Publications by authors named "Tomoki Matsuda"

86 Publications

Multicolor Bioluminescence Imaging of Subcellular Structures and Multicolor Calcium Imaging in Single Living Cells.

Methods Mol Biol 2021 ;2350:229-237

SANKEN (The Institute of Scientific and Industrial Research), Osaka University, Osaka, Ibaraki, Japan.

The recent development of the bright luciferase NanoLuc (Nluc) has greatly improved the sensitivity of bioluminescence imaging, enabling real-time cellular imaging with high spatial resolution. However, the limited color variants of Nluc have restricted its wider application to multicolor imaging of biological phenomena. To address this issue, we developed five new spectral variants of the bright bioluminescent protein with emissions across the visible spectrum. In this chapter, we describe the following two protocols for single-cell bioluminescence imaging: (a) multicolor bioluminescence imaging of subcellular structures and (b) multicolor calcium imaging in single living cells.
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http://dx.doi.org/10.1007/978-1-0716-1593-5_14DOI Listing
September 2021

Automatic anatomical classification of colonoscopic images using deep convolutional neural networks.

Gastroenterol Rep (Oxf) 2021 Jun 7;9(3):226-233. Epub 2020 Dec 7.

Tada Tomohiro Institute of Gastroenterology and Proctology, Saitama, Japan.

Background: A colonoscopy can detect colorectal diseases, including cancers, polyps, and inflammatory bowel diseases. A computer-aided diagnosis (CAD) system using deep convolutional neural networks (CNNs) that can recognize anatomical locations during a colonoscopy could efficiently assist practitioners. We aimed to construct a CAD system using a CNN to distinguish colorectal images from parts of the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum.

Method: We constructed a CNN by training of 9,995 colonoscopy images and tested its performance by 5,121 independent colonoscopy images that were categorized according to seven anatomical locations: the terminal ileum, the cecum, ascending colon to transverse colon, descending colon to sigmoid colon, the rectum, the anus, and indistinguishable parts. We examined images taken during total colonoscopy performed between January 2017 and November 2017 at a single center. We evaluated the concordance between the diagnosis by endoscopists and those by the CNN. The main outcomes of the study were the sensitivity and specificity of the CNN for the anatomical categorization of colonoscopy images.

Results: The constructed CNN recognized anatomical locations of colonoscopy images with the following areas under the curves: 0.979 for the terminal ileum; 0.940 for the cecum; 0.875 for ascending colon to transverse colon; 0.846 for descending colon to sigmoid colon; 0.835 for the rectum; and 0.992 for the anus. During the test process, the CNN system correctly recognized 66.6% of images.

Conclusion: We constructed the new CNN system with clinically relevant performance for recognizing anatomical locations of colonoscopy images, which is the first step in constructing a CAD system that will support us during colonoscopy and provide an assurance of the quality of the colonoscopy procedure.
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http://dx.doi.org/10.1093/gastro/goaa078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8309686PMC
June 2021

Method for Detecting Emission Spectral Change of Bioluminescent Ratiometric Indicators by a Smartphone.

Methods Mol Biol 2021 ;2274:295-304

The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Ibaraki, Japan.

The application of smartphones as detectors is essential to achieve ubiquitous measurement targeting biomolecules. Because bioluminescence (BL), as a tag for a target sample, does not require an excitation light source, it can be combined with a smartphone to constitute a compact and mobile measurement system. A method was recently established to detect the spectral change of ratiometric indicators based on bioluminescence resonance energy transfer with a smartphone camera. For example, it was possible to detect changes in the BL color of the Ca indicator quantitatively and easily calculate the concentration of free Ca by setting appropriate image acquisition conditions in a smartphone application. In this paper, we describe techniques to obtain scientifically relevant and reliable BL data with such a convenient instrument. This protocol expands the potential of the smartphone as a personal imaging device with high mobility that can be used anywhere.
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http://dx.doi.org/10.1007/978-1-0716-1258-3_25DOI Listing
June 2021

Genetically Encoded Photosensitizer for Destruction of Protein or Cell Function.

Adv Exp Med Biol 2021 ;1293:265-279

The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Osaka, Japan.

There are several paths when excited molecules return to the ground state. In the case of fluorescent molecules, the dominant path is fluorescence emission that is greatly contributing to bioimaging. Meanwhile, photosensitizers transfer electron or energy from chromophore to the surrounding molecules, including molecular oxygen. Generated reactive oxygen species has potency to attack other molecules by oxidation. In this chapter, we introduce the chromophore-assisted light inactivation (CALI) method using a photosensitizer to inactivate proteins in a spatiotemporal manner and development of CALI tools, which is useful for investigation of protein functions and dynamics, by inactivation of the target molecules. Moreover, photosensitizers with high efficiency make it possible optogenetic control of cell ablation in living organisms and photodynamic therapy. Further development of photosensitizers with different excitation wavelengths will contribute to the investigation of multiple proteins or cell functions through inactivation in the different positions and timings.
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http://dx.doi.org/10.1007/978-981-15-8763-4_16DOI Listing
February 2021

Hyperspectral two-photon excitation microscopy using visible wavelength.

