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1.  A Case of Mild Encephalopathy with a Reversible Splenial Lesion Associated with G5P[6]Rotavirus Infection 
Case Reports in Pediatrics  2013;2013:197163.
We report a case of mild encephalopathy with a reversible splenial lesion (MERS) associated with acute gastroenteritis caused by rotavirus (RV) infection. The patient (male, 4 years and 3 months old) was admitted to our hospital for diarrhea and afebrile seizures. Head MRI revealed a hyperintense signal in the splenium of the corpus callosum on DWI and a hypointense signal on the ADC-map. After awakening from sedation, the patient's disturbance of consciousness improved. On day 5 after admission of the illness, the patient was discharged from the hospital in a good condition. Electroencephalography on day 2 after admission was normal. On day 8 of admission, head MRI revealed that the splenial lesion had disappeared. RV antigen-positive stools suggested that RV had caused MERS. This RV genotype was considered to be G5P[6]; it may have spread to humans as a strain reassortment through substitution of porcine RV into human RV gene segments. This extremely rare genotype was detected first in Japan and is not covered by existing vaccines; this is the first sample isolated from encephalopathy patients. Few reports have investigated RV genotypes in encephalopathy; we believe that this case is valuable for studying the relationship between genotypes and clinical symptoms.
PMCID: PMC3845239  PMID: 24324908
2.  Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid 
Journal of Synchrotron Radiation  2013;20(Pt 6):923-928.
The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate.
Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.
PMCID: PMC3795557  PMID: 24121341
X-ray structure; FABP3–ANS complex; human-heart fatty-acid-binding protein
3.  NFIL3 deficient mice develop microbiota dependent, IL-12/23 driven spontaneous colitis 
NFIL3 (nuclear factor, IL-3 regulated) is a transcription factor that regulates multiple immunologic functions. In myeloid cells, NFIL3 is IL-10 inducible, and has a key role as a repressor of IL-12p40 transcription. NFIL3 is a susceptibility gene for the human inflammatory bowel diseases. Here we describe spontaneous colitis in Nfil3−/− mice. Mice lacking both Nfil3 and Il10 (NIDKO) had severe early-onset colitis, suggesting NFIL3 and IL-10 independently regulate mucosal homeostasis. Lymphocytes were necessary for colitis, as Nfil3/Rag1 double knockout (NRDKO) mice were protected from disease. However, NRDKO mice adoptively transferred with wild type CD4+ T cells developed severe colitis compared to Rag1−/− recipients, suggesting that colitis was linked to defects in innate immune cells. Colitis was abrogated in Nfil3/Il12b double-deficient mice, identifying Il12b dysregulation as a central pathogenic event. Finally, germ-free Nfil3−/− mice do not have colonic inflammation. Thus, NFIL3 is a microbiota-dependent, IL-10-independent regulator of mucosal homeostasis via IL-12p40.
PMCID: PMC3948213  PMID: 24442434
Cells: Monocytes/Macrophages; Diseases: Autoimmunity; Molecules: Cytokines; Processes: Inflammation; Tissues: Mucosa
4.  Involvement of dachshund and Distal-less in distal pattern formation of the cricket leg during regeneration 
Scientific Reports  2015;5:8387.
Cricket nymphs have the remarkable ability to regenerate a functional leg following amputation, indicating that the regenerating blastemal cells contain information for leg morphology. However, the molecular mechanisms that underlie regeneration of leg patterns remain poorly understood. Here, we analyzed phenotypes of the tibia and tarsus (three tarsomeres) obtained by knockdown with regeneration-dependent RNA interference (rdRNAi) against Gryllus dachshund (Gb'dac) and Distal-less (Gb'Dll). We found that depletion of Gb'Dll mRNA results in loss of the tarsal segments, while rdRNAi against Gb'dac shortens the tibia at the two most distal tarsomeres. These results indicate that Gb'Dll expression is indispensable for formation of the tarsus, while Gb'dac expression is necessary for elongation of the tibia and formation of the most proximal tarsomere. These findings demonstrate that mutual transcriptional regulation between the two is indispensable for formation of the tarsomeres, whereas Gb'dac is involved in determination of tibial size through interaction with Gb'ds/Gb'ft.
