Publications by authors named "Yohei Nishikawa"

27 Publications

  • Page 1 of 1

Unified magnifying endoscopic classification for esophageal, gastric and colonic lesions: a feasibility pilot study.

Endosc Int Open 2021 Sep 16;9(9):E1306-E1314. Epub 2021 Aug 16.

Digestive Diseases Center, Showa University Koto Toyosu Hospital, Tokyo, Japan.

Image-enhanced magnifying endoscopy allows optimization of the detection and diagnosis of lesions found in the gastrointestinal tract. Current organ-specific classifications are well-accepted by specialized endoscopists but may pose confusion for general gastroenterologists. To address this, our group proposed the Unified Magnifying Endoscopic Classification (UMEC) which can be applied either in esophagus, stomach, or colon. The aim of this study was to evaluate the diagnostic performance and clinical applicability of UMEC. A single-center, feasibility pilot study was conducted. Two endoscopists with experience in magnifying narrow band imaging (NBI), blinded to white-light and non-magnifying NBI findings as well as histopathological diagnosis, independently reviewed and diagnosed all images based on UMEC. In brief, UMEC is divided into three categories: non-neoplasia, intramucosal neoplasia, and deep submucosal invasive cancer. The diagnostic performance of UMEC was assessed while using the gold standard histopathology as a reference. A total of 303 gastrointestinal lesions (88 esophageal squamous lesions, 90 gastric lesions, 125 colonic lesions) were assessed. The overall accuracy for both endoscopists in the diagnosis of esophageal squamous cell cancer, gastric cancer, and colorectal cancer were 84.7 %, 89.5 %, and 83.2 %, respectively. The interobserver agreement for each organ, Kappa statistics of 0.51, 0.73, and 0.63, was good. UMEC appears to be a simple and practically acceptable classification, particularly to general gastroenterologists, due to its good diagnostic accuracy, and deserves further evaluation in future studies.
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http://dx.doi.org/10.1055/a-1499-6638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8367430PMC
September 2021

Diagnosis of congenital esophageal stenosis in adults and treatment with peroral endoscopic myotomy.

Ann Gastroenterol 2021 Jul-Aug;34(4):493-500. Epub 2021 Mar 23.

Digestive Diseases Center, Showa University Koto Toyosu Hospital, Tokyo, Japan (Haruo Ikeda, Haruhiro Inoue, Mary Raina Angeli Abad, Yusuke Fujiyoshi, Yohei Nishikawa, Akiko Toshimori, Mayo Tanabe, Yuto Shimamura, Kazuya Sumi, Yugo Iwaya, Manabu Onimaru).

Background: Congenital esophageal stenosis (CES) in adults is a rare disorder that can present as achalasia, particularly in the distal esophagus. We describe the salient features of CES in adults and identify the feasibility and short-term outcomes of peroral endoscopic myotomy (POEM) for CES.

Methods: In this retrospective, single-center case series, we included 6 patients with a "misdiagnosis" of achalasia established elsewhere, ultimately diagnosed with CES and referred to our institution for POEM. Symptom improvement (clinical success rate), defined as an Eckardt Symptom Score (ESS) of <3 at 2-month follow up was assessed.

Results: Six patients (median age: 40 [range: 18-58] years; 4 males) were included. A long-standing history of dysphagia, ring-shaped stenosis on endoscopic examination, "lopsided hourglass" sign on barium esophagogram, and high-resolution manometry findings indicated by a compartmentalized intrabolus pressure pattern with distinction between the stenotic area and the lower esophageal sphincter were the salient features identified. POEM could not be completed in the first 2 cases due to technical challenges. All subsequent 4 patients who underwent successful POEM, exhibited improved ESS of ≤3 (clinical success rate 100%) at 2 months post-POEM.