Opt Lett 2021 Jan;46(1):37-40

We demonstrate hyperspectral imaging by visible-wavelength two-photon excitation microscopy using line illumination and slit-confocal detection. A femtosecond pulsed laser light at 530 nm was used for the simultaneous excitation of fluorescent proteins with different emission wavelengths. The use of line illumination enabled efficient detection of hyperspectral images and achieved simultaneous detection of three fluorescence spectra in the observation of living HeLa cells with an exposure time of 1 ms per line, which is equivalent to about 2 µs per pixel in point scanning, with 160 data points per spectrum. On combining linear spectral unmixing techniques, localization of fluorescent probes in the cells was achieved. A theoretical investigation of the imaging property revealed high-depth discrimination property attained through the combination of nonlinear excitation and slit detection.
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http://dx.doi.org/10.1364/OL.413526DOI Listing
January 2021

Smartphone-Based Portable Bioluminescence Imaging System Enabling Observation at Various Scales from Whole Mouse Body to Organelle.

Sensors (Basel) 2020 Dec 14;20(24). Epub 2020 Dec 14.

The Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan.

Current smartphones equipped with high-sensitivity and high-resolution sensors in the camera can respond to the needs of low-light imaging, streaming acquisition, targets of various scales, etc. Therefore, a smartphone has great potential as an imaging device even in the scientific field and has already been introduced into biomolecular imaging using fluorescence tags. However, owing to the necessity of an excitation light source, fluorescence methods impair its mobility. Bioluminescence does not require illumination; therefore, imaging with a smartphone camera is compact and requires minimal devices, thus making it suitable for personal and portable imaging devices. Here, we report smartphone-based methods to observe biological targets in various scales using bioluminescence. In particular, we demonstrate, for the first time, that bioluminescence can be observed in an organelle in a single living cell using a smartphone camera by attaching a detachable objective lens. Through capturing color changes with the camera, changes in the amount of target molecules was detected using bioluminescent indicators. The combination of bioluminescence and a mobile phone makes possible a compact imaging system without an external light source and expands the potential of portable devices.
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http://dx.doi.org/10.3390/s20247166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764933PMC
December 2020

Cancer of unknown primary inside the gastric wall identified by endoscopic submucosal dissection.

Clin J Gastroenterol 2021 Apr 18;14(2):506-510. Epub 2020 Nov 18.

Department of Gastroenterology, Sendai Kousei Hospital, 4-15, Hirose-machi, Aoba-ku, Sendai, Miyagi, 980-0873, Japan.

We report the rare and interesting case of cancer of unknown primary (CUP) detected by endoscopic submucosal dissection (ESD). A 67-year-old man with a gastric adenoma was referred to our hospital for endoscopic treatment. Esophagogastroduodenoscopy revealed a 15-mm submucosal tumor (SMT) at the lesser curvature of the lower gastric body, near the gastric adenoma. Both lesions were resected by ESD. Pathological examination showed that the SMT was a poorly differentiated adenocarcinoma with lymphatic tissue. Additional surgical resection was performed, and the lymph nodes were found to have the same pathological findings as the SMT. These lesions were diagnosed as CUP because the obvious primary site was not detected with additional examination. The patient has been followed up for 24 months without recurrence.
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http://dx.doi.org/10.1007/s12328-020-01297-zDOI Listing
April 2021

Palmitoylated CKAP4 regulates mitochondrial functions through an interaction with VDAC2 at ER-mitochondria contact sites.

J Cell Sci 2020 11 10;133(21). Epub 2020 Nov 10.

Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan

Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces the alteration of mitochondrial structures and increases the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys of CKAP4 was required for the binding between CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca concentration increased and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, cancer cell proliferation under low-glucose conditions and xenograft tumor formation. The phenotypes were not rescued by expression of a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.249045DOI Listing
November 2020

LC3 lipidation is essential for TFEB activation during the lysosomal damage response to kidney injury.

Nat Cell Biol 2020 10 28;22(10):1252-1263. Epub 2020 Sep 28.

Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan.

Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)-a master transcriptional regulator of lysosomal biogenesis and autophagy-is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.
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http://dx.doi.org/10.1038/s41556-020-00583-9DOI Listing
October 2020

The risks and characteristics of the delayed bleeding after endoscopic submucosal dissection for early gastric carcinoma in cases with anticoagulants.

Scand J Gastroenterol 2020 Oct 14;55(10):1253-1260. Epub 2020 Sep 14.

Department of Gastroenterology, Sendai Kousei Hospital, Sendai, Japan.

Background: Endoscopic submucosal dissection (ESD) is a minimally invasive treatment for early gastric carcinoma. Vitamin K antagonists and direct oral anticoagulants (DOAC) were reported to increase the risk of delayed bleeding after ESD. However, the evaluation of ESD cases taking anticoagulants is scarce. We analyzed the risk and characteristics of delayed bleeding after gastric ESD in patients on anticoagulants.

Methods: We performed a retrospective observational study at a single center. Consecutive patients who underwent ESD for early gastric carcinoma and took anticoagulants, including warfarin, rivaroxaban, dabigatran, apixaban, and edoxaban, between January 2012 and December 2018, were analyzed. We also calculated delayed bleeding rates for those without anticoagulants.