PMCID: PMC4323655  PMID: 25669615
5.  Efficacy and Safety of Sitagliptin in Japanese Patients With Type 2 Diabetes 
The aim of this study was to investigate the clinical efficacy and safety of sitagliptin in Japanese patients with type 2 diabetes.
A total of 3,247 subjects treated with sitagliptin were retrospectively recruited. Glucose parameters were collected at baseline, and 1, 3 and 6 months after initiation of sitagliptin. In addition, we explored factors that can be used to predict sitagliptin-induced reduction in HbA1c using linear mixed effect model. Factors associated with hypoglycemic events were examined by logistic analyses.
We analyzed the available data of 3,201 subjects (1,287 females). Treatment of sitagliptin significantly reduced HbA1c level from 7.44±1.20% at baseline to 6.73±0.99% at 6 months (P < 0.0001). Linear mixed effect model analyses demonstrated that reduction of HbA1c was associated with higher baseline HbA1c level, younger age, lower BMI and sitagliptin monotherapy. During this study, 82 cases of hypoglycemia were recorded. Logistic analyses indicated that hypoglycemic events were more frequent in female patients, and patients with low BMI, long history of type 2 diabetes, high HbA1c and on combination therapy experienced. Other adverse events were rare and mild.
Sitagliptin is effective for diabetic management and generally well tolerated in Japanese patients with type 2 diabetes. This trial was registered with UMIN (no. 000004121).
PMCID: PMC4330012
DPP-4 inhibitor; Treatment efficacy; Treatment safety
6.  Disease Severity Is Associated with Differential Gene Expression at the Early and Late Phases of Infection in Nonhuman Primates Infected with Different H5N1 Highly Pathogenic Avian Influenza Viruses 
Journal of Virology  2014;88(16):8981-8997.
Occasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome.
IMPORTANCE Highly pathogenic avian H5N1 influenza viruses sometimes transmit to humans and cause severe pneumonia with ca. 60% lethality. The continued circulation of these viruses poses a pandemic threat; however, their pathogenesis in mammals is not fully understood. We, therefore, investigated the pathogenicity of six H5N1 viruses and compared the host responses of cynomolgus macaques to the virus infection. We identified differences in the viral replicative ability of and in disease severity caused by these H5N1 viruses. A comparison of global host responses between severe and mild disease cases identified the limited upregulation of interferon-stimulated genes early in infection in severe cases. The dynamics of the host responses indicated that the limited response early in infection failed to suppress virus replication and led to hyperinduction of pathological condition-related genes late in infection. These findings provide insight into the pathogenesis of H5N1 viruses in mammals.
PMCID: PMC4136255  PMID: 24899188
7.  A mindfulness-based stress management program and treatment with omega-3 fatty acids to maintain a healthy mental state in hospital nurses (Happy Nurse Project): study protocol for a randomized controlled trial 
Trials  2015;16:36.
It is reported that nursing is one of the most vulnerable jobs for developing depression. While they may not be clinically diagnosed as depressed, nurses often suffer from depression and anxiety symptoms, which can lead to a low level of patient care. However, there is no rigorous evidence base for determining an effective prevention strategy for these symptoms in nurses. After reviewing previous literature, we chose a strategy of treatment with omega-3 fatty acids and a mindfulness-based stress management program for this purpose. We aim to explore the effectiveness of these intervention options for junior nurses working in hospital wards in Japan.
A factorial-design multi-center randomized trial is currently being conducted. A total of 120 nurses without a managerial position, who work for general hospitals and gave informed consent, have been randomly allocated to a stress management program or psychoeducation using a leaflet, and to omega-3 fatty acids or identical placebo pills. The stress management program has been developed according to mindfulness cognitive therapy and consists of four 30-minute individual sessions conducted using a detailed manual. These sessions are conducted by nurses with a managerial position. Participants allocated to the omega-3 fatty acid groups are provided with 1,200 mg/day of eicosapentaenoic acid and 600 mg/day of docosahexaenoic acid for 90 days.
The primary outcome is the change in the total score of the Hospital Anxiety and Depression Scale (HADS), determined by a blinded rater via the telephone at week 26. Secondary outcomes include the change in HADS score at 13 and 52 weeks; presence of a major depressive episode; severity of depression, anxiety, insomnia, burnout, and presenteeism; utility scores and adverse events at 13, 26 and 52 weeks.
