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.

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.

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.

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.

Combined treatment with a selective PDE10A inhibitor TAK-063 and either haloperidol or olanzapine at subeffective doses produces potent antipsychotic-like effects without affecting plasma prolactin levels and cataleptic responses in rodents.

Activation of indirect pathway medium spiny neurons (MSNs) via promotion of cAMP production is the principal mechanism of action of current antipsychotics with dopamine D2 receptor antagonism. TAK-063 [1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one] is a novel phosphodiesterase 10A inhibitor that activates both direct and indirect pathway MSNs through increasing both cAMP and cGMP levels by inhibition of their degradation. The activation of indirect pathway MSNs through the distinct mechanism of action of these drugs raises the possibility of augmented pharmacological effects by combination therapy. In this study, we evaluated the potential of combination therapy with TAK-063 and current antipsychotics, such as haloperidol or olanzapine after oral administration. Combined treatment with TAK-063 and either haloperidol or olanzapine produced a significant increase in phosphorylation of glutamate receptor subunit 1 in the rat striatum. An electrophysiological study using rat corticostriatal slices showed that TAK-063 enhanced N-methyl-D -aspartic acid receptor-mediated synaptic responses in both direct and indirect pathway MSNs to a similar extent. Further evaluation using pathway-specific markers revealed that coadministration of TAK-063 with haloperidol or olanzapine additively activated the indirect pathway, but not the direct pathway. Combined treatment with TAK-063 and either haloperidol or olanzapine at subeffective doses produced significant effects on methamphetamine- or MK-801-induced hyperactivity in rats and MK-801-induced deficits in prepulse inhibition in mice. TAK-063 at 0.1 mg/kg did not affect plasma prolactin levels and cataleptic response from antipsychotics in rats. Thus, TAK-063 may produce augmented antipsychotic-like activities in combination with antipsychotics without effects on plasma prolactin levels and cataleptic responses in rodents.

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.

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.

Combining Fluorescence and Bioluminescence Microscopy to Study the Series of Events from Cellular Signal Transduction to Gene Expression.

The molecular interactions and translocation of signal transduction factors in individual cells can be imaged by fluorescence microscopy. Alternatively, downstream promoter activity in single cells can be imaged by bioluminescence microscopy. However, the same stimuli can lead to different gene expression responses in individual cells. For this reason, it is desirable to simultaneously image signal transduction and gene expression events in the same cells. Here, we describe a method that combines fluorescence and bioluminescence microscopy to image protein kinase C (PKC) translocation from the cytosol to the plasma membrane and the expression of nuclear factor kappa-light polypeptide B (NF-κB)-regulated genes. © 2017 by John Wiley & Sons, Inc.

Colonoscopy reduces colorectal cancer mortality: A multicenter, long-term, colonoscopy-based cohort study.

BACKGROUND: There are limited colonoscopy-based cohort data concerning the effectiveness of colonoscopy in reducing colorectal cancer deaths. The aim of this study was to clarify whether colonoscopy reduces colorectal cancer mortality. METHODS: A cohort of 18,816 patients who underwent colonoscopy without a diagnosis of colorectal cancer between 2001 and 2010 at high colonoscopy procedure volume centers was selected. Patient characteristics and colonoscopy findings were assessed. The main endpoint was colorectal cancer death (all, right-sided, and left-sided cancers), and data were censored at the time of the final visit or the final colonoscopy. The standardized all colorectal, colon, and rectal cancer mortality rates were estimated with reference to those of the general Japanese population. Additional outcome was all- cause death and the standardized all-cause mortality rate was also estimated. RESULTS: The total observed person-year mortality for colorectal cancer was 67,119. Of these, 4, 3, and 1 patients died from colorectal, colon, and rectal cancers, respectively; these values were significantly lower than the number of expected deaths in the general population, estimated to be 53.1, 34.0, and 19.1, respectively. The standardized mortalities for all colorectal, colon, and rectal cancers were 0.08 (95% confidence interval (CI), 0.02-0.17), 0.09 (95% CI, 0.02-0.22), and 0.05 (95% CI, 0.0002-0.21), respectively. There were 586 all-cause deaths (3.11%) during the observation period. The standardized all-cause mortality ratios were 0.22 (95% CI, 0.206-0.23). CONCLUSIONS: The colorectal cancer mortality of patients who received colonoscopy without colorectal cancer diagnosis decreased significantly compared with that of individuals in the general population. These results were compatible even in patients with right-sided colon cancer.

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.