Results: Of 1855 eligible patients who underwent gastric ESDs, 143 took anticoagulants. Delayed bleeding occurred in 30 (21.0%) cases taking anticoagulants, with 15 (19.5%) cases in the DOAC group [rivaroxaban, seven cases (21.2%); dabigatran, four cases (20.0%); apixaban, four cases (23.5%); and edoxaban, zero cases (0%)] and 15 cases (22.7%) in the warfarin group. Furthermore, 43/344 (12.5%) patients taking antiplatelets and 76/1368 (5.6%) patients without antithrombic drugs experienced delayed bleeding. Multivariable logistic analysis revealed post-heart valve replacement (OR, 6.56; 95% CI, 1.75-24.7;  < .05) as a risk for delayed bleeding in warfarin-taking patients, while no statistically significant factor was found in DOAC-taking patients.

Conclusions: Anticoagulants were associated with a high incidence of severe delayed bleeding. Careful attention should be paid to patients on anticoagulants after gastric ESD, especially those on warfarin after heart valve replacement.
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http://dx.doi.org/10.1080/00365521.2020.1817542DOI Listing
October 2020

Massive esophageal hemorrhage after a MitraClip procedure successfully treated by balloon compression.

Clin J Gastroenterol 2020 Oct 25;13(5):693-696. Epub 2020 Jun 25.

Department of Gastroenterology, Sendai Kousei Hospital, 4-15, Hirose-machi, Aoba-ku, Sendai, Miyagi, 980-0873, Japan.

MitraClip procedure is an effective treatment for mitral regurgitation, performed globally. During the MitraClip procedure, transesophageal echocardiography is an essential modality to perform the operation safely. Although a few transesophageal echocardiography-related complications, such as esophageal hematoma, have been reported, there are no reports on massive esophageal bleeding after the MitraClip procedure. We present the first case of massive esophageal hemorrhage caused by transesophageal echocardiography after a MitraClip procedure and the successful treatment by balloon compression using a Sengstaken-Blakemore tube.
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http://dx.doi.org/10.1007/s12328-020-01160-1DOI Listing
October 2020

Bioluminescent Ratiometric Indicator for Analysis of Water Hardness in Household Water.

Sensors (Basel) 2020 Jun 2;20(11). Epub 2020 Jun 2.

The Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan.

Water hardness (WH) is a useful parameter for testing household water, such as drinking, cooking, and washing water. Many countries around the world use pipeline water in their houses, but there is a need to monitor the WH because hard water has a negative impact on appliances. Currently, WH is often measured using chemical dye-based WH indicators, and these techniques require expensive equipment, and trained personnel. Therefore, a low-cost and simple measurement method has been desired. Here, we report LOTUS-W, which consists of a luciferase, Nanoluc, a yellow fluorescent protein Venus, and a Ca/Mg detection domain of human centrin 3. The binding of Ca/Mg to this indicator changes the conformation of human centrin 3, and induces bioluminescence resonance energy transfer (BRET) from Nanoluc to Venus, which changes its emission spectrum about 140%. The dissociation constants of LOTUS-W for Ca/Mg are approximately several mM, making it suitable for measuring WH in the household water. With this indicator in combination with a smartphone, we have demonstrated that it is possible to evaluate WH easily and quickly. This novel indicator has the potential to be used for measuring not only household water but also water used in the food industry, etc.
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http://dx.doi.org/10.3390/s20113164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308811PMC
June 2020

A case of gastric adenocarcinoma considered to originate from a sporadic fundic gland polyp in a Helicobacter pylori-uninfected stomach.

Clin J Gastroenterol 2020 Oct 28;13(5):740-745. Epub 2020 May 28.

Department of Gastroenterology, Sendai Kousei Hospital, 4-15 Hirosemachi, Aoba-ku, Sendai-shi, Miyagi-ken, 980-0873, Japan.

We encountered a rare case of gastric adenocarcinoma considered to arise from a sporadic fundic gland polyp (FGP). A woman in her 70 s, who had been prescribed a proton pump inhibitor for 5 years, was referred to our institution for further investigation and treatment of a gastric lesion. White light endoscopy showed numerous isochromatic FGPs in the greater curvature of the gastric body and a 15-mm reddish polypoid lesion with uneven surface characteristics. Magnifying endoscopy with narrow band imaging revealed an irregular granular microsurface structure with irregular microvessels, which is suggestive of cancer. The absence of atrophic changes in the entire gastric mucosa was confirmed endoscopically and histologically, and multiple Helicobacter pylori (HP) tests were negative. An en bloc resection was performed by polypectomy. The specimen showed adenocarcinoma that was thought to arise from an FGP. The lesion consisted of cystically dilated fundic glands in the basal part and neoplastic cells with nuclear atypia and high nuclear-cytoplasmic ratio in the foveolar part; on the basis of these findings, noninvasive adenocarcinoma was diagnosed. Although both adenocarcinoma in an HP-uninfected stomach and epithelial high-grade dysplasia in a sporadic FGP are extremely rare, this possibility should be considered when performing esophagogastroduodenoscopy.
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http://dx.doi.org/10.1007/s12328-020-01139-yDOI Listing
October 2020

Automatic detection of various abnormalities in capsule endoscopy videos by a deep learning-based system: a multicenter study.