An effective preventive intervention may not only lead to the maintenance of a healthy mental state in nurses, but also to better quality of care for inpatients. This paper outlines the background and methods of a randomized trial that evaluates the possible additive value of omega-3 fatty acids and a mindfulness-based stress management program for reducing depression in nurses.
Trial registration NCT02151162 (registered on 27 May 2014).
PMCID: PMC4326519  PMID: 25636180
Anxiety; Behavior therapy; Depression; Fatty acids; Omega-3; Mindfulness; Prevention and control
8.  Sentinel node navigation surgery for gastric cancer: Overview and perspective 
The sentinel node (SN) technique has been established for the treatment of some types of solid cancers to avoid unnecessary lymphadenectomy. If node disease were diagnosed before surgery, minimal surgery with omission of lymph node dissection would be an option for patients with early gastric cancer. Although SN biopsy has been well ascertained in the treatment of breast cancer and melanoma, SN navigation surgery (SNNS) in gastric cancer has not been yet universal due to the complicated lymphatic flow from the stomach. Satisfactory establishment of SNNS will result in the possible indication of minimally invasive surgery of gastric cancer. However, the results reported in the literature on SN biopsy in gastric cancer are widely divergent and many issues are still to be resolved, such as the collection method of SN, detection of micrometastasis in SN, and clinical benefit. The difference in the procedural technique and learning phase of surgeons is also varied the accuracy of SN mapping. In this review, we outline the current status of application for SNNS in gastric cancer.
PMCID: PMC4300912  PMID: 25625004
Sentinel node navigation surgery; Gastric cancer; Micrometastasis; Minimal surgery; Review
9.  African Green Monkeys Recapitulate the Clinical Experience with Replication of Live Attenuated Pandemic Influenza Virus Vaccine Candidates 
Journal of Virology  2014;88(14):8139-8152.
Live attenuated cold-adapted (ca) H5N1, H7N3, H6N1, and H9N2 influenza vaccine viruses replicated in the respiratory tract of mice and ferrets, and 2 doses of vaccines were immunogenic and protected these animals from challenge infection with homologous and heterologous wild-type (wt) viruses of the corresponding subtypes. However, when these vaccine candidates were evaluated in phase I clinical trials, there were inconsistencies between the observations in animal models and in humans. The vaccine viruses did not replicate well and immune responses were variable in humans, even though the study subjects were seronegative with respect to the vaccine viruses before vaccination. Therefore, we sought a model that would better reflect the findings in humans and evaluated African green monkeys (AGMs) as a nonhuman primate model. The distribution of sialic acid (SA) receptors in the respiratory tract of AGMs was similar to that in humans. We evaluated the replication of wt and ca viruses of avian influenza (AI) virus subtypes H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of AGMs. All of the wt viruses replicated efficiently, while replication of the ca vaccine viruses was restricted to the upper respiratory tract. Interestingly, the patterns and sites of virus replication differed among the different subtypes. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3, and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than when it was delivered intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects and that AGMs may be a useful model to evaluate the replication of ca vaccine candidates.
IMPORTANCE Ferrets and mice are commonly used for preclinical evaluation of influenza vaccines. However, we observed significant inconsistencies between observations in humans and in these animal models. We used African green monkeys (AGMs) as a nonhuman primate (NHP) model for a comprehensive and comparative evaluation of pairs of wild-type and pandemic live attenuated influenza virus vaccines (pLAIV) representing four subtypes of avian influenza viruses and found that pLAIVs replicate similarly in AGMs and humans and that AGMs can be useful for evaluation of the protective efficacy of pLAIV.
PMCID: PMC4097805  PMID: 24807726
10.  Continuous low-dose irradiation by I-125 seeds induces apoptosis of gastric cancer cells regardless of histological origin 
Cancer Biology & Therapy  2013;15(1):81-88.