Conclusions: Along with identification of salient features on several diagnostic modalities, a differential diagnosis of CES in adults must be considered in patients presenting with long-standing history of dysphagia arising from childhood and persisting into adulthood. Although favorable short-term outcomes of POEM were achieved, further evaluation is still warranted, and an inexperienced operator should not attempt POEM on CES patients due to its technical difficulties.
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http://dx.doi.org/10.20524/aog.2021.0618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276369PMC
March 2021

Long-term clinical results of per-oral endoscopic myotomy (POEM) for achalasia: First report of more than 10-year patient experience as assessed with a questionnaire-based survey.

Endosc Int Open 2021 Mar 19;9(3):E409-E416. Epub 2021 Feb 19.

Digestive Diseases Center, Showa University Koto Toyosu Hospital, Tokyo, Japan.

Since per-oral endoscopic myotomy (POEM) was introduced in 2010, it has become accepted as one of the standard treatments for esophageal achalasia worldwide. This study aimed to present long-term clinical results of POEM over 10 years and evaluate the technique and outcomes at the institution where it was first used in clinical settings. Questionnaire-based surveys were sent to patients who received POEM in our institution from September 2008 to May 2010. Patient demographics and procedural outcomes and open-ended questions were posed about the postoperative courses, including symptom improvement and recurrence, additional treatments, and post-POEM gastroesophageal reflux disease (GERD) symptoms. Achalasia symptoms and post-POEM GERD symptoms were evaluated with Eckhardt scores and GerdQ systems, respectively.  Thirty-six consecutive POEMs were performed in that period and 10-year follow-up data were obtained from 15 patients (41.7 %). Although four cases (26.7 %) required additional pneumatic balloon dilatation (PBD), reduction in post-Eckardt scores were observed in 14 cases (93.3 %). GerdQ score was positive in one patient (6.7 %). Proton pump inhibitors (PPI) were taken by four patients (26.7 %) and their symptoms were well-controlled.  Clinical results of POEM over 10 years were favorable regardless of various factors. Symptoms improved even in patients who required additional treatments, suggesting that POEM plays a significant role in treatment of achalasia.
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http://dx.doi.org/10.1055/a-1333-1883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895648PMC
March 2021

Simplified endoscopic pressure study integrated system for the diagnosis of gastroesophageal reflux disease.

Dig Endosc 2021 May 22;33(4):663-667. Epub 2021 Mar 22.

Digestive Diseases Center, Showa University Koto Toyosu Hospital, Tokyo, Japan.

Endoscopic pressure study integrated system (EPSIS) is a novel tool for the diagnosis of gastroesophageal reflux disease. It enables the evaluation of the function of the lower esophageal sphincter by monitoring intragastric pressure (IGP) while insufflating the stomach during esophagogastroduodenoscopy. EPSIS can predict abnormal acid reflux with high accuracy based on previous studies. IGP was measured by inserting through the working channel of the scope an intragastric catheter connected to a pressure measuring device. Herein, we assess the feasibility of an updated EPSIS system, which can be performed just by connecting a flush tube to the working channel. This method does not require inserting foreign objects in the stomach and spares catheter insertion in order to simplify the procedure and reduce costs. A single-center pilot study was conducted to evaluate the association between catheter-based EPSIS and the updated EPSIS. The results of EPSIS in 20 patients who underwent both methods were assessed. In all cases, the waveform pattern of IGP measured by catheter-based EPSIS and updated EPSIS was consistent with 15 uphill pattern and five flat pattern. Intraobserver agreement of waveform pattern was perfect between two examiners with kappa value = 1. Intraclass correlation coefficient (ICC) for intraobserver reliability for maximum IGP was excellent with 0.91 (95% confidence interval [CI] of 0.77 < ICC < 0.96) and for pressure gradient was also good with 0.89 (95% CI of 0.71 < ICC < 0.95). In conclusion, our study suggests that the updated EPSIS can be performed without the use of a catheter.
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http://dx.doi.org/10.1111/den.13947DOI Listing
May 2021

A novel endoscopic purse-string suture technique, "loop 9", for gastrointestinal defect closure: a pilot study.