Gastrointest Endosc 2021 01 15;93(1):165-173.e1. Epub 2020 May 15.

AI Medical Service Inc, Tokyo, Japan; Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Tada Tomohiro Institute of Gastroenterology and Proctology, Saitama, Japan.

Background And Aims: A deep convolutional neural network (CNN) system could be a high-level screening tool for capsule endoscopy (CE) reading but has not been established for targeting various abnormalities. We aimed to develop a CNN-based system and compare it with the existing QuickView mode in terms of their ability to detect various abnormalities.

Methods: We trained a CNN system using 66,028 CE images (44,684 images of abnormalities and 21,344 normal images). The detection rate of the CNN for various abnormalities was assessed per patient, using an independent test set of 379 consecutive small-bowel CE videos from 3 institutions. Mucosal breaks, angioectasia, protruding lesions, and blood content were present in 94, 29, 81, and 23 patients, respectively. The detection capability of the CNN was compared with that of QuickView mode.

Results: The CNN picked up 1,135,104 images (22.5%) from the 5,050,226 test images, and thus, the sampling rate of QuickView mode was set to 23% in this study. In total, the detection rate of the CNN for abnormalities per patient was significantly higher than that of QuickView mode (99% vs 89%, P < .001). The detection rates of the CNN for mucosal breaks, angioectasia, protruding lesions, and blood content were 100% (94 of 94), 97% (28 of 29), 99% (80 of 81), and 100% (23 of 23), respectively, and those of QuickView mode were 91%, 97%, 80%, and 96%, respectively.

Conclusions: We developed and tested a CNN-based detection system for various abnormalities using multicenter CE videos. This system could serve as an alternative high-level screening tool to QuickView mode.
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http://dx.doi.org/10.1016/j.gie.2020.04.080DOI Listing
January 2021

Automatic detection and classification of protruding lesions in wireless capsule endoscopy images based on a deep convolutional neural network.

Gastrointest Endosc 2020 07 19;92(1):144-151.e1. Epub 2020 Feb 19.

AI Medical Service Inc., Tokyo, Japan; Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Tada Tomohiro Institute of Gastroenterology and Proctology, Saitama, Japan.

Background And Aims: Protruding lesions of the small bowel vary in wireless capsule endoscopy (WCE) images, and their automatic detection may be difficult. We aimed to develop and test a deep learning-based system to automatically detect protruding lesions of various types in WCE images.

Methods: We trained a deep convolutional neural network (CNN), using 30,584 WCE images of protruding lesions from 292 patients. We evaluated CNN performance by calculating the area under the receiver operating characteristic curve (AUC), sensitivity, and specificity, using an independent set of 17,507 test images from 93 patients, including 7507 images of protruding lesions from 73 patients.

Results: The developed CNN analyzed 17,507 images in 530.462 seconds. The AUC for detection of protruding lesions was 0.911 (95% confidence interval [Cl], 0.9069-0.9155). The sensitivity and specificity of the CNN were 90.7% (95% CI, 90.0%-91.4%) and 79.8% (95% CI, 79.0%-80.6%), respectively, at the optimal cut-off value of 0.317 for probability score. In a subgroup analysis of the category of protruding lesions, the sensitivities were 86.5%, 92.0%, 95.8%, 77.0%, and 94.4% for the detection of polyps, nodules, epithelial tumors, submucosal tumors, and venous structures, respectively. In individual patient analyses (n = 73), the detection rate of protruding lesions was 98.6%.

Conclusion: We developed and tested a new computer-aided system based on a CNN to automatically detect various protruding lesions in WCE images. Patient-level analyses with larger cohorts and efforts to achieve better diagnostic performance are necessary in further studies.
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http://dx.doi.org/10.1016/j.gie.2020.01.054DOI Listing
July 2020

Hierarchical Development of Motile Polarity in Durotactic Cells Just Crossing an Elasticity Boundary.

Cell Struct Funct 2020 Feb 27;45(1):33-43. Epub 2019 Dec 27.

Laboratory of Biomedical and Biophysical Chemistry, Institute for Materials Chemistry and Engineering, Kyushu University.

Cellular durotaxis has been extensively studied in the field of mechanobiology. In principle, asymmetric mechanical field of a stiffness gradient generates motile polarity in a cell, which is a driving factor of durotaxis. However, the actual process by which the motile polarity in durotaxis develops is still unclear. In this study, to clarify the details of the kinetics of the development of durotactic polarity, we investigated the dynamics of both cell-shaping and the microscopic turnover of focal adhesions (FAs) for Venus-paxillin-expressing fibroblasts just crossing an elasticity boundary prepared on microelastically patterned gels. The Fourier mode analysis of cell-shaping based on a persistent random deformation model revealed that motile polarity at a cell-body scale was established within the first few hours after the leading edges of a moving cell passed through the boundary from the soft to the stiff regions. A fluorescence recovery after photobleaching (FRAP) analysis showed that the mobile fractions of paxillin at FAs in the anterior part of the cells exhibited an asymmetric increase within several tens of minutes after cells entered the stiff region. The results demonstrated that motile polarity in durotactic cells is established through the hierarchical step-wise development of different types of asymmetricity in the kinetics of FAs activity and cell-shaping with a several-hour time lag.Key words: Microelasticity patterned gel, durotaxis, cell polarity, focal adhesions, paxillin.
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http://dx.doi.org/10.1247/csf.19040DOI Listing
February 2020

Automatic detection of blood content in capsule endoscopy images based on a deep convolutional neural network.