Genetic deletion of GPR52 enhances the locomotor-stimulating effect of an adenosine A2A receptor antagonist in mice: A potential role of GPR52 in the function of striatopallidal neurons.

G protein-coupled receptor 52 (GPR52) is largely co-expressed with dopamine D2 receptor (DRD2) in the striatum and nucleus accumbens, and this expression pattern is similar to that of adenosine A2A receptor (ADORA2A). GPR52 has been proposed as a therapeutic target for positive symptoms of schizophrenia, based on observations from pharmacological and transgenic mouse studies. However, the physiological role of GPR52 in dopaminergic functions in the basal ganglia remains unclear. Here, we used GPR52 knockout (KO) mice to examine the role of GPR52 in dopamine receptor-mediated and ADORA2A-mediated locomotor activity and dopamine receptor signaling. High expression of GPR52 protein in the striatum, nucleus accumbens, and lateral globus pallidus of wild type (WT) littermates was confirmed by immunohistochemical analysis. GPR52 KO and WT mice exhibited almost identical locomotor responses to the dopamine releaser methamphetamine and the N-methyl-d-aspartate antagonist MK-801. In contrast, the locomotor response to the ADORA2A antagonist istradefylline was significantly augmented in GPR52 KO mice compared to WT mice. Gene expression analysis revealed that striatal expression of DRD2, but not of dopamine D1 receptor and ADORA2A, was significantly decreased in GPR52 KO mice. Moreover, a significant reduction in the mRNA expression of enkephalin, a marker of the activity of striatopallidal neurons, was observed in the striatum of GPR52 KO mice, suggesting that GPR52 deletion could enhance DRD2 signaling. Taken together, these results imply the physiological relevance of GPR52 in modulating the function of striatopallidal neurons, possibly by interaction of GPR52 with ADORA2A and DRD2.

Efficacy of repeat review with flexible spectral imaging color enhancement in patients with no findings by capsule endoscopy.

BACKGROUND/AIM: The efficacy of flexible spectral imaging color enhancement (FICE) ch. 1 (F1) for the detection of ulcerative lesions and angioectasias in the small intestine with capsule endoscopy (CE) has been reported. In the present study, we evaluated whether F1 could detect incremental findings in patients with no findings in a standard review mode. PATIENTS AND METHODS: In total, 52 patients (age: 60.1 ± 15.3 years; 30 males) with obscure gastrointestinal bleeding (OGIB) who underwent CE and in whom no lesion was detected in the small intestine in the standard mode (first review) were enrolled. Two experienced endoscopists independently reviewed CE videos again by F1 (second review). The following findings were defined to be significant: Ulcers, erosions, aphthas, angioectasias, tumors, and bleeding. Incremental findings at the second review were checked at F1 and in standard mode by the two reviewers (third review). Finally, the findings were confirmed by the agreement of the two reviewers at the third review. RESULTS: F1 detected five significant lesions in three patients with overt OGIB; three erosions, one aphtha, and one angioectasia. For nonsignificant lesions, F1 detected 12 red mucosas and 16 red spots. Moreover, 29 patients with 71 findings were considered false positives. CONCLUSION: F1 detected incremental significant findings in a small percentage of patients with no findings in the standard review mode. In addition, F1 showed many false-positive findings. The incremental effect of a repeated review by F1 in patients with no findings in the first review is limited.

Vonoprazan versus conventional proton pump inhibitor-based triple therapy as first-line treatment against Helicobacter pylori: A multicenter retrospective study in clinical practice.

OBJECTIVE: Vonoprazan is a potassium-competitive acid blocker, a new type of acid-suppressing drug, and has recently become available for peptic ulcers, gastroesophageal reflux disease, and Helicobacter pylori (H. pylori) eradication. Its efficacy for H. pylori eradication has been reported. However, the evidence for its efficacy and feasibility remains limited. We aimed to compare the feasibility, effectiveness and safety of vonoprazan-based triple therapy with conventional proton pump inhibitor (PPI)-based triple therapy in multicenter clinical practice. METHODS: We performed a multicenter retrospective study on patients receiving first-line H. pylori eradication therapy between March 2013 and November 2015 with either vonoprazan-based triple therapy or conventional PPI-based triple therapy. RESULTS: A total of 2715 patients aged 63.0 ± 12.1 years (1412 [52.0%] males) were analyzed. Eradication rates were 87.2% (368/422) for vonoprazan-based therapy and 72.4% (1661/2293) for conventional PPI-based therapy (P < 0.01). Among the former group, there were 10 cases of diarrhea, six of nausea/vomiting, and five of rash, but the rates of these adverse events were similar to those in the conventional PPI group. CONCLUSION: Vonoprazan-based triple therapy is feasible, and has a higher rate for H. pylori eradication than conventional PPI as a first-line regimen.