Endoscopy 2021 Jan 20. Epub 2021 Jan 20.

Digestive Diseases Center, Showa University Koto Toyosu Hospital, Tokyo, Japan.

BACKGROUND : This study aimed to assess the feasibility and efficacy of the novel loop 9 method of gastrointestinal (GI) defect closure. METHODS : 20 patients underwent a GI procedure that required defect closure. Loop 9 can be delivered through a single instrument channel (3.2 mm) and released at the defect site. After it has been anchored by two clips positioned on opposite sides of the defect edge, the loop 9 is tightened by pulling the end of the suture intraluminally using biopsy forceps. Additional clips are placed to achieve complete closure. The primary outcome was complete closure rate. The secondary outcomes were closure time, sustained closure rate, and adverse events. RESULTS : Complete closure was achieved in 100 % of cases. The mean size of the mucosal defects was 17.5 mm (range 10-55 mm). The median closure time was 14 minutes. The sustained closure rate was 90 %. No adverse events were noted. CONCLUSIONS : The loop 9 technique is feasible and effective in achieving complete and sustained closure of therapeutic endoscopy-related GI defects.
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http://dx.doi.org/10.1055/a-1364-4160DOI Listing
January 2021

MBC2019: Marine Biotechnology Conference 2019.

Mar Biotechnol (NY) 2020 12;22(6):725-726

Computational Bio Big-Data Open Innovation Laboratory, AIST-Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo, 169-0072, Japan.

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http://dx.doi.org/10.1007/s10126-020-10009-0DOI Listing
December 2020

High-Quality Draft Genome Sequence of a Bacterium Found in Coral from Okinawa, Japan.

Microbiol Resour Announc 2020 Nov 25;9(48). Epub 2020 Nov 25.

Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan

-like organisms are important for the survival and functioning of corals, prompting an investigation of their complete genomes. Earlier reports of the genomes of these organisms remain incomplete. Here, we report a novel draft genome of bacterial strain SESOKO1, found in coral, using single-cell genome technology.
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http://dx.doi.org/10.1128/MRA.00848-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686419PMC
November 2020

Peroral endoscopic myotomy with diverticulum resection.

VideoGIE 2020 Nov 26;5(11):534-538. Epub 2020 Sep 26.

Digestive Diseases Center, Showa University Koto Toyosu Hospital, Showa University, Tokyo, Japan.

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http://dx.doi.org/10.1016/j.vgie.2020.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652700PMC
November 2020

Draft Genome Sequence of sp. Strain KiyG1, Assembled from Single-Amplified Genomes Collected from Cape Kiyan, Okinawa, Japan.

Microbiol Resour Announc 2020 Nov 12;9(46). Epub 2020 Nov 12.

Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan

The genus is a globally distributed group of microorganisms that live in shallow seabed regions. These organisms play several environmentally important roles and are also known producers of several active secondary metabolites with potential human applications. Here, we present a draft genome of sp. strain KiyG1 (92.7% completeness) that was assembled from four single-amplified genomes.
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http://dx.doi.org/10.1128/MRA.00837-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660993PMC
November 2020

Do Polyethylene Supra-Macroparticles Lead to Pseudotumor Formation in Metal-on-Polyethylene Total Hip Arthroplasty?

Arthroplast Today 2020 Sep 23;6(3):526-531. Epub 2020 Jul 23.

Department of Orthopedic Surgery, Tokyo Medical University, Tokyo, Japan.

We describe 2 cases of pseudotumors induced by an unusual size of polyethylene wear particle after metal-on-polyethylene total hip arthroplasty (MoP THA). The supra-macroparticles of size >100 μm originated from a polyethylene liner with relatively small cup anteversion, potentially leading to excessive loading and increased wear of the anterior edge of the polyethylene liner. Histopathology showed a foreign-body reaction to the polyethylene particles without an adverse reaction to metal debris and with no severe signs of corrosion at the head-neck junction, which have been noted in past reports of pseudotumors in MoP THA. It has been suggested that the large polyethylene wear particles might be the cause of pseudotumor formation in MoP THA.
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http://dx.doi.org/10.1016/j.artd.2020.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387676PMC
September 2020

Importance of second-look endoscopy after per-oral endoscopic myotomy for safe postoperative management.