J Gastroenterol Hepatol 2020 Jul 27;35(7):1196-1200. Epub 2019 Dec 27.

AI Medical Service Inc., Tokyo, Japan.

Background And Aim: Detecting blood content in the gastrointestinal tract is one of the crucial applications of capsule endoscopy (CE). The suspected blood indicator (SBI) is a conventional tool used to automatically tag images depicting possible bleeding in the reading system. We aim to develop a deep learning-based system to detect blood content in images and compare its performance with that of the SBI.

Methods: We trained a deep convolutional neural network (CNN) system, using 27 847 CE images (6503 images depicting blood content from 29 patients and 21 344 images of normal mucosa from 12 patients). We assessed its performance by calculating the area under the receiver operating characteristic curve (ROC-AUC) and its sensitivity, specificity, and accuracy, using an independent test set of 10 208 small-bowel images (208 images depicting blood content and 10 000 images of normal mucosa). The performance of the CNN was compared with that of the SBI, in individual image analysis, using the same test set.

Results: The AUC for the detection of blood content was 0.9998. The sensitivity, specificity, and accuracy of the CNN were 96.63%, 99.96%, and 99.89%, respectively, at a cut-off value of 0.5 for the probability score, which were significantly higher than those of the SBI (76.92%, 99.82%, and 99.35%, respectively). The trained CNN required 250 s to evaluate 10 208 test images.

Conclusions: We developed and tested the CNN-based detection system for blood content in CE images. This system has the potential to outperform the SBI system, and the patient-level analyses on larger studies are required.
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http://dx.doi.org/10.1111/jgh.14941DOI Listing
July 2020

Fluorescent Protein-Based Indicators for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells.

Int J Mol Sci 2019 Nov 17;20(22). Epub 2019 Nov 17.

The Institute of Scientific and Industrial Research, Osaka University, Ibaraki 567-0047, Japan.

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.
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http://dx.doi.org/10.3390/ijms20225784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887983PMC
November 2019

Visible-wavelength two-photon excitation microscopy with multifocus scanning for volumetric live-cell imaging.

J Biomed Opt 2019 11;25(1):1-5

Osaka Univ., Japan.

Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.
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http://dx.doi.org/10.1117/1.JBO.25.1.014502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008499PMC
November 2019

Colon Capsule Endoscopy versus CT Colonography in Patients with Large Non-Polypoid Tumours: A Multicentre Prospective Comparative Study (4CN Study).

Digestion 2020 1;101(5):615-623. Epub 2019 Oct 1.

Department of Coloproctology, Aizu Medical Center, Fukushima Medical University, Aizuwakamatsu, Fukushima, Japan,

Background/aims: Non-polypoid colon lesions compared with polypoid lesions has a high malignant potential. The diagnostic performance of colon capsule endoscopy (CCE) and CT colonography (CTC) for large colorectal non-polypoid tumours, that is, laterally spreading tumours is still unclear. The aim of this study is to evaluate the performance of CCE and CTC for the diagnosis of large non-polypoid tumours.

Methods: Thirty patients referred for endoscopic submucosal dissection of non-polypoid tumours measuring ≥20 mm were enrolled. Patients first underwent CCE, then colonoscopy (without resection) and CTC on the same day. An experienced gastroenterologist in a third hospital evaluated the CCE and recorded the location, size and morphology of all lesions detected, blinded to the colonoscopic findings. An experienced radiologist read the CTC under the same conditions. Colonoscopic findings were defined as the reference.

Results: A total of 30 lesions (T1 cancer: 3, Tis cancer: 7, adenoma: 14, sessile serrated adenoma/polyp: 6) in 27 patients were observed for evaluation. The capsule excretion rate within 8 h was 85% (23/27), and all capsules went beyond the target lesions. Non-polypoid tumours tend to be depicted as polypoid on CCE. Per patient sensitivities were 0.89 (24/27) by CCE and 0.70 (19/27) by CTC (p = 0.0253, McNemar), and per lesion sensitivities were 0.87 (26/30) and 0.67 (20/30) respectively (p = 0.0143). Most lesions missed by both modalities were located in the proximal colon.

Conclusion: Eighty-seven per cent of non-polypoid tumours were detected by CCE, and the sensitivity using CCE was higher than that obtained using CTC (UMIN0000014772).
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http://dx.doi.org/10.1159/000501609DOI Listing
June 2021

Clinical usefulness of a deep learning-based system as the first screening on small-bowel capsule endoscopy reading.

Dig Endosc 2020 May 2;32(4):585-591. Epub 2019 Oct 2.

Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Background And Aim: To examine whether our convolutional neural network (CNN) system based on deep learning can reduce the reading time of endoscopists without oversight of abnormalities in the capsule-endoscopy reading process.