The efficacy of conventional radiation therapy for gastric cancer is controversial. In this study, we evaluated the in vitro and in vivo effects of continuous low-dose-rate irradiation by I-125 seeds on different histological types of gastric cancer cell lines. Three human gastric cancer cell lines (MKN74, MKN45, and NUGC4) were treated with or without continuous low-dose irradiation by I-125 seeds in vitro and in vivo. Cell viability, apoptosis, caspase-3 assay, and cell-cycle distribution were examined in vitro. Body weight and tumor volumes of BALB/c nude mice bearing MKN74, MKN45, and NUGC4 gastric cancer xenografts were measured, and in vivo cell proliferation and apoptosis assays were performed by Ki67 and TUNEL staining, respectively. Continuous low-dose-rate irradiation by I-125 seeds reduced cell viability and induced cell apoptosis through the activation of caspase-3, and led to the accumulation of cells in the G2/M phase in vitro. It also suppressed the growth of gastric cancer xenografts in nude mice, while inhibiting cell proliferation and inducing apoptosis as demonstrated by Ki67 and TUNEL staining. Therefore, our data suggest that continuous low-dose-rate irradiation by I-125 seeds could be a promising new option for gastric cancer treatment, regardless of histological origin.
PMCID: PMC3938526  PMID: 24149371
I-125 seed irradiation; gastric cancer; apoptosis; cell cycle; caspase
12.  Plant Omics Data Center: An Integrated Web Repository for Interspecies Gene Expression Networks with NLP-Based Curation 
Plant and Cell Physiology  2014;56(1):e9.
Comprehensive integration of large-scale omics resources such as genomes, transcriptomes and metabolomes will provide deeper insights into broader aspects of molecular biology. For better understanding of plant biology, we aim to construct a next-generation sequencing (NGS)-derived gene expression network (GEN) repository for a broad range of plant species. So far we have incorporated information about 745 high-quality mRNA sequencing (mRNA-Seq) samples from eight plant species (Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, Sorghum bicolor, Vitis vinifera, Solanum tuberosum, Medicago truncatula and Glycine max) from the public short read archive, digitally profiled the entire set of gene expression profiles, and drawn GENs by using correspondence analysis (CA) to take advantage of gene expression similarities. In order to understand the evolutionary significance of the GENs from multiple species, they were linked according to the orthology of each node (gene) among species. In addition to other gene expression information, functional annotation of the genes will facilitate biological comprehension. Currently we are improving the given gene annotations with natural language processing (NLP) techniques and manual curation. Here we introduce the current status of our analyses and the web database, PODC (Plant Omics Data Center;, now open to the public, providing GENs, functional annotations and additional comprehensive omics resources.
PMCID: PMC4301748  PMID: 25505034
Correspondence analysis; Database; Gene expression network; Manual curation; Natural language processing (NLP); Omics
13.  Development of a High-Yield Live Attenuated H7N9 Influenza Virus Vaccine That Provides Protection against Homologous and Heterologous H7 Wild-Type Viruses in Ferrets 
Journal of Virology  2014;88(12):7016-7023.
Live attenuated H7N9 influenza vaccine viruses that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the newly emerged wild-type (wt) A/Anhui/1/2013 (H7N9) and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (AA ca) were generated by reverse genetics. The reassortant virus containing the original wt A/Anhui/1/2013 HA and NA sequences replicated poorly in eggs. Multiple variants with amino acid substitutions in the HA head domain that improved viral growth were identified by viral passage in eggs and MDCK cells. The selected vaccine virus containing two amino acid changes (N133D/G198E) in the HA improved viral titer by more than 10-fold (reached a titer of 108.6 fluorescent focus units/ml) without affecting viral antigenicity. Introduction of these amino acid changes into an H7N9 PR8 reassortant virus also significantly improved viral titers and HA protein yield in eggs. The H7N9 ca vaccine virus was immunogenic in ferrets. A single dose of vaccine conferred complete protection of ferrets from homologous wt A/Anhui/1/2013 (H7N9) and nearly complete protection from heterologous wt A/Netherlands/219/2003 (H7N7) challenge infection. Therefore, this H7N9 live attenuated influenza vaccine (LAIV) candidate has been selected for vaccine manufacture and clinical evaluation to protect humans from wt H7N9 virus infection.
IMPORTANCE In response to the recent avian H7N9 influenza virus infection in humans, we developed a live attenuated H7N9 influenza vaccine (LAIV) with two amino acid substitutions in the viral HA protein that improved vaccine yield by 10-fold in chicken embryonated eggs, the substrate for vaccine manufacture. The two amino acids also improved the antigen yield for inactivated H7N9 vaccines, demonstrating that this finding could great facilitate the efficiency of H7N9 vaccine manufacture. The candidate H7N9 LAIV was immunogenic and protected ferrets against homologous and heterologous wild-type H7 virus challenge, making it suitable for use in protecting humans from H7 infection.