Dig Endosc 2021 Mar 2;33(3):364-372. Epub 2020 Sep 2.

Digestive Diseases Center, Showa University Koto Toyosu Hospital, Tokyo, Japan.

Objectives: Per-oral endoscopic myotomy (POEM) is a safe and effective treatment for achalasia and esophageal motility disorders. The role of second-look endoscopy (SE) on postoperative day 1 has not been examined. This study aimed to evaluate the findings and need of SE after POEM.

Methods: This is a single-center, retrospective study. All consecutive patients who underwent POEM and SE on postoperative day 1 between December 2017 and September 2019 were included. The primary endpoint was the rate of newly-detected adverse events (nAE) during SE that required endoscopic intervention or deviation from the normal postoperative course. Multivariate logistic regression was used to identify predictors of nAE.

Results: Four-hundred-ninety-seven patients (mean age, 50.3 years; female, 49.9%) were included. SE identified abnormal findings in a total of 71 patients (14.3%). nAE which required endoscopic intervention or deviation from the normal postoperative course were identified in 12 patients (2.4%): eight (1.6%) entry site dehiscence; two (0.4%) submucosal hemorrhage or hematoma; and two (0.4%) dehiscence of an intraoperative perforation site after endoclip closure. Other findings such as mucosal thermal damage without perforation and small submucosal hematoma were found in 54 patients (10.9%) and five patients (1.0%), respectively. Multivariate analysis showed that longer operation time and intraoperative adverse events (AE) were associated with clinically significant nAE during SE.

Conclusions: Second-look endoscopy can detect and treat nAE that may lead to severe AE. Thus, SE should be highly considered before starting oral ingestion in all cases, and especially in those who present an intraoperative AE and longer operation time.
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http://dx.doi.org/10.1111/den.13770DOI Listing
March 2021

Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota.

Microbiome 2020 01 23;8(1). Epub 2020 Jan 23.

Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsucho, Shinjuku-ku, Tokyo, 162-8480, Japan.

Background: The gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel single-cell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota.

Results: Inulin-feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., resulting in the production of abundant succinate in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning 2 phyla, 4 classes, 7 orders, and 14 families, and 31 different strains of SAGs were graded as high- and medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin-responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production.

Conclusions: Our single-cell genomics approach generated a massive amount of SAGs, enabling a functional analysis of uncultured bacteria in the intestinal microbiome. This enabled us to estimate metabolic lineages involved in the bacterial fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota and could be exploited to improve human and animal health. Video abstract.
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http://dx.doi.org/10.1186/s40168-019-0779-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6977353PMC
January 2020

High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics.

Sci Rep 2019 07 29;9(1):10920. Epub 2019 Jul 29.

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.

Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.
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http://dx.doi.org/10.1038/s41598-019-47388-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662714PMC
July 2019

Correction to: A CCR5 memory subset within HIV-1-infected primary resting CD4 T cells is permissive for replication-competent, latently infected viruses in vitro.

BMC Res Notes 2019 06 10;12(1):322. Epub 2019 Jun 10.

Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

After publication of the original article [1], the authors became aware of a miscalculation in the original Fig. 2d.
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http://dx.doi.org/10.1186/s13104-019-4357-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556951PMC
June 2019

A CCR5 memory subset within HIV-1-infected primary resting CD4 T cells is permissive for replication-competent, latently infected viruses in vitro.

BMC Res Notes 2019 Apr 29;12(1):242. Epub 2019 Apr 29.

Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

Objective: Resting CD4 T cells are major reservoirs of latent HIV-1 infection, and may be formed during the early phase of the infection. Although CCR5-tropic (R5) HIV-1 is highly transmissible during the early phase, newly infected individuals have usually been exposed to a mixture of R5 and CXCR4-tropic (X4) viruses, and X4 viral DNA is also detectable in the host. Our aim was to identify which subsets of resting CD4 T cells contribute to forming the latent reservoir in the presence of both X4 and R5 viruses.

Results: Primary resting CD4 naïve T (T) cells, CCR5 memory T (T) cells, and CCR5 T cells isolated by flow cytometry were infected simultaneously with X4 and R5 HIV-1, which harbored different reporter genes, and were cultured in the resting condition. Flow cytometry at 3 days post-infection demonstrated that X4 HIV-1 cells were present in all three subsets of cells, whereas R5 HIV-1 cells were present preferentially in CCR5 T cells, but not in T cells. Following CD3/CD28-mediated activation at 3 days post-infection, numbers of R5 HIV-1 cells and X4 HIV-1 cells increased significantly only in the CCR5 T subset, suggesting that it provides a major reservoir of replication-competent, latently infected viruses.
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http://dx.doi.org/10.1186/s13104-019-4281-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6489248PMC
April 2019

Obtaining high-quality draft genomes from uncultured microbes by cleaning and co-assembly of single-cell amplified genomes.

Sci Rep 2018 02 1;8(1):2059. Epub 2018 Feb 1.

Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-8480, Japan.

Single-cell genomics is a straightforward approach to obtain genomes from uncultured microbes. However, sequence reads from a single-cell amplified genome (SAG) contain significant bias and chimeric sequences. Here, we describe Cleaning and Co-assembly of a Single-Cell Amplified Genome (ccSAG), a novel analytical workflow to obtain composite single-cell genomes with elimination of sequence errors. By the integration of ccSAG with a massively parallel single-cell genome amplification platform based on droplet microfluidics, we can generate multiple SAGs and effectively integrate them into the composite genomes quality equivalent to the data obtained from bulk DNA. We obtained two novel draft genomes from single gut microbial cells with high completeness (>96.6%) and extremely low contamination (<1.25%). Moreover, we revealed the presence of single nucleotide polymorphisms in the specific gene by sequence comparison at the single-cell level. Thus, the workflow yields near-complete genomes from uncultured microbes, and enables analyses of genetic heterogeneity within identical strains.
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http://dx.doi.org/10.1038/s41598-018-20384-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794965PMC
February 2018

Modulation of Stimulator of Interferon Genes (STING) Expression by Interferon-γ in Human Keratinocytes.

Biochem Genet 2018 Apr 16;56(1-2):93-102. Epub 2017 Nov 16.

Department of Dermatology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.

Infection of microbial pathogen triggers the innate immune system, and the induction of interferons (IFNs) play a vital role in host antiviral response. Stimulator of interferon genes (STING) was identified as a crucial regulator of the DNA sensing pathway, and activates both nuclear factor-κB and interferon regulatory factor 3 transcription pathways to evoke IFNs production. In this study, we studied the upregulation of STING mRNA expression, induced by IFN-γ in human keratinocytes (HaCaT). STING promoter assays clarified that a gamma-activated sequence (GAS), located at - 7 to - 15-bp, is required for IFN-γ-upregulated promoter activity. Furthermore, an electrophoretic mobility shift assay showed that signal transducers and activators of transcription 1 (STAT1) attach to the GAS motif on the human STING promoter region. This indicates that IFN-γ/Janus kinases/STAT1 signaling is essential for the STING upregulation in human keratinocytes.
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http://dx.doi.org/10.1007/s10528-017-9832-7DOI Listing
April 2018

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

Sci Rep 2017 07 12;7(1):5199. Epub 2017 Jul 12.

Research Organization for Nano & Life Innovation, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo, 162-0041, Japan.

