Etiology and long-term rebleeding of endoscopic ulcerative lesions in the small bowel in patients with obscure gastrointestinal bleeding: A multicenter cohort study.

BACKGROUND: Among patients with obscure gastrointestinal bleeding (OGIB), endoscopic ulcerative lesions in the small bowel have diverse etiologies and often cause rebleeding. Certain characteristics of patients or ulcerations may be reasonable indications for diagnostic balloon-assisted endoscopy (BAE) to assess etiology and may be risks of rebleeding; however, these characteristics are unclear. We aimed to elucidate appropriate indications for diagnostic BAE and predictors of long-term rebleeding in patients with small bowel ulcerative lesions. METHODS: We conducted a multicenter retrospective cohort study of 68 patients with OGIB, in whom small bowel ulcerative lesions were detected by capsule endoscopy (n = 60) and/or BAE (n = 43). Patients' characteristics, including medications and endoscopic findings, were evaluated. Predictors of the need for diagnostic BAE to determine ulceration etiology were identified by logistic regression analysis. Rebleeding risks were evaluated using Cox proportional hazards analysis. RESULTS: Single ulcerations were diagnosed in 26 patients, and multiple ulcerations were diagnosed in 42 patients. Among 43 patients who underwent BAE, ulceration etiology was identified in 12 (28%) patients. In the etiology identification, BAE was more useful for a single ulceration than for multiple ulcerations (P < 0.001). Among the 68 patients, rebleeding occurred in 14 (21%) patients during a mean follow-up period of 17 months. Aspirin use and multiple ulcerations were significant predictors of rebleeding (P < 0.05). CONCLUSIONS: When we manage small bowel ulcerative lesions in OGIB patients, a single ulceration is a reasonable indication for the diagnostic BAE. The rebleeding rate was lower for single ulcerations than for multiple ulcerations.

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real-time bioluminescence imaging.

It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging. It was found that several chemical chaperones, such as ferulic acid (FA), silybin, and rutin, affected the activity. We visualized EVs from cancer cells using bioluminescence imaging and showed that several EVs could be observed when using CD63 fused with NanoLuc luciferase, which has a much smaller molecular weight and higher intensity than conventional firefly luciferase. We then examined the effects of the chemical chaperones on EVs from cancer cells by bioluminescence imaging and quantified the expression of CD63 in these EVs. It was found that the chemical chaperones examined in this study affected CD63 levels in EVs. These results showed that imaging at the level of the single cell using bioluminescence is a powerful tool and could be used to evaluate chemical chaperones and EVs from cancer cells. This approach may produce new information in this field when taken together with conventional and classical methods.

FTBMT, a Novel and Selective GPR52 Agonist, Demonstrates Antipsychotic-Like and Procognitive Effects in Rodents, Revealing a Potential Therapeutic Agent for Schizophrenia.

GPR52 is a Gs-coupled G protein-coupled receptor that is predominantly expressed in the striatum and nucleus accumbens (NAc) and was recently proposed as a potential therapeutic target for schizophrenia. In the current study, we investigated the in vitro and in vivo pharmacologic activities of a novel GPR52 agonist, 4-(3-(3-fluoro-5-(trifluoromethyl)benzyl)-5-methyl-1H-1,2,4-triazol-1-yl)-2-methylbenzamide (FTBMT). FTBMT functioned as a selective GPR52 agonist in vitro and in vivo, as demonstrated by the activation of Camp signaling in striatal neurons. FTBMT inhibited MK-801-induced hyperactivity, an animal model for acute psychosis, without causing catalepsy in mice. The c-fos expression also revealed that FTBMT preferentially induced neuronal activation in the shell of the Nac compared with the striatum, thereby supporting its antipsychotic-like activity with less catalepsy. Furthermore, FTBMT improved recognition memory in a novel object-recognition test and attenuated MK-801-induced working memory deficits in a radial arm maze test in rats. These recognitive effects were supported by the results of FTBMT-induced c-fos expression in the brain regions related to cognition, including the medial prefrontal cortex, entorhinal cortex, and hippocampus. Taken together, these findings suggest that FTBMT shows antipsychotic and recognitive properties without causing catalepsy in rodents. Given its unique pharmacologic profile, which differs from that of current antipsychotics, FTBMT may provide a new therapeutic option for the treatment of positive and cognitive symptoms of schizophrenia.

