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Find video protocols related to scientific articles indexed in Pubmed.
Can sonographic features be efficacy predictors of robotic massage treatment for masseter and temporal muscle in patients with temporomandibular disorder with myofascial pain?
Cranio
PUBLISHED: 11-18-2014
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Objective: The aim of this study was to detect sonographic predictors for the efficacy of massage treatment of masseter and temporal muscle in temporomandibular disorders (TMDs) patients with myofascial pain. Methods: Thirty-seven TMD patients with myofascial pain (6 men and 31 women, a median age of 45 years) were enrolled. An oral rehabilitation robot massaged the patient's masseter and temporal muscles with a standard massage pressure of 10 N for 16 min. The standard treatment protocol was set five sessions every 2 weeks. The median total duration of treatment was 9·5 weeks. Efficacy of treatment was evaluated based on maximum mouth opening and visual analog scale scores of muscle pain and daily life impediments. The intramuscular echogenic bands and elasticity index ratios of the masseter muscles were evaluated on sonographic or sonoelastographic images obtained before treatment and after the third and last treatment sessions. Results: The sonographic features detected different changes after the third treatment session between the therapy-effective and therapy-ineffective groups: in the therapy-effective group, the frequency of visibility of the distinct echogenic bands increased, and the elasticity index ratio decreased. Conclusion: Sonographic features after the third treatment session may be useful as predictors of therapeutic efficacy.
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Potential clinical application of masseter and temporal muscle massage treatment using an oral rehabilitation robot in temporomandibular disorder patients with myofascial pain.
Cranio
PUBLISHED: 11-12-2014
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Objectives: To investigate the safety, suitable treatment regimen, and efficacy of masseter and temporal muscle massage treatment using an oral rehabilitation robot. Methods: Forty-one temporomandibular disorder (TMD) patients with myofascial pain (8 men, 33 women, median age: 46 years) were enrolled. The safety, suitable massage regimen, and efficacy of this treatment were investigated. Changes in masseter muscle thickness were evaluated on sonograms. Results: No adverse events occurred with any of the treatment sessions. Suitable massage was at pressure of 10 N for 16 minutes. Five sessions were performed every 2 weeks. Total duration of treatment was 9·5 weeks in median. Massage treatment was effective in 70·3% of patients. Masseter muscle thickness decreased with treatment in the therapy-effective group. Conclusion: This study confirmed the safety of massage treatment, and established a suitable regimen. Massage was effective in 70·3% of patients and appeared to have a potential as one of the effective treatments for myofascial pain.
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Visible-light photodecomposition of acetaldehyde by TiO2-coated gold nanocages: plasmon-mediated hot electron transport via defect states.
Chem. Commun. (Camb.)
PUBLISHED: 10-30-2014
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Skeletal gold nanocages (Au NCs) are synthesized and coated with TiO2 layers (TiO2-Au NCs). The TiO2-Au NCs exhibit enhanced photodecomposition activity toward acetaldehyde under visible light (>400 nm) illumination because hot electrons are generated over the Au NCs by local surface plasmon resonance (LSPR) and efficiently transported across the metal/semiconductor interface via the defect states of TiO2.
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Light and Intermittent Smoking Among California Black, Hispanic/Latino, and Non-Hispanic White Men and Women.
Nicotine Tob. Res.
PUBLISHED: 10-21-2014
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The prevalence of light and intermittent smoking (LITS) is increasing as the prevalence of heavier smoking continues to fall. The purpose of this study was to examine changes in LITS over time among Blacks, Hispanic/Latinos (Latinos) and non-Hispanic Whites (Whites).
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Increase of TRPV1-Immunoreactivity in Dorsal Root Ganglia Neurons Innervating the Femur in a Rat Model of Osteoporosis.
Yonsei Med. J.
PUBLISHED: 10-18-2014
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Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel, which can be activated by capsaicin and other noxious stimuli. Recently, an association between bone pain and TRPV1 has been reported. However, the influence of osteoporosis on TRPV1 in the sensory system innervating the femur has not been reported.