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.
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http://dx.doi.org/10.1038/s41598-017-05436-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507899PMC
July 2017

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification.

PLoS One 2015 21;10(9):e0138733. Epub 2015 Sep 21.

Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Research Organization for Nano & Life Innovation, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan; Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138733PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577099PMC
May 2016

Droplet-based microfluidics for high-throughput screening of a metagenomic library for isolation of microbial enzymes.

Biosens Bioelectron 2015 May 27;67:379-85. Epub 2014 Aug 27.

Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Institute for Nanoscience and Nanotechnology, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan; Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. Electronic address:

This paper proposes a high-throughput, function-based screening approach of a metagenomic library for isolating novel microbial enzymes by droplet-based microfluidics. We used gel microdroplets (GMDs) dispersed in oil as picoliter-volume reaction vessels for lipolytic enzyme by encapsulating cells in individual GMDs. Using this approach, we monitored the growth of individual cells encapsulated in GMDs and assessed the enzyme reaction activities at the level of an individual GMD. We then applied this method to screen lipolytic enzyme genes from the metagenomic library constructed from soil collected from a quercus serrate forest of Mount Tsukuba, Ibaraki, Japan. In the workflow presented in this study, metagenomic library clones were encapsulated in 100-pL GMDs with a fluorogenic reporter substrate. A total of 67,000 metagenomic library clones can be screened in only 24 h with reduced consumption of reagents (i.e., <10 μL). As a result, we identified a novel lipolytic enzyme, EstT1, belonging to the EstD2 family of esterases and containing a putative signal peptide, which facilitates enzyme export and catalyzation of substrates in the periplasm. Our study demonstrates the potential of microfluidic GMDs as an efficient tool for metagenomic library screening of industrially relevant enzymes with the potential of significantly reducing the cost and time factors involved in successful practical application of microbial enzymes.
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http://dx.doi.org/10.1016/j.bios.2014.08.059DOI Listing
May 2015

Characterization of retinoic acid-inducible gene-I (RIG-I) expression corresponding to viral infection and UVB in human keratinocytes.

J Dermatol Sci 2012 Apr 21;66(1):64-70. Epub 2012 Feb 21.

Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Background: Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic protein that recognizes viral double-stranded RNA to induce the type I interferon (IFN) response. In human keratinocytes, RIG-I is induced by IFN-γ and tumor necrosis factor-α stimulation, and is abundantly expressed in psoriatic keratinocytes of the spinous and basal layers.

Objective: This study investigated the effects of extraneous stimuli including viral infection and UVB exposure on RIG-I expression in human keratinocytes.

Methods: Human skin keratinocytes (HaCaT cells) were stimulated by polyinosinic-polycytidylic acid (poly(I:C)), which mimics viral infection, and UVB exposure. We assessed the expression of RIG-I and IFN-regulatory factor (IRF)-1 in HaCaT cells by RT-PCR and Western blot analysis. Moreover, we investigated the effect of IRF-1 binding site of RIG-I gene promoter on the regulation of RIG-I expression by luciferase promoter assay and electrophoretic mobility shift assay.

Results: Poly(I:C) induced RIG-I expression, while UVB inhibited basal RIG-I expression and the poly(I:C)-induced RIG-I overexpression in HaCaT cells. IRF-1, which binds to a regulatory element located on the RIG-I gene promoter, was required for both inductions of RIG-I expression. IRF-1 expression was enhanced three hours after the poly(I:C) stimulation, consistent with the RIG-I response to poly(I:C), and thereafter was suppressed. Moreover, UVB exposure promptly decreased IRF-1 expression, resulting in decreased IRF-1 protein binding to the RIG-I promoter, and consequently, decreased RIG-I expression.

Conclusion: Thus, suppression of RIG-I and IRF-1 expression caused by UVB exposure may partly explain the inhibition of skin-based immune responses, leading to viral infection and recrudescence.
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http://dx.doi.org/10.1016/j.jdermsci.2012.02.006DOI Listing
April 2012

Late-onset, eruptive syringoma in an elderly man: correlation with carbamazepine.