PMCID: PMC4054345  PMID: 24719414
14.  Integrated image navigation system using head-mounted display in “RoboSurgeon” endoscopic radical prostatectomy 
The safety and efficacy of minimally invasive surgery relies on visual information. We aimed to develop an integrated image navigation system (RoboSurgeon System) that combines head-mounted displays (HMDs) with multiple image modalities, and assessed its feasibility in 5 prostate cancer patients who underwent gasless single-port endoscopic radical prostatectomy. A robotically manipulated transrectal ultrasound (TRUS) system was used. In all cases, preoperative magnetic resonance (MR) images and intraoperative real-time images of an endoscope, TRUS, and HMD-mounted camera were integrated and displayed synchronously on each HMD in a four-split screen mode during the entire process. The TRUS helped identify the boundary with the adjacent structures endoscopically in reference to MR images. There were no negative incidents in intraoperative or postoperative courses. Integrated image navigation using HMDs as individualized monitors is feasible in the natural ergonomic position and may be beneficial to identify correct dissection planes. The efficacy of the RoboSurgeon System deserves further evaluation.
PMCID: PMC4280404  PMID: 25562001
head-mounted display; image navigation; minimally invasive surgery; prostate cancer; radical prostatectomy; transrectal ultrasound
15.  A three-dimensional head-mounted display system (RoboSurgeon system) for gasless laparoendoscopic single-port partial cystectomy 
We developed a new three-dimensional (3D) head-mounted display (HMD) system (RoboSurgeon system) that combines a high-definition 3D organic electroluminescent HMD with a high-definition 3D endoscope and applies it to minimally invasive surgery. This system presents the surgeon with a higher quality of magnified 3D imagery in front of the eyes, regardless of head position. We report 5 cases of RoboSurgeon gasless laparoendoscopic single-port partial cystectomy, which is carried out as part of our selective bladder-sparing protocol, with a technique utilizing both an intravesical and extravesical approach. While carrying out the surgery, the system provides the surgeon with both excellent 3D imagery of the operative field and clear imagery of the cystoscopy. All procedures were safely completed and there were no complications except for a case of postoperative lymphorrhea. Our experience shows that the 3D HMD system might facilitate maneuverability and safety in various minimally invasive procedures.
PMCID: PMC4280413  PMID: 25562007
head-mounted display; minimally invasive surgery; three-dimensional high-definition endoscope; cystoscope; bladder cancer; partial cystectomy
16.  Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in vivo in human multiple myeloma 
BMC Cancer  2014;14(1):882.
Human multiple myeloma (MM) is an incurable hematological malignancy for which novel therapeutic agents are needed. Calmodulin (CaM) antagonists have been reported to induce apoptosis and inhibit tumor cell invasion and metastasis in various tumor models. However, the antitumor effects of CaM antagonists on MM are poorly understood. In this study, we investigated the antitumor effects of naphthalenesulfonamide derivative selective CaM antagonists W-7 and W-13 on MM cell lines both in vitro and in vivo.
The proliferative ability was analyzed by the WST-8 assay. Cell cycle was evaluated by flow cytometry after staining of cells with PI. Apoptosis was quantified by flow cytometry after double-staining of cells by Annexin-V/PI. Molecular changes of cell cycle and apoptosis were determined by Western blot. Intracellular calcium levels and mitochondrial membrane potentials were determined using Fluo-4/AM dye and JC-10 dye, respectively. Moreover, we examined the in vivo anti-MM effects of CaM antagonists using a murine xenograft model of the human MM cell line.
Treatment with W-7 and W-13 resulted in the dose-dependent inhibition of cell proliferation in various MM cell lines. W-7 and W-13 induced G1 phase cell cycle arrest by downregulating cyclins and upregulating p21cip1. In addition, W-7 and W-13 induced apoptosis via caspase activation; this occurred partly through the elevation of intracellular calcium levels and mitochondrial membrane potential depolarization and through inhibition of the STAT3 phosphorylation and subsequent downregulation of Mcl-1 protein. In tumor xenograft mouse models, tumor growth rates in CaM antagonist-treated groups were significantly reduced compared with those in the vehicle-treated groups.