Methods: Twenty videos of the entire small-bowel capsule endoscopy procedure were prepared, each of which included 0-5 lesions of small-bowel mucosal breaks (erosions or ulcerations). At another institute, two reading processes were compared: (A) endoscopist-alone readings and (B) endoscopist readings after the first screening by the proposed CNN. In process B, endoscopists read only images detected by CNN. Two experts and four trainees independently read 20 videos each (10 for process A and 10 for process B). Outcomes were reading time and detection rate of mucosal breaks by endoscopists. Gold standard was findings at the original institute by two experts.

Results: Mean reading time of small-bowel sections by endoscopists was significantly shorter during process B (expert, 3.1 min; trainee, 5.2 min) compared to process A (expert, 12.2 min; trainee, 20.7 min) (P < 0.001). For 37 mucosal breaks, detection rate by endoscopists did not significantly decrease in process B (expert, 87%; trainee, 55%) compared to process A (expert, 84%; trainee, 47%). Experts detected all eight large lesions (>5 mm), but trainees could not, even when supported by the CNN.

Conclusions: Our CNN-based system for capsule endoscopy videos reduced the reading time of endoscopists without decreasing the detection rate of mucosal breaks. However, the reading level of endoscopists should be considered when using the system.
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http://dx.doi.org/10.1111/den.13517DOI Listing
May 2020

Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions.

Cell Chem Biol 2019 10 15;26(10):1469-1479.e6. Epub 2019 Aug 15.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan; The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan. Electronic address:

Reversibly switchable fluorescent proteins (RSFPs) are crucial tags for super-resolution observation of protein localization and dynamics inside living cells. However, due to the high fluorescence pK (∼5-6) of most RSFPs, their usage in acidic conditions (pH 4.5-6.0) has been limited. Here, we investigated a new photochromic mechanism in Gamillus, a recently developed green fluorescent protein with acid tolerance. Gamillus exhibits negative switching with especially high contrast in acidic conditions, and its off switching is caused by trans-to-cis isomerization of the chromophore hydroxyphenyl ring that accompanies protonation. Through a combination of rational design and saturation mutagenesis, we developed two variants with enhanced switching contrasts and off-switching speeds, designated rsGamillus-S and rsGamillus-F, respectively. The fluorescence intensity, off-switching speed, and switching contrast of the rsGamillus variants are only slightly affected by changes in pH between 4.5 and 7.5. Exploiting these properties, we succeeded in high-contrast super-resolution imaging of cellular architectures in acidic conditions.
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http://dx.doi.org/10.1016/j.chembiol.2019.07.012DOI Listing
October 2019

Artificial intelligence using a convolutional neural network for automatic detection of small-bowel angioectasia in capsule endoscopy images.

Dig Endosc 2020 Mar 2;32(3):382-390. Epub 2019 Oct 2.

AI Medical Service Inc., Tokyo, Japan.

Background And Aim: Although small-bowel angioectasia is reported as the most common cause of bleeding in patients and frequently diagnosed by capsule endoscopy (CE) in patients with obscure gastrointestinal bleeding, a computer-aided detection method has not been established. We developed an artificial intelligence system with deep learning that can automatically detect small-bowel angioectasia in CE images.

Methods: We trained a deep convolutional neural network (CNN) system based on Single Shot Multibox Detector using 2237 CE images of angioectasia. We assessed its diagnostic accuracy by calculating the area under the receiver operating characteristic curve (ROC-AUC), sensitivity, specificity, positive predictive value, and negative predictive value using an independent test set of 10 488 small-bowel images, including 488 images of small-bowel angioectasia.

Results: The AUC to detect angioectasia was 0.998. Sensitivity, specificity, positive predictive value, and negative predictive value of CNN were 98.8%, 98.4%, 75.4%, and 99.9%, respectively, at a cut-off value of 0.36 for the probability score.

Conclusions: We developed and validated a new system based on CNN to automatically detect angioectasia in CE images. This may be well applicable to daily clinical practice to reduce the burden of physicians as well as to reduce oversight.
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http://dx.doi.org/10.1111/den.13507DOI Listing
March 2020

Genetically Encoded Fluorescence/Bioluminescence Bimodal Indicators for Ca Imaging.

ACS Sens 2019 07 5;4(7):1825-1834. Epub 2019 Jul 5.

Department of Biotechnology, Graduate School of Engineering , Osaka University , 2-1 Yamadaoka , Suita 565-0871 , Japan.

Fluorescent and bioluminescent genetically encoded Ca indicators (GECIs) are an indispensable tool for monitoring Ca dynamics in numerous cellular events. Although fluorescent GECIs have a high spatiotemporal resolution, their application is often confined to short-term imaging due to the external illumination that causes phototoxicity and autofluorescence from specimens. Bioluminescent GECIs overcome these pitfalls with enhanced compatibility to optogenetic manipulation and photophysiological processes; however, they are compromised for spatiotemporal resolution. Therefore, there has been a push toward the use of Ca indicators that possess the advantages of both fluorescent and bioluminescent GECI for a wide range of applications. To address this, we developed a high-affinity bimodal GECI, GLICO, using a single fluorescent protein-based GECI combined with a split luciferase. Through this novel design, the fusion protein becomes bimodal and possesses Ca sensing properties similar to those of its fluorescent ancestor and confers bioluminescence-based Ca imaging. GLICO in bioluminescence mode has the highest dynamic range (2200%) of all bioluminescent GECIs. We demonstrated the performance of GLICO in studying cytosolic Ca dynamics in different cultured cells in each mode. With the purpose of Ca imaging in high Ca content organelle, we also created a low-affinity variant, ReBLICO and performed Ca imaging of the ER in both fluorescence and bioluminescence modes. The ability to switch between fluorescence and bioluminescence modes with a single indicator would benefit transgenic applications by presenting an opportunity for a wide range of live Ca imaging in physiological and pathophysiological conditions.
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http://dx.doi.org/10.1021/acssensors.9b00531DOI Listing
July 2019

The sub-classification of type B2 vessels according to the magnifying endoscopic classification of the Japan Esophageal Society.