Characterization of a new small bowel adenocarcinoma cell line and screening of anti-cancer drug against small bowel adenocarcinoma.

Small bowel adenocarcinoma (SBA) is a rare, aggressive malignancy with a poor prognosis, and the mechanisms of carcinogenesis in SBA remain unclear. Our aims were to investigate the molecular mechanisms underlying SBA and to identify treatments by establishing and characterizing an SBA cell line and performing anti-cancer drug screening. SIAC1 cells, established from jejunal SBA, showed epithelial characteristics and formed organoids in 3D culture. SIAC1 cells had a heterozygous ß-catenin deletion mutation, resulting in a stable ß-catenin protein with enhanced Wnt/ß-catenin activity. SIAC1 cells lacked MLH1 and MSH6 expression, and target genes such as TGFBR2 and ACVR2 showed frameshift mutations. Among 10 clinical SBA samples, 2 (20%) had interstitial deletions in ß-catenin, expression of mismatch repair protein was aberrant in 4 (40%), and heterozygous frameshift mutations of three target genes were found in all 10 samples. On screening assay using 140 compounds, eribulin significantly inhibited SIAC1 cell growth both in vitro and in vivo by inhibition of the Wnt/ß-catenin pathway via enhanced degradation of ß-catenin. In conclusion, we established an SBA cell line with molecular characteristics similar to those of clinical SBA samples, including ß-catenin deletion and mismatch repair protein deficiency, that will be useful for SBA research. Eribulin might be a candidate for SBA treatment due to its inhibitory effect on Wnt/ß-catenin signaling.

Inhibition of autophagy exerts anti-colon cancer effects via apoptosis induced by p53 activation and ER stress.

BACKGROUND: Although some molecularly targeted drugs for colorectal cancer are used clinically and contribute to a better prognosis, the current median survival of advanced colorectal cancer patients is not sufficient. Autophagy, a basic cell survival mechanism mediated by recycling of cellular amino acids, plays an important role in cancer. Recently, autophagy has been highlighted as a promising new molecular target. The unfolded protein response (UPR) reportedly act in complementary fashion with autophagy in intestinal homeostasis. However, the roles of UPR in colon cancer under autophagic inhibition remain to be elucidated. We aim to clarify the inhibitory effect of autophagy on colon cancer. METHODS: We crossed K19 (CreERT) and Atg5 (flox/flox) mice to generate Atg5 (flox/flox)/K19 (CreERT) mice. Atg5 (flox/flox)/K19 (CreERT) mice were first treated with azoxymethane/dextran sodium sulfate and then injected with tamoxifen to inhibit autophagy in CK19-positive epithelial cells. To examine the anti-cancer mechanisms of autophagic inhibition, we used colon cancer cell lines harboring different p53 gene statuses, as well as small interfering RNAs (siRNAs) targeting Atg5 and immunoglobulin heavy-chain binding protein (BiP), a chaperone to aid folding of unfolded proteins. RESULTS: Colon tumors in Atg5 (flox/flox)/K19 (CreERT) mice showed loss of autophagic activity and decreased tumor size (the total tumor diameter was 28.1 mm in the control and 20.7 mm in Atg5 (flox/flox)/K19 (CreERT) mice, p = 0.036). We found that p53 and UPR/endoplasmic reticulum (ER) stress-related proteins, such as cleaved caspase 3, and CAAT/enhancer-binding protein homologous protein, are up-regulated in colon tumors of Atg5 (flox/flox)/K19 (CreERT) mice. Although Atg5 and BiP silencing, respectively, increased apoptosis in p53 wild type cells, Atg5 silencing alone did not show the same effect on apoptosis in p53 mutant cells. However, co-transfection of Atg5 and BiP siRNAs led to increased apoptosis in p53 mutant cells. CONCLUSIONS: Blocking autophagy has potential in the treatment of colon cancer by inducing apoptosis via p53 and ER stress, and suppressing the UPR pathway is a valid strategy to overcome resistance to autophagic inhibition.