Our results demonstrate that CaM antagonists induce cell cycle arrest, induce apoptosis via caspase activation, and inhibit tumor growth in a murine MM model and raise the possibility that inhibition of CaM might be a useful therapeutic strategy for the treatment of MM.
PMCID: PMC4258255  PMID: 25424011
Calmodulin; Multiple myeloma; Cell cycle; Apoptosis
17.  The gut microbiota and inflammatory bowel disease 
Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gut. Although the precise cause of IBD remains unknown, the most accepted hypothesis of IBD pathogenesis to date is that an aberrant immune response against the gut microbiota is triggered by environmental factors in a genetically susceptible host. The advancement of next-generation sequencing technology has enabled identification of various alterations of the gut microbiota composition in IBD. While some results related to dysbiosis in IBD are different between studies owing to variations of sample type, method of investigation, patient profiles, and medication, the most consistent observation in IBD is reduced bacterial diversity, a decrease of Firmicutes, and an increase of Proteobacteria. It has not yet been established how dysbiosis contributes to intestinal inflammation. Many of the known IBD susceptibility genes are associated with recognition and processing of bacteria, which is consistent with a role of the gut microbiota in the pathogenesis of IBD. A number of trials have shown that therapies correcting dysbiosis, including fecal microbiota transplantation and probiotics, are promising in IBD.
PMCID: PMC4281375  PMID: 25420450
Inflammatory bowel disease; Ulcerative colitis; Crohn’s disease; Dysbiosis
18.  gone early, a Novel Germline Factor, Ensures the Proper Size of the Stem Cell Precursor Pool in the Drosophila Ovary 
PLoS ONE  2014;9(11):e113423.
In order to sustain lifelong production of gametes, many animals have evolved a stem cell–based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. Here we show that a novel germline factor, Gone early (Goe), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell–cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment.
PMCID: PMC4242634  PMID: 25420147
19.  Analysis of pain level in cases treated with Invisalign aligner: comparison with fixed edgewise appliance therapy 
Progress in Orthodontics  2014;15(1):64.
The aim of this study was to evaluate and compare the difference in the level of pain using the visual analog scale (VAS) between cases treated with the edgewise appliance and Invisalign. In addition, the cause of pain and discomfort in the Invisalign cases was identified.
The sample consisted of 145 cases for the edgewise group (EG; n = 55), Invisalign group (IG; n = 38), and edgewise and Invisalign group (EIG; n = 52). VAS scores were collected during the first three stages (first stage: 0 to 7 days, second stage: 14 to 21 days, and third stage: 28 to 35 days) and at the end of the treatment (overall VAS score). Evaluation of the cause of pain was categorized into three different types of problem (category 1: non-smoothed marginal ridge or missing materials, category 2: deformation of attachments, and Category 3: deformation of the tray). Statistical comparison of VAS scores between groups was performed by two-way analysis of variance.
A significantly higher VAS score was observed at 3 and 4 days after, at 1, 2, and 3 days after, and at 2 and 3 days after in stages 1, 2, and 3, respectively, in EG compared to EIG and IG. A significant difference was observed in overall VAS scores between EG and IG in intensity of pain, number of days that pain lasted, and discomfort level. Only intensity of pain resulted in a significant difference between EG and EIG. Most of the causes of problem in the Invisalign cases were deformation of the tray.
Invisalign may offer less pain compared to the edgewise appliance during the initial stages of treatment. In the use of Invisalign, deformation of tray must be carefully checked to avoid pain and discomfort for the patients.
PMCID: PMC4240829  PMID: 25416143
Invisalign; Pain; Tooth movement; Edgewise appliance
20.  Characterization of Time-Course Morphological Features for Efficient Prediction of Osteogenic Potential in Human Mesenchymal Stem Cells 
Biotechnology and Bioengineering  2014;111(7):1430-1439.