Dig Endosc 2020 Jan 20;32(1):49-55. Epub 2019 Aug 20.

Department of Gastroenterology, Sendai Kousei Hospital, Miyagi, Japan.

Objectives: Guidelines for magnified endoscopic diagnosis of esophageal squamous cell carcinoma (SCC) have been proposed by the Japan Esophageal Society. Type B1, B2, and B3 reflect increasing tumor invasion depths (within mucosal epithelium or into lamina propria mucosa [T1a-EP/LPM], into muscularis mucosa or superficial invasion into submucosa [T1a-MM/T1b-SM1], and into submucosa [T1b-SM2], respectively). The diagnostic accuracy of type B1 and B3 is high, but accuracy of type B2 is low. We aimed to improve the diagnostic accuracy of type B2.

Methods: We retrospectively reviewed 248 SCC lesions treated with endoscopic submucosal dissection between January 2012 and July 2018 and identified the B2 lesions. The maximum diameter of the area presenting B2 was measured and evaluated in relation to tumor invasion, for which receiver-operating characteristic (ROC) curves were generated. The optimal area size for distinguishing T1a-EP/LPM from T1a-MM or deeper invasion was determined.

Results: There were 78 lesions with B2, of which 26 (33%) were T1a-MM or T1b-SM1 SCCs. ROC curve analysis indicated that the optimal cut-off for the target area showing B2 was 4 mm. The invasion depth (EP/LPM: MM/SM1: SM2) of B2 observed in an area with a diameter <4 mm (B2-Narrow) and those with diameter ≥4 mm (B2-Broad) was 46:11:1 and 1:15:4, respectively. To predict T1a-MM or deeper invasion, B2-Broad had a sensitivity, specificity, positive predictive value, and negative predictive value of 61%, 98%, 95%, and 79%, respectively.

Conclusion: The diagnostic accuracy of type B2 was improved by evaluating the area of type B2.
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http://dx.doi.org/10.1111/den.13459DOI Listing
January 2020

Impact of Urgent Double-Balloon Enteroscopy on the Short-Term and Long-Term Outcomes in Overt Small Bowel Bleeding.

Dig Dis Sci 2019 10 17;64(10):2933-2938. Epub 2019 Apr 17.

Department of Gastroenterology, Sendai Kousei Hospital, Sendai, 980-0873, Japan.

Background: Double-balloon enteroscopy (DBE) is a safe and useful procedure for managing small bowel bleeding. However, there are limited studies regarding the preferable timing of DBE and its impact on long-term outcomes.

Aim: We aimed to evaluate the association between the timing of DBE and the long-term outcomes of patients suspected of having overt small bowel bleeding who underwent DBE.

Methods: We retrospectively reviewed a prospectively collected database of patients who underwent DBE procedures between May 2004 and April 2016. The electronic medical records were reviewed, and interviews were conducted via mail and telephone.

Results: One-hundred sixty-five patients could be followed up. The bleeding source was detected during the initial DBE (DBE-positive group) for 102 patients. Sixty-three patients had no definite lesion during the initial DBE (DBE-negative group). Urgent DBE (DBE within 24 h after the last bleeding episode) was performed more often for the DBE-positive group (50/102; 49.0%) than for the DBE-negative group (10/63; 16.1%) (p < 0.0001). Nine patients in the DBE-positive group underwent curative surgery after diagnosis. Among the remaining DBE-positive patients, 38 of 93 (40.9%) had recurrent bleeding during 2675 days of follow-up. Twenty-one of 63 patients (33.3%) in the DBE-negative group had recurrent bleeding during 2490 days of follow-up. There was no significant difference between the two groups in terms of intervals without rebleeding (p = 0.17).

Conclusion: Urgent DBE at the initial bleeding episode was useful for detecting lesions. However, the rebleeding rate was not dependent on the initial DBE results.
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http://dx.doi.org/10.1007/s10620-019-05627-1DOI Listing
October 2019

Automatic detection of erosions and ulcerations in wireless capsule endoscopy images based on a deep convolutional neural network.

Gastrointest Endosc 2019 02 25;89(2):357-363.e2. Epub 2018 Oct 25.

AI Medical Service Inc., Tokyo, Japan; Tada Tomohiro Institute of Gastroenterology and Proctology, Saitama, Japan; Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Background And Aims: Although erosions and ulcerations are the most common small-bowel abnormalities found on wireless capsule endoscopy (WCE), a computer-aided detection method has not been established. We aimed to develop an artificial intelligence system with deep learning to automatically detect erosions and ulcerations in WCE images.