Acta Derm Venereol 2012 Jan;92(1):87-8

Department of Dermatology, Hirosaki University Graduate School of Medicine, Aomori, Japan.

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http://dx.doi.org/10.2340/00015555-1156DOI Listing
January 2012

Dermoscopy of eccrine poroma with calcification.

J Dermatol Case Rep 2009 Nov;3(3):38-40

Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki 036-8182, Japan.

Background: Eccrine poromas are relatively common slow-growing benign solitary adnexal tumors originating from the intraepidermal portion of the eccrine sweat duct (acrosyringium). Dystrophic calcification is rarely found in lesions of eccrine poroma, and only 2 cases of eccrine poroma with calcification have been reported thus far. In the present report, we describe another case of eccrine poroma with calcification occurring in the palm of the hand. Also, we show dermoscopic features of this case.

Main Observations: A 73-year-old man with hemiparesis, who had a 10-year history of tumor on his right palm, which was occasionally injured by a walking crutch, causing bleeding and ulceration. Physical examination revealed a pigmented dome-shaped tumor. Dermoscopic analysis revealed glomerular vessels, multiple pink-white structureless areas, and lacunae. Histological examination revealed that the tumor was composed of cords of tumor cells extending from the epidermis into the dermis. These were uniformly cuboidal cells with round, basophilic nuclei and dense vascular stromas with telangiectasia. The tumor showed cystic structures and calcification. The patient was diagnosed with Pinkus-type eccrine poroma on the basis of histological findings.

Conclusions: Although cutaneous neoplasms commonly associated with calcification are of follicular origin, it is known that dystrophic calcification may be triggered also in tumors of eccrine origin by multiple factors, including mechanical injury. Dermoscopy may be helpful in establishing clinical diagnosis of calcified eccrine poromas.
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http://dx.doi.org/10.3315/jdcr.2009.1033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157797PMC
November 2009

Electric field-assisted alignment of self-assembled fibers composed of hydrogen-bonded molecules having laterally fluorinated mesogens.

J Am Chem Soc 2009 May;131(19):6763-7

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

Aligned fibrous aggregates of amide compounds having laterally fluorinated aromatic mesogens have been successfully obtained by the application of the alternating current electric field (1.0 V/microm, 1 kHz) in dodecylbenzene. In contrast, randomly entangled fibers are formed in the solvent without electric fields. For the analogous compounds without fluorine substituent, no aligned fibrous aggregates have been obtained under the electric fields. The electric field alignment of the fibers should be assisted by the fluorinated rod-shaped mesogens that exhibit negative dielectric anisotropy.
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http://dx.doi.org/10.1021/ja8093718DOI Listing
May 2009

A ferroelectrically switchable columnar liquid crystal phase with achiral molecules: superstructures and properties of liquid crystalline ureas.

J Am Chem Soc 2005 Mar;127(8):2565-71

Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.

Novel columnar liquid crystalline compounds N,N'-bis(3,4,5-trialkoxylphenyl)ureas 1a-c (R = n-C(8)H(17), n-C(12)H(25), and n-C(16)H(33)) were synthesized, and their phase transitions were measured by differential scanning calorimetery. The superstructures were investigated by X-ray diffraction, polarized light optical microscopy, and IR spectroscopy. The compounds exhibited both rectangular and hexagonal columnar phases in which the urea molecules in each column were stacked in one direction with strong hydrogen bonds. To confirm the ferroelectric switching, optoelectronic experiments were carried out, and the hexagonal columnar phases of 1b and 1c gave a sharp peak of spontaneous polarization in response to an applied triangular wave electric field (0.1-18 Hz). This is the first example of ferroelectrically switchable columnar liquid crystal phases generated by achiral molecules.
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http://dx.doi.org/10.1021/ja046100cDOI Listing
March 2005
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