures and is expected to be performed more frequently, adverse events, such as that described in this case, should be anticipated.

A new continuous cell line from the pest insect, Anomala cuprea (Coleoptera; Scarabaeidae): emergence of contractile cells.

Insect contractile cells frequently appear at an early phase of cell culture, but in most cases, they disappear before a continuous cell line is established, so the cell line ceases to contract. Continuous contractile insect cell lines are currently available from only one species each of Hymenoptera and Diptera. Here, we obtained a new cell line that contracted long after being established as a continuous cell line. The cell line contracted for a short period at an early phase of insect cell culture before a continuous cell line was established, but then did not contract again for several years. After this cell line entered the continuous growth phase, it produced spontaneously contractile tissues for about 4 mo but stopped contracting again. This is the first instance of a cell line that contracted after its establishment as a non-contractile continuous cell line. It is unclear whether the contractile cells survive or die after contraction ceases at an early phase of cell culture, and our results indicate that potential contractile cells survive for years after they stop to contract. The cells of this line sometimes produced complex contractile structures, such as sheet-like tissues. Only a few continuous cell lines have been derived from scarabaeid beetles. The new continuous cell line was derived from the culture of the fat bodies of the scarab beetle Anomala cuprea, which is a pest in the agriculture and forestry of Japan. The population doubling time of the new cell line was 2.5 d and thus it grows very rapidly among coleopteran continuous cell lines. Our new cell line will facilitate research on the physiology and pathology of Coleoptera, including scarab beetles, and may also contribute to research on invertebrate muscles.

Evaluation of the visibility of bleeding points using red dichromatic imaging in endoscopic hemostasis for acute GI bleeding (with video).

BACKGROUND AND AIMS: We aimed to clarify whether red dichromatic imaging (RDI), a new type of image-enhanced endoscopy, improves the visibility of bleeding points in acute GI bleeding (AGIB) compared with white-light imaging (WLI). METHODS: Images and videos of bleeding points acquired with WLI and RDI during endoscopic hemostasis for AGIB were retrospectively compared. In images, the color difference between bleeding points and surrounding blood was analyzed. In videos, 4 expert and 4 trainee endoscopists evaluated the visibility on a scale of 1 (undetectable) to 4 (easily detectable). Furthermore, the correlation between the color difference and visibility score was evaluated. RESULTS: We analyzed 64 lesions. The color difference was significantly higher in RDI (13.11 ± 4.02) than in WLI (7.38 ± 3.68, P < .001). The mean visibility score for all endoscopists was significantly higher in RDI (3.12 ± .51) compared with WLI (2.72 ± .50, P < .001); this was also observed in experts (3.18 ± .51 vs 2.79 ± .54, P < .001) and trainees (3.05 ± .54 vs 2.64 ± .47, P < .001). The color difference and visibility score were moderately correlated for all endoscopists (γ = .56, P < .001) and for experts (γ = .53, P < .001) and trainees (γ = .57, P < .001). CONCLUSIONS: RDI improves the visibility of bleeding points in AGIB compared with WLI. RDI can help endoscopists at all levels of experience to recognize bleeding points by enhancing the color contrast relative to surrounding blood.

Implementation and Evaluation of Life-planning Lecture to Enhance Perspective-Taking among High School Students: A School-based Nonrandomized Waitlist Intervention Study in Japan.

INTRODUCTION: The Australian "empathy session," which is a parenting program aimed at alleviating postpartum depression by increasing empathy among expecting couples, was adapted to a life-planning education program for Japanese high school students. In this present study, we aimed to assess changes in high school students' empathy levels. METHODS: A nonrandomized, controlled, waitlist intervention was performed in 210 first-year students. They were divided into intervention and waitlist control groups. The life-planning lecture consisted of two parts: (1) reproductive health and (2) empathy and communication skills. The main outcome indicator was the Perspective-Taking subscale of an empathy index. Logistic regression was used to examine the association between the intervention and change in the Perspective-Taking scale score controlling for background factors. RESULTS: As per our findings, a significant difference was noted in the scale scores of Perspective-Taking before and after the program within the intervention group (3.76 ± 0.61 before the lecture and 3.86 ± 0.64 after the lecture; P = 0.01). In the between-group analysis, the likelihood of an increase in the scale score of Perspective-Taking was significantly higher in the intervention group (OR = 2.29, 95 % confidence interval = 1.23-4.26). CONCLUSIONS: Japanese high school students' Perspective-Taking improved through learning reproductive life-planning and communication skills.