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Public attitudes toward practice by medical students: a nationwide survey in Japan.
Teach Learn Med
PUBLISHED: 10-16-2014
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It is essential for medical students to interact directly with patients. However, patients may be reluctant to be seen by medical students in settings in which they may also be seen by senior staff.
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Phase II Study of Concurrent Chemoradiotherapy with S-1 in Patients with Stage II (T2N0M0) Squamous Cell Carcinoma of the Pharynx or Larynx.
Jpn. J. Clin. Oncol.
PUBLISHED: 10-01-2014
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The goals of treatment for head and neck cancer are cure and organ-function preservation. For organ preservation, primary treatment via radiotherapy alone is thought to be insufficient for Stage II squamous cell carcinoma of the larynx, oropharynx or hypopharynx. The objective of the present study was to investigate the efficacy and safety of concurrent chemoradiotherapy with S-1 for patients with Stage II squamous cell carcinoma of the pharynx or larynx for primary organ preservation.
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Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato.
Plant Cell
PUBLISHED: 09-12-2014
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Potatoes (Solanum tuberosum) contain ?-solanine and ?-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts and green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) from potato as a key enzyme in the biosynthesis of cholesterol and related SGAs. Using in vitro enzyme activity assays, we determined that potato SSR2 (St SSR2) reduces desmosterol and cycloartenol to cholesterol and cycloartanol, respectively. These reduction steps are branch points in the biosynthetic pathways between C-24 alkylsterols and cholesterol in potato. Similar enzymatic results were also obtained from tomato SSR2. St SSR2-silenced potatoes or St SSR2-disrupted potato generated by targeted genome editing had significantly lower levels of cholesterol and SGAs without affecting plant growth. Our results suggest that St SSR2 is a promising target gene for breeding potatoes with low SGA levels.
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Influence of adverse drug events on morbidity and mortality in intensive care units: the JADE study.
Int J Qual Health Care
PUBLISHED: 09-05-2014
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To identify the influence of adverse drug events (ADEs) on morbidity and mortality in intensive care units (ICUs).
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An Individual Patient Data Meta-Analysis on Factors Associated with Adverse Drug Events in Surgical and Nonsurgical Inpatients.
Br J Clin Pharmacol
PUBLISHED: 09-02-2014
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The incidence of adverse drug events (ADEs) in surgical and non-surgical patients may differ. This IPDMA identifies patient characteristics and types of medication most associated with patients experiencing ADEs and suggests target areas for reducing harm and implement focused interventions.
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Down syndrome-associated haematopoiesis abnormalities created by chromosome transfer and genome editing technologies.
Sci Rep
PUBLISHED: 08-27-2014
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Infants with Down syndrome (DS) are at a high risk of developing transient abnormal myelopoiesis (TAM). A GATA1 mutation leading to the production of N-terminally truncated GATA1 (GATA1s) in early megakaryocyte/erythroid progenitors is linked to the onset of TAM and cooperated with the effect of trisomy 21 (Ts21). To gain insights into the underlying mechanisms of the progression to TAM in DS patients, we generated human pluripotent stem cells harbouring Ts21 and/or GATA1s by combining microcell-mediated chromosome transfer and genome editing technologies. In vitro haematopoietic differentiation assays showed that the GATA1s mutation blocked erythropoiesis irrespective of an extra chromosome 21, while Ts21 and the GATA1s mutation independently perturbed megakaryopoiesis and the combination of Ts21 and the GATA1s mutation synergistically contributed to an aberrant accumulation of skewed megakaryocytes. Thus, the DS model cells generated by these two technologies are useful in assessing how GATA1s mutation is involved in the onset of TAM in patients with DS.
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Impact of the early detection of esophageal neoplasms in hypopharyngeal cancer patients treated with concurrent chemoradiotherapy.
Asia Pac J Clin Oncol
PUBLISHED: 08-21-2014
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We examined the risk factors and prognostic factors for synchronous esophageal neoplasia (SEN) by comparing the characteristics of hypopharyngeal cancer (HPC) patients with and without SEN.