Irsogladine improves small-intestinal injuries in regular users of nonsteroidal anti-inflammatory drugs.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) cause a high frequency of mucosal injuries in the small intestine. However, no reliable intervention, other than cessation of NSAIDs, has been established. OBJECTIVE: To evaluate whether irsogladine maleate reduces these injuries while continuing NSAID therapy. DESIGN: Prospective, interventional, endoscopist-blinded, randomized, controlled trial (RCT). SETTING: University hospital. PATIENTS: Patients regularly taking conventional NSAIDs for more than 4 weeks. INTERVENTIONS: We initially examined small-intestinal mucosal injuries by capsule endoscopy (CE) and screened participants for the RCT. In the RCT, patients with any mucosal injury were randomly assigned to the irsogladine group (4 mg/day) or the control group. MAIN OUTCOME MEASUREMENTS: The primary endpoint was the rate of mucosal injury improvement after 4 weeks of treatment monitored with a second CE. RESULTS: Sixty-one patients were evaluated with the first CE. Small intestine mucosal injuries were found in 41 patients (67.2%) and erosive or ulcerative lesions in 21 patients (34.4%). The injury prevalence was not different with gastroprotective drug treatment. Of 41 patients enrolled, 39 (19 patients in the irsogladine group and 20 in the control group) completed the study. The improvement rate was significantly higher in the irsogladine group (16/19 patients; 84.2%) than in the control group (9/20 patients; 45.0%; P = .02). LIMITATIONS: Asymptomatic lesions, single-institution data, and single-blind setting. CONCLUSION: Irsogladine maleate was effective for reducing NSAID-induced small-intestinal mucosal injury. (University Hospital Medical Information Network Clinical Trials Registry number UMIN000001507.).

Bioluminescence imaging to track real-time armadillo promoter activity in live Drosophila embryos.

We established a method for bioluminescence imaging (BLI) to track real-time gene expression in live Drosophila embryos. We constructed a transgenesis vector containing multiple cloning sites and enhanced green-emitting luciferase (ELuc; Emerald Luc), a brighter and pH-insensitive luciferase for promoter analysis. To evaluate the utility of BLI using an ELuc reporter together with an optimized microscope system, we visualized the expression pattern of armadillo (arm), a member of the Wnt pathway in Drosophila, throughout embryogenesis. We generated transgenic flies carrying the arm:: ELuc fusion gene, and successfully performed BLI continuously for 22 h in the same embryos. Our study showed, for the first time, that arm::Eluc expression was dramatically increased in the anterior midgut rudiment, myoblasts of the dorsal/lateral musculature, and the posterior spiracle after stage 13, and the cephalic region at stage 17. To further demonstrate the application of our BLI system, we revealed that arm transcriptional activity in embryos was modulated inversely by treatment with ionomycin or 6-bromoindirubin-3-oxime (BIO), an inhibitor and activator of Wnt/ß-catenin signaling, respectively. Therefore, our microscopic BLI system is useful for monitoring gene expression in live Drosophila embryos, and for investigating regulatory mechanisms by using chemicals and mutations that might affect expression.

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single-cell bioluminescence imaging.

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single-cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single-cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay.

Sonographic detection of a patency capsule prior to capsule endoscopy: case report.

To avoid retention of the capsule used in capsule endoscopy (CE), the patency capsule (PC), a self-disintegrating sham capsule, is administered prior to CE in patients suspected of small intestinal stenosis. If the PC is excreted intact within 30 hours of ingestion, the patient can undergo CE without retention. However, if the PC is not excreted within 30 hours, its location must be confirmed as in either the small intestine or the colon because of the potential for small intestinal stenosis in the former case. It is often difficult to confirm the location of the PC by abdominal radiograph. We report the case of one patient who did not excrete the PC within 30 hours and for whom it was difficult to distinguish whether the PC was in the small intestine or the colon on abdominal series. Abdominal sonography revealed the PC in the colon and subsequent CE was performed without complication.