A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis.

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace's insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

Horizontally transmitted parasitoid killing factor shapes insect defense to parasitoids.

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor (pkf), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts.

Correction to: Establishment and characterization of novel cell lines derived from six lepidopteran insects collected in the field.

This article was originally published with the final word of the title, "field", omitted.

Establishment and characterization of novel cell lines derived from six lepidopteran insects collected in the field.

Insect cell lines are used to study cellular interactions and gene functions in vitro in several research areas. However, suitable cell lines for experiments are not always available, especially in non-model species. Here, we established novel cell lines derived from fat bodies of six lepidopteran insects: Cydia kurokoi (named NARO-Cyku), Cephonodes hylas (NARO-Cehy), Haritalodes basipunctalis (NARO-Haba), Theretra oldenlandiae (NARO-Thol), Lymantria dispar (NARO-Lydi), and Hyphantria cunea (NARO-Hycu) collected in the field. The larval fat body was a promising tissue for the starting material when samples were limited due to field collection. It was critical that the medium volume was kept to a minimum for primary culture to maintain adherence of the fat body cells to the flask. The flask was coated with poly-L-lysine for effective induction of adherence and cell division. The identities of cell lines were confirmed using DNA barcoding with the mitochondrial cytochrome c oxidase I gene after cultures were passaged over 50 times. All lines except for NARO-Lydi and NARO-Hycu are adherent cells, and population doubling time of six cell lines ranged from 1.03 to 2.49. Induction of gene expression was practicable in the four adherent cell lines as revealed by transfection of expression vectors and found the immediate early 2 and the Bombyx actin 3 were effective gene promoters. The results suggest that these cell lines are capable of gene functional analysis. Thus, establishments of cell line using our methods for non-model lepidopterans could make a practical contribution to pest management and insect utilization.

Characteristics and changes in the mental health indicators of expecting parents in a couple-based parenting support program in Japan.

Postpartum depression is a serious problem not only for mothers, but also for their children and families. Evidence is scarce on the effectiveness of couple-targeted antenatal interventions aimed at preventing postpartum depression in Asia. Therefore, we implemented an antenatal parenting support program from Australia ("empathy program") at three sites in Japan, and assessed the characteristics of participating couples and changes in their mental health indicators before and after the program (during pregnancy and 6 weeks postpartum, respectively). In this program, participant couples discussed concerns during pregnancy and a "difficult day" scenario with other same-gender participants and then with their own partners. In total, 100 couples attended the program. Among 60 participating mothers, 20% screened positive for maternal depression symptoms (defined as an Edinburgh Postnatal Depression Scale greater than 9). Changes in paternal empathy and maternal antenatal Edinburgh Postnatal Depression Scale score were significantly associated with maternal postpartum Edinburgh Postnatal Depression Scale score in multivariable analyses. Our results indicate that a key in preventing postpartum depression is paternal involvement and long-term support from antenatal phase.

Concurrent chemoradiotherapy with intravenous cisplatin and docetaxel for advanced oral cancer.