Human bone marrow mesenchymal stem cells (hBMSCs) represents one of the most frequently applied cell sources for clinical bone regeneration. To achieve the greatest therapeutic effect, it is crucial to evaluate the osteogenic differentiation potential of the stem cells during their culture before the implantation. However, the practical evaluation of stem cell osteogenicity has been limited to invasive biological marker analysis that only enables assaying a single end-point. To innovate around invasive quality assessments in clinical cell therapy, we previously explored and demonstrated the positive predictive value of using time-course images taken during differentiation culture for hBMSC bone differentiation potential. This initial method establishes proof of concept for a morphology-based cell evaluation approach, but reveals a practical limitation when considering the need to handle large amounts of image data. In this report, we aimed to scale-down our proposed method into a more practical, efficient modeling scheme that can be more broadly implemented by physicians on the frontiers of clinical cell therapy. We investigated which morphological features are critical during the osteogenic differentiation period to assure the performance of prediction models with reduced burden on image acquisition. To our knowledge, this is the first detailed characterization that describes both the critical observation period and the critical number of time-points needed for morphological features to adequately model osteogenic potential. Our results revealed three important observations: (i) the morphological features from the first 3 days of differentiation are sufficiently informative to predict bone differentiation potential, both activities of alkaline phosphatase and calcium deposition, after 3 weeks of continuous culture; (ii) intervals of 48 h are sufficient for measuring critical morphological features; and (iii) morphological features are most accurately predictive when early morphological features from the first 3 days of differentiation are combined with later features (after 10 days of differentiation). Biotechnol. Bioeng. 2014;111: 1430–1439.
PMCID: PMC4231256  PMID: 24420699
image-based analysis; mesenchymal stem cell; non-invasive analysis; osteogenic differentiation; prediction
21.  Association of a genetic variant of the ZPR1 zinc finger gene with type 2 diabetes mellitus 
Biomedical Reports  2014;3(1):88-92.
Various loci and genes that confer susceptibility to coronary heart disease (CHD) have been identified in Caucasian populations by genome-wide association studies (GWASs). As type 2 diabetes mellitus (DM) is an important risk factor for CHD, we hypothesized that certain polymorphisms may contribute to the genetic susceptibility to CHD through affecting the susceptibility to type 2 DM. The purpose of the present study was to examine a possible association of type 2 DM in Japanese individuals with 29 polymorphisms identified as susceptibility loci for CHD by meta-analyses of the GWASs. The study subjects comprised of 3,757 individuals (1,444 subjects with type 2 DM and 2,313 controls). The polymorphism genotypes were determined by the multiplex bead-based Luminex assay, which combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. To compensate for multiple comparisons of genotypes, the criterion of a false discovery rate (FDR) ≤0.05 was adopted for testing the statistical significance of the association. The comparisons of allele frequencies by the χ2 test revealed that the rs964184 (C→G) of the ZPR1 zinc finger gene (ZPR1) was significantly associated (P=0.0017; FDR=0.050) with type 2 DM. Multivariable logistic regression analysis with adjustment for age, gender and body mass index revealed that rs964184 of ZPR1 was significantly associated (P=0.0012; odds ratio, 1.25; dominant model) with type 2 DM with the minor G allele representing a risk factor for this condition. Fasting plasma glucose levels (P=0.0076) and blood glycosylated hemoglobin contents (P=0.0132) significantly differed among ZPR1 genotypes with the G allele associated with increases in these parameters. ZPR1 may thus be a susceptibility locus for type 2 DM in Japanese individuals.
PMCID: PMC4251148  PMID: 25469254
diabetes mellitus; genetics; polymorphism; ZPR1 zinc finger gene
22.  Characterization of Reverse Genetics-Derived Cold-Adapted Master Donor Virus A/Leningrad/134/17/57 (H2N2) and Reassortants with H5N1 Surface Genes in a Mouse Model 
Live attenuated influenza vaccines (LAIV) offer significant advantages over subunit or split inactivated vaccines to mitigate an eventual influenza pandemic, including simpler manufacturing processes and more cross-protective immune responses. Using an established reverse genetics (rg) system for wild-type (wt) A/Leningrad/134/1957 and cold-adapted (ca) A/Leningrad/134/17/1957 (Len17) master donor virus (MDV), we produced and characterized three rg H5N1 reassortant viruses carrying modified HA and intact NA genes from either A/Vietnam/1203/2004 (H5N1, VN1203, clade 1) or A/Egypt/321/2007 (H5N1, EG321, clade 2) virus. A mouse model of infection was used to determine the infectivity and tissue tropism of the parental wt viruses compared to the ca master donor viruses as well as the H5N1 reassortants. All ca viruses showed reduced replication in lungs and enhanced replication in nasal epithelium. In addition, the H5N1 HA and NA enhanced replication in lungs unless it was restricted by the internal genes of the ca MDV. Mice inoculated twice 4 weeks apart with the H5N1 reassortant LAIV candidate viruses developed serum hemagglutination inhibition HI and IgA antibody titers to the homologous and heterologous viruses consistent with protective immunity. These animals remained healthy after challenge inoculation with a lethal dose with homologous or heterologous wt H5N1 highly pathogenic avian influenza (HPAI) viruses. The profiles of viral replication in respiratory tissues and the immunogenicity and protective efficacy characteristics of the two ca H5N1 candidate LAIV viruses warrant further development into a vaccine for human use.