Genome assembly of Genji firefly (Nipponoluciola cruciata) reveals novel luciferase-like luminescent proteins without peroxisome targeting signal.

The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662 Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.

Development of DNA aptamers universally bound to single-chain fragment variables and their applications in bioprocess monitoring.

Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with KD = 2.1 µM or KD = 1.6 µM toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and anti-hemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10-500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real-time and versatile monitoring of large-scale scFv production.

Dual-color-emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy.

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long-pass emission filter with single-wavelength excitation, which is technically easier to use than dual-emission fluorescent proteins that require dual-wavelength excitation.

Feasibility of spiral enteroscopy in Japanese patients: study in two tertiary hospitals.

BACKGROUND AND AIM: Despite recent advances in enteroscopy, such as balloon enteroscopy, accessing the small intestine remains challenging. Spiral enteroscopy is a novel technique in which an endoscope is fitted with a rotating overtube that has a soft spiral fin at the tip. Whereas spiral enteroscopy is beginning to be carried out in Western countries, it is not common in many Asian countries. The aim of the present study was to evaluate the efficacy and safety of spiral enteroscopy in Japanese patients. METHODS: We prospectively conducted spiral enteroscopy in patients with suspected or known small bowel disease. All procedures were carried out using a spiral overtube. The main outcome measurements of the study were diagnosis rate, endoscopic intervention rate, and complication rate. RESULTS: Thirty-two patients underwent spiral enteroscopy. Spiral enteroscopy diagnosed 16 patients (50%) with small intestinal lesions, including six malignant lymphomas (19%), three erosions or ulcers (9%), three polyps (9%), two angioectasias (6%), one carcinoma (3%), and one submucosal tumor (3%). Additionally, four patients underwent endoscopic interventions (13%). Mallory-Weiss syndrome occurred in one patient (3%). No perforation occurred in any patient (0%). CONCLUSIONS: Our initial experience of spiral enteroscopy suggests that it can be introduced safely, but it is relatively invasive and technically demanding. More experience is needed to conduct spiral enteroscopy easily and safely.

Acute Liver Failure Due to Hypereosinophilic Syndrome Accompanied by Duodenal Perforation.

A 78-year-old woman presenting with severe acute liver failure was admitted to our hospital. On screening for the etiology of acute liver failure, it was diagnosed as being due to idiopathic hypereosinophilic syndrome (eosinophil count reported as 4766/µL; 33.8% of the white blood cells). Her medical history included marked eosinophilia, as observed six months prior to this admission. Corticosteroid therapy was initiated. During the clinical course, duodenal perforation occurred but was managed promptly by appropriate surgery. A liver biopsy, following the initiation of corticosteroid therapy, revealed degenerating hepatic cells with mild eosinophilic infiltration. With corticosteroid therapy, the liver function improved.

Single-agent gemcitabine in elderly patients with unresectable biliary tract cancer.

BACKGROUND/AIMS: This study examined the effect of systemic chemotherapy with gemcitabine (GEM) on survival in elderly patients (aged > or = 70 years) with unresectable biliary tract cancer as compared with best supportive care (BSC). METHODOLOGY: We conducted a retrospective study of consecutive patients with unresectable biliary tract cancer administered GEM (800-1,000 mg/m2) on days 1, 8 and 15 every 4 weeks as a first-line treatment. Eligibility included age 70 years and over, and bile duct carcinoma or gallbladder cancer. RESULTS: Twenty-eight patients were enrolled: 13 (46.4%) received chemotherapy with GEM and 15 (53.6%) received BSC. No cases of complete or partial response were observed. Stable and progressive disease was observed in 9 (69.2%) and 2 patients (15.4%), respectively. Disease control rate was 69.2%. The median overall survival time of patients treated with GEM and BSC was 9.1 and 2.9 months, and the 1-year survival rates were 15.4% and 6.7%, respectively. Grade 3/4 neutropenia occurred in three (23.1%), leukopenia in two (15.4%) and anemia in one patient (7.7%). Grade 3 non-hematologic toxicities were constipation (7.7%) and fatigue (7.7%). CONCLUSIONS: Chemotherapy with single-agent GEM is a safe and well tolerated regimen for elderly patients with unresectable biliary tract cancer.