Concurrent chemoradiotherapy (CCRT) is a common treatment for advanced oral cancer, and its efficacy has been reported in many reviews. We have performed concurrent CCRT with intravenous cisplatin and docetaxel in patients with advanced oral cancer. The purpose of this report was to evaluate this treatment and to compare the outcome of this treatment with that of standard CCRT treatments for advanced head and neck cancer using intravenous administration. The patients were treated for primary advanced oral squamous cell carcinoma in our department between February 2003 and November 2015. In all, 17 patients (14 men, 3 women) with stage III (2 patients) stage IVA (10 patients), and stage IVB (5 patients) oral cancer were treated. The patient ages ranged from 44 to 87 years (average age: 65.4 years). The follow-up duration ranged from 5 to 117 months (average follow-up duration: 41 months, median follow-up duration: 39 months). The primary cancer sites were the maxillary gingiva (7 cases), mandible gingiva (3 cases), buccal mucosa (3 cases), tongue (3 cases), and floor of the mouth (1 case). The 3-year and 5-year survival rates were 52.9% and 33.0%, respectively, and both the 3-year and 5-year locoregional control rates were 50.9% as determined by the Kaplan-Meier method. The response rate was 94% (CR: 8 cases: 47% and PR: 8 cases: 47%). The incidences of toxicity greater than grade 3 included dermatitis and stomatitis in 9 cases each (52.9%), anemia in 3 cases (18.7%) and liver dysfunction in 1 case (6.2%). We found that the results of this therapy were equivalent to those of standard CCRT treatments for advanced head and neck cancer using intravenous administration, and the incidences of toxicity were lower than those of standard treatments. These findings suggested that this treatment is safe and useful for advanced oral cancer.

Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term preservation at room temperature and retain proliferation potential after rehydration.

Pv11, a cell line derived from the anhydrobiotic insect, Polypedilum vanderplanki, was preserved in a dry form (only 6% residual moisture) at room temperature for up to 251 days and restarted proliferating after rehydration. A previous study already reported survival of Pv11 cells after desiccation, but without subsequent proliferation. Here, the protocol was improved to increase survival and achieve proliferation of Pv11 cells after dry storage. The method basically included preincubation, desiccation and rehydration processes and each step was investigated. So far, preincubation in a 600 mM trehalose solution for 48 h before dehydration was the most favourable preconditioning to achieve successful dry preservation of Pv11 cells, allowing about 16% of survival after rehydration and subsequent cell proliferation. Although the simple air-dry method established for Pv11 cells here was not applicable for successful dry-preservation of other insect cell lines, Pv11 is the first dry-preservable animal cell line and will surely contribute not only to basic but also applied sciences.

A new cell line from the fat body of Spodoptera litura (Lepidoptera, Noctuidae) and detection of lysozyme activity release upon immune stimulation.

A new cell line, designated NIAS-SL64, was established from the fat body of the fifth instar larvae of the common cutworm Spodoptera litura. NIAS-SL64 cells grew as spindle-shaped and non-adherent cells in the insect-specific cell culture medium MGM-450 supplemented with 10% fetal bovine serum. Criterions for the establishment of the NIAS-SL64 cell line is spindle shape and length (30~90 µm) stabilized after 100 passages. The doubling time of the cells was 24 h at 25°C. Lipopolysaccharide significantly stimulated the release of lysozyme activity by NIAS-SL64 cells. Lysozyme is one of the components of the innate immunity and plays important role as lytic enzyme in infection. Lysozyme activity released from NIAS-SL64 would be a marker for immune response. The released lysozyme activity critically depends on morphology of the cells and would be a criterion of the establishment of the cell line. Lysozyme activity was suppressed in a dose-dependent manner by the immunosuppressive agent cyclosporin A.

Endoscopic hemostasis for bleeding gastric varices treated by combination of variceal ligation and sclerotherapy with N-butyl-2-cyanoacrylate.