TiLIA: a software package for image analysis of firefly flash patterns.

As flash signaling patterns of fireflies are species specific, signal-pattern analysis is important for understanding this system of communication. Here, we present time-lapse image analysis (TiLIA), a free open-source software package for signal and flight pattern analyses of fireflies that uses video-recorded image data. TiLIA enables flight path tracing of individual fireflies and provides frame-by-frame coordinates and light intensity data. As an example of TiLIA capabilities, we demonstrate flash pattern analysis of the fireflies Luciola cruciata and L. lateralis during courtship behavior.

TAK-063, a PDE10A Inhibitor with Balanced Activation of Direct and Indirect Pathways, Provides Potent Antipsychotic-Like Effects in Multiple Paradigms.

Phosphodiesterase 10A (PDE10A) inhibitors are expected to be novel drugs for schizophrenia through activation of both direct and indirect pathway medium spiny neurons. However, excess activation of the direct pathway by a dopamine D1 receptor agonist SKF82958 canceled antipsychotic-like effects of a dopamine D2 receptor antagonist haloperidol in methamphetamine (METH)-induced hyperactivity in rats. Thus, balanced activation of these pathways may be critical for PDE10A inhibitors. Current antipsychotics and the novel PDE10A inhibitor TAK-063, but not the selective PDE10A inhibitor MP-10, produced dose-dependent antipsychotic-like effects in METH-induced hyperactivity and prepulse inhibition in rodents. TAK-063 and MP-10 activated the indirect pathway to a similar extent; however, MP-10 caused greater activation of the direct pathway than did TAK-063. Interestingly, the off-rate of TAK-063 from PDE10A in rat brain sections was faster than that of MP-10, and a slower off-rate PDE10A inhibitor with TAK-063-like chemical structure showed an MP-10-like pharmacological profile. In general, faster off-rate enzyme inhibitors are more sensitive than slower off-rate inhibitors to binding inhibition by enzyme substrates. As expected, TAK-063 was more sensitive than MP-10 to binding inhibition by cyclic nucleotides. Moreover, an immunohistochemistry study suggested that cyclic adenosine monophosphate levels in the direct pathway were higher than those in the indirect pathway. These data can explain why TAK-063 showed partial activation of the direct pathway compared with MP-10. The findings presented here suggest that TAK-063's antipsychotic-like efficacy may be attributable to its unique pharmacological properties, resulting in balanced activation of the direct and indirect striatal pathways.

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.

Risk factors for small bowel angioectasia: The impact of visceral fat accumulation.

AIM: To investigate visceral fat accumulation in association with the risk of small bowel angioectasia. METHODS: We retrospectively investigated 198 consecutive patients who underwent both capsule endoscopy and CT for investigation of obscure gastrointestinal bleeding (OGIB) from January 2009 to September 2013. The visceral fat area (VFA) and subcutaneous fat area were measured by CT, and information on comorbidities, body mass index, and medications was obtained from their medical records. Logistic regression analysis was used to evaluate associations. RESULTS: Capsule endoscopy revealed small bowel angioectasia in 18/198 (9.1%) patients with OGIB. Compared to patients without small bowel angioectasia, those with small bowel angioectasia had a significantly higher VFA (96 ± 76.0 cm(2) vs 63.4 ± 51.5 cm(2), P = 0.016) and a higher prevalence of liver cirrhosis (61% vs 22%, P < 0.001). The proportion of patients with chronic renal failure was higher in patients with small bowel angioectasia (22% vs 9%, P = 0.11). There were no significant differences in subcutaneous fat area or waist circumference. The prevalence of small bowel angioectasia progressively increased according to the VFA. Multivariate analysis showed that the VFA [odd ratio (OR) for each 10-cm(2) increment = 1.1; [95% confidence interval (CI): 1.02-1.19; P = 0.021] and liver cirrhosis (OR = 6.1, 95%CI: 2.2-18.5; P < 0.001) were significant risk factors for small bowel angioectasia. CONCLUSION: VFA is positively associated with the prevalence of small bowel angioectasia, for which VFA and liver cirrhosis are independent risk factors in patients with OGIB.

Combining fluorescence and bioluminescence microscopy.

Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 µM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.