Single-agent gemcitabine in elderly patients with unresectable biliary tract cancer.

BACKGROUND/AIMS: This study examines the effect of systemic chemotherapy with gemcitabine (GEM) on survival in elderly patients (aged > or =70 years) with unresectable biliary tract cancer and compares it with best supportive care (BSC). METHODOLOGY: We conducted a retrospective study of consecutive patients aged > or =70 years, with unresectable biliary tract cancer who were administered GEM (800-1000 mg/m2) on days 1, 8, and 15 every 4 weeks as a first-line treatment. RESULTS: Twenty-eight patients were enrolled: 13 (46.4%) received chemotherapy with GEM and 15 (53.6%) received BSC. No cases of complete or partial response were observed. Stable disease was observed in 9 patients (69.2%) and progressive disease in 2 patients (15.4%). Disease control rate was 69.2%. The median overall survival time of patients treated with GEM and BSC was 9.1 and 2.9 months, and the 1-year survival rates were 15.4% and 6.7% respectively. Grade 3/4 neutropenia occurred in three patients (23.1%), leukopenia in two patients (15.4%) and anemia in one patient (7.7%). Grade 3 non-hematologic toxicities were constipation (7.7%) and fatigue (7.7%). CONCLUSIONS: Chemotherapy with single-agent GEM is a safe and well tolerated regimen for elderly patients with unresectable biliary tract cancer.

Cloning and mutagenetic modification of the firefly luciferase gene and its use for bioluminescence microscopy of engrailed expression during Drosophila metamorphosis.

Bioluminescence microscopy is an area attracting considerable interest in the field of cell biology because it offers several advantages over fluorescence microscopy, including no requirement for excitation light and being phototoxicity free. This method requires brighter luciferase for imaging; however, suitable genetic resource material for this purpose is not available at present. To achieve brighter bioluminescence microscopy, we developed a new firefly luciferase. Using the brighter luciferase, a reporter strain of Drosophila Gal4-UAS (Upstream Activating Sequence) system was constructed. This system demonstrated the expression pattern of engrailed, which is a segment polarity gene, during Drosophila metamorphosis by bioluminescence microscopy, and revealed drastic spatiotemporal change in the engrailed expression pattern during head eversion in the early stage of pupation.

Evaluation of the population structure and phylogeography of the Japanese Genji firefly, Luciola cruciata, at the nuclear DNA level using RAD-Seq analysis.

The Genji firefly, Luciola cruciata, is widely distributed throughout the major Japanese islands (Honshu, Shikoku, and Kyushu) and distinguished into two ecological types on the basis of the flash interval of the mate-seeking males (4-sec slow-flash or 2-sec fast-flash intervals). The boundary of the ecological types corresponds to the Fossa Magna, a great rupture zone that separates eastern and western Japan. Although the degree of genetic differentiation of the two types has been evaluated using allozyme and mitochondrial DNA sequence data, it has not been evaluated using genome-wide data. Based on the genome-wide data obtained using single-end restriction-site-associated DNA (RAD-Seq), principal component, gene-level phylogenetic tree, admixture, and Wright's fixation index analyses, we identified three phylogenetic groups in L. cruciata: East-Honshu, West-Honshu, and Kyushu. This grouping corresponds to the ecological types: East-Honshu to the slow-flash type and West-Honshu and Kyushu to the fast-flash type. Although introgression was exceptionally observed around adjacent or artificially transplanted areas, gene flow among the groups was almost absent in the natural populations. The phylogenetic tree under the coalescent model also evaluated differentiation among the East-Honshu, West-Honshu and Kyushu groups. Furthermore, because the distribution patterns of the three groups are consistent with the geological history of Japanese islands, a vicariant speciation scenario of L. cruciata is concluded. In addition, we identified genetic markers that can be used to distinguish the three genetic groups for genetic management of firefly transplantation in nature conservation and regeneration.