Methods: We trained a deep convolutional neural network (CNN) system based on a Single Shot Multibox Detector, using 5360 WCE images of erosions and ulcerations. We assessed its performance by calculating the area under the receiver operating characteristic curve and its sensitivity, specificity, and accuracy using an independent test set of 10,440 small-bowel images including 440 images of erosions and ulcerations.

Results: The trained CNN required 233 seconds to evaluate 10,440 test images. The area under the curve for the detection of erosions and ulcerations was 0.958 (95% confidence interval [CI], 0.947-0.968). The sensitivity, specificity, and accuracy of the CNN were 88.2% (95% CI, 84.8%-91.0%), 90.9% (95% CI, 90.3%-91.4%), and 90.8% (95% CI, 90.2%-91.3%), respectively, at a cut-off value of 0.481 for the probability score.

Conclusions: We developed and validated a new system based on CNN to automatically detect erosions and ulcerations in WCE images. This may be a crucial step in the development of daily-use diagnostic software for WCE images to help reduce oversights and the burden on physicians.
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http://dx.doi.org/10.1016/j.gie.2018.10.027DOI Listing
February 2019

Kappa light-chain amyloidosis of gastrointestinal tract.

Ann Gastroenterol 2019 Jan-Feb;32(1):109. Epub 2018 Oct 3.

Department of Gastroenterology, Sendai Kousei Hospital, Japan.

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http://dx.doi.org/10.20524/aog.2018.0319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302194PMC
October 2018

False-negative double-balloon enteroscopy in overt small bowel bleeding: long-term follow-up after negative results.

Surg Endosc 2019 08 5;33(8):2635-2641. Epub 2018 Nov 5.

Department of Gastroenterology, Sendai Kousei Hospital, Sendai, Japan.

Background And Aim: Double-balloon enteroscopy (DBE) performed to investigate overt small bowel bleeding can miss the source of bleeding. We investigated the clinical outcomes of patients with negative DBE results for suspected overt small bowel bleeding, which is defined in the current guidelines as obscure gastrointestinal bleeding.

Methods: We reviewed the prospectively collected medical records of patients who underwent DBE at our hospital between May 1, 2004 and April 30, 2016. During this period, 297 patients underwent DBE for suspected overt small bowel bleeding. The first DBE yielded negative results for 83 patients (27.9%). Written interviews, telephone interviews, and medical records of these patients were reviewed in April 2017. Follow-up data were collected for 63 patients (75.9%).

Results: During a mean follow-up period of 83.5 months, re-bleeding occurred in 21 of 63 patients (33.3%) after a mean of 23.0 months after the first DBE yielded negative results. The bleeding source was identified in 19 of 21 patients (90.5%). In 15 of these 19 patients (78.9%), the source was the small intestine. Among these 15 patients, 14 (93.3%) had bleeding sites within reach of the first DBE and 3 (20%) experienced their first incidence of re-bleeding more than 3 years after the first DBE. The need for transfusion for the first bleeding episode was a predictor of re-bleeding (odds ratio 7.5; 95% confidence interval 1.7-33.0).

Conclusions: False-negative DBE results for overt small bowel bleeding are not rare, and the first re-bleeding episode can occur 3 years later. Repeat DBE when re-bleeding occurs should be considered, even if the first DBE results were negative.
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http://dx.doi.org/10.1007/s00464-018-6561-xDOI Listing
August 2019

Inhibitory effect of lidocaine on colonic spasm during colonoscopy: A multicenter double-blind, randomized controlled trial.

Dig Endosc 2019 Mar 17;31(2):173-179. Epub 2018 Oct 17.

Department of Coloproctology, Aizu Medical Center, Fukushima Medical University, Fukushima, Japan.

Objectives: Colonic spasm can interfere with colonoscopy, but antispasmodic agents can cause complications. This study aimed to assess the inhibitory effect of topical lidocaine compared with a placebo control.

Methods: In five tertiary-care hospitals in Japan, 128 patients requiring endoscopic resection of a colorectal lesion were enrolled and randomly and double-blindly allocated to colonoscopy with topical administration of 2% lidocaine solution 20mL (LID, n = 64) or normal saline 20mL (control, n = 64). During colonoscopy, the assigned solution was applied with a spray catheter near the lesion and the area was observed for three minutes. primary endpoint was the inhibitory effect at three time-points (1, 2 and 3 minutes after dispersion), using a three-point scale (excellent, fair, poor). Secondary endpoints were rebound spasm and adverse events. All endpoints were scored in real time. Serum lidocaine levels were measured in 32 patients (LID 16, control 16).

Results: There were no significant differences between groups in patient demographics. At all time-points, the proportion of patients with "excellent" scores was greater in LID group than control group, with significant differences observed at 2 minutes (p = 0.02) and 3 minutes (p = 0.02). In LID group, the rate of "excellent" scores increased by 12.5% at 2 minutes and was maintained at 3 minutes. Rebound spasm did not occur in LID group, compared with 15.6% of control group (p = 0.001). There were no adverse events in LID group. All serum lidocaine levels were below detectable levels.

Conclusions: Topical lidocaine is an effective and safe method for suppressing colorectal spasm during colonoscopy (UMIN000024733).
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http://dx.doi.org/10.1111/den.13272DOI Listing
March 2019
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