PMCID: PMC4018889  PMID: 24648485
24.  Effective palliative radiofrequency ablation for tumors causing pain, numbness and motor function disorders: case series 
BMC Research Notes  2014;7(1):765.
We present a case series of a palliative radiofrequency ablation (RFA) for the tumors that lead to the resolution of pain and motor function disorders. RFA is widely used on tumors in various organs and often reported in good outcome. There are some reports that RFA was performed as a palliative treatment but a few reports of RFA that performed for lung tumor as a palliative treatment. This case series includes two cases, palliative RFA for a sacrum and a lung tumor. The results of this case series presented that a palliative RFA is effective in improving the symptoms of patients.
Case presentation
Case 1. A 64-year-old Japanese woman with a chordoma at her sacrum presented with pain in her left leg and claudication. Though operations, radiation therapy and GS-TAE (gelatin sponge–transarterial embolization, via the L5 lumbar artery) were performed, the size of the tumor leading pain and claudication increased. RFA was performed for the sacral tumor, and these symptoms resolved one year after the procedure.
Case 2. A 68-year-old Japanese man with a leiomyosarcoma at the apex of left lung presented with pain and motor function disorders of the left upper limb. Dissemination in the pleura was appeared after the operation for a leiomyosarcoma at the mediastinum. Though radiation therapy and a second operation were performed, the tumor at the apex of the left lung increased and pain and numbness of the left upper limb were appeared after the second operation. RFA was performed for the left lung tumor, and the symptoms resolved 3 months after RFA.
RFA is effective as a palliative treatment and has a potential to salvage the patients from the symptoms of the tumors when conventional palliative treatments such as surgery, radiation therapy, and chemotherapy are difficult or contraindicated.
PMCID: PMC4226904  PMID: 25348892
Radiofrequency ablation; Palliative treatment; Carcinomatous neuropathy
25.  In vivo Sonothrombolysis of Ear Marginal Vein of Rabbits Monitored with High-frequency Ultrasound Needle Transducer 
Ultrasound (US) is known to enhance thrombolysis when thrombolytic agents and/or microbubbles are injected into the targeted vessels. In this research, high-intensity US (1 MHz, 7 W/cm2, 30 % duty cycle) was applied in vivo to the ear marginal vein of three rabbits which was occluded by either laser photothrombosis or thrombin, right after the injection of 0.3~0.6 cc of microbubbles (13 × 108 bubbles/ml of concentration) through the other ear vein without using any thrombolytic agent. To determine the effect of the sonothrombolysis, the blood flow velocity near the occlusion site in the vein was measured by a custom-made 40-MHz US needle transducer and its corresponding Doppler US system. The Doppler spectra show that the blood flow velocity recovered from total occlusion after three 10-minute high-intensity US treatments. Fluorescein angiography was employed to confirm the opening of the vessel occlusion. A control study of three rabbits with only the microbubble injection showed no recovery on the occlusion in 3 hours. The results show that the sonothrombolysis in the rabbit ear marginal vein can be achieved with microbubbles only. The results of cavitation measurements indicate that the mechanism of sonothrombolysis is probably due to the cavitation induced by the microbubbles. Without the need of applying any thrombolytic agent, high-intensity US has high potential for therapies targeting on small blood vessels.
PMCID: PMC4209747  PMID: 25356073
Therapeutic ultrasound; Sonothrombolysis; Rabbit ear marginal vein; Microbubbles

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