BACKGROUND: Bleeding from esophagogastric varices is a serious complication of portal hypertension. The aim of the present study was to determine whether endoscopic sclerotherapy with injection of N-butyl-2-cyanoacrylate combined with variceal ligation was useful for hemostasis of bleeding gastric varices. METHODS: Twenty-seven patients with bleeding gastric varices underwent endoscopic treatment with variceal ligation followed by sclerotherapy with N-butyl-2-cyanoacrylate from November 1995 to November 2000. Patients underwent endoscopic variceal ligation only for the bleeding spot just before sclerotherapy. Injection was continued until varices were engorged. After these therapies, patients were followed for at least for 5 years. Retreatment was applied as necessary. RESULTS: Among these patients, 11 had active bleeding and 16 had recent bleeding within 24 h with white or red plaques on gastric varices. All varices presented as nodular or tumorous forms. The hemostasis rate at 1 week after treatment with N-butyl-2-cyanoacrylate was 88.9% (24/27). Among the patients achieving hemostasis at 1 week, 33.3% (8/24) experienced recurrent bleeding between 5 and 53 months after the initial treatment. Five patients with rebleeding were treated with N-butyl-2-cyanoacrylate, and the other three patients were treated by other procedures. The final hemostasis rate was 81.5% (22/27). The 5-year survival rate after initial hemostasis was 63.0% (17/27). CONCLUSIONS: This study showed that endoscopic variceal ligation combined with sclerotherapy might become a recommended choice for effective treatment of bleeding gastric varices.

Clinical outcomes of EMR for gastric tumors: historical pilot evaluation between endoscopic submucosal dissection and conventional mucosal resection.

BACKGROUND: EMR is currently a standard treatment for mucosal gastric tumors. Endoscopic submucosal dissection (ESD) has been developed for en bloc resection. OBJECTIVE: We evaluated the clinical outcomes of ESD compared with conventional EMR. DESIGN: Not applicable. SETTING: A historical control study was performed between EMR and ESD. PATIENTS: EMR of 245 gastric tumors was performed in 229 patients. Lesions were divided into two groups. Conventional EMR was performed in group A from February 1999 to June 2001, and ESD was performed in group B from July 2001 to March 2004. Group B was divided into subgroups: subgroup B-1 underwent ESD from July 2001 to March 2003 and subgroup B-2 from April 2003 to March 2004. INTERVENTIONS: All lesions were resected with conventional EMR or with ESD. MAIN OUTCOME MEASUREMENTS: En bloc resection rate, rate in completeness of resection, required time, remnant ratio, and complications were evaluated. RESULTS: With regard to lesions >10 mm in size, the en bloc resection rate and the rate in completeness of resection of group B was significantly higher than that of group A (p < 0.01). Although the required time was longer in group B than A (p < 0.01), it was shorter in subgroup B-2 compared with B-1 (p < 0.05) with lesions < or =10 mm in size. The remnant ratio and perforation rate were not different between groups. LIMITATIONS: Not applicable. CONCLUSIONS: The en bloc resection rate was better with ESD than with conventional EMR. The required time was longer in ESD, but this disadvantage might be improved with experience.

Dysphagia associated with gastroesophageal reflux disease is improved by proton pump inhibitor.

This study aimed to determine whether dysphagia associated with gastroesophageal reflux disease was effectively treated with rabeprazole, a proton pump inhibitor. Sixty-eight outpatients with gastroesophageal reflux-associated dysphagia were enrolled in this study. Endoscopic esophagitis was confirmed in 52 of 68 subjects. The proton pump inhibitor rabeprazole was administered at 20 mg daily for 8 weeks. Rabeprazole was administered for a further 6 months to 16 subjects whose dysphagia was improved (10 mg/day) and 5 of these underwent 24-hr esophageal pH monitoring before and after treatment. Dysphagia was completely resolved in 40 of 68 subjects, which were categorized in Group I. Dysphagia improved partially in 20 subjects and was unchanged in 8 subjects. These 28 subjects were categorized into Group II. Comparison was made between Group I and Group II and multivariate analysis demonstrated that the only factor that correlated with the effect of rabeprazole on dysphagia was "improvement in heartburn symptoms." There were no relapses of symptoms during the 6-month follow-up period, and pH monitoring showed sustained suppression of acid secretion. The results indicate that rabeprazole is effective in the treatment of dysphagia associated with gastroesophageal reflux disease. We were, however, unable to elicit any factors that could predict the therapeutic effect of rabeprazole before commencing treatment.