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Beta cell regeneration after single-round immunological destruction in a mouse model.
Diabetologia
PUBLISHED: 08-21-2014
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Achieving a better understanding of beta cell regeneration after immunological destruction is crucial for the development of immunotherapy approaches for type 1 diabetes. In previous type 1 diabetes models, sustained immune activation eliminates regenerating beta cells, thus limiting the study of the regenerative capacity of beta cells upon immunological destruction. Here, we employed an adeno-associated virus 8 (AAV8) vector for beta cell-targeted overexpression of a foreign antigen to induce single-round immunological destruction of existing beta cells.
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EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step.
J. Cell Biol.
PUBLISHED: 08-06-2014
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Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) in which Htm1-mediated mannose trimming from the oligosaccharide Man8GlcNAc2 to Man7GlcNAc2 is the rate-limiting step in yeast. In contrast, the roles of the three Htm1 homologues (EDEM1/2/3) in mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or as lectins. We therefore conducted transcription activator-like effector nuclease-mediated gene knockout analysis in human cell line and found that all endogenous EDEMs possess mannosidase activity. Mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 is performed mainly by EDEM3 and to a lesser extent by EDEM1. Most surprisingly, the upstream mannose trimming from Man9GlcNAc2 to Man8GlcNAc2 is conducted mainly by EDEM2, which was previously considered to lack enzymatic activity. Based on the presence of two rate-limiting steps in mammalian gpERAD, we propose that mammalian cells double check gpERAD substrates before destruction by evolving EDEM2, a novel-type Htm1 homologue that catalyzes the first mannose trimming step from Man9GlcNAc2.
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Application of oocyte cryopreservation technology in TALEN-mediated mouse genome editing.
Exp. Anim.
PUBLISHED: 08-01-2014
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Reproductive engineering techniques, such as in vitro fertilization (IVF) and cryopreservation of embryos or spermatozoa, are essential for preservation, reproduction, and transportation of genetically engineered mice. However, it has not yet been elucidated whether these techniques can be applied for the generation of genome-edited mice using engineered nucleases such as transcription activator-like effector nucleases (TALENs). Here, we demonstrate the usefulness of frozen oocytes fertilized in vitro using frozen sperm for TALEN-mediated genome editing in mice. We examined side-by-side comparisons concerning sperm (fresh vs. frozen), fertilization method (mating vs. IVF), and fertilized oocytes (fresh vs. frozen) for the source of oocytes used for TALEN injection; we found that fertilized oocytes created under all tested conditions were applicable for TALEN-mediated mutagenesis. In addition, we investigated whether the ages in weeks of parental female mice can affect the efficiency of gene modification, by comparing 5-week-old and 8-12-week-old mice as the source of oocytes used for TALEN injection. The genome editing efficiency of an endogenous gene was consistently 95-100% when either 5-week-old or 8-12-week-old mice were used with or without freezing the oocytes. Thus, our report describes the availability of freeze-thawed oocytes and oocytes from female mice at various weeks of age for TALEN-mediated genome editing, thus boosting the convenience of such innovative gene targeting strategies.
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Targeted gene disruption by use of transcription activator-like effector nuclease (TALEN) in the water flea Daphnia pulex.
BMC Biotechnol.
PUBLISHED: 07-26-2014
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BackgroundThe cosmopolitan microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have its complete genome sequenced, an unprecedented ca. 36% of which has no known homologs with any other species. Moreover, D. pulex is ideally suited for experimental manipulation because of its short reproductive cycle, large numbers of offspring, synchronization of oocyte maturation, and other life history characteristics. However, existing gene manipulation techniques are insufficient to accurately define gene functions. Although our previous investigations developed an RNA interference (RNAi) system in D. pulex, the possible time period of functional analysis was limited because the effectiveness of RNAi is transient. Thus, in this study, we developed a genome editing system for D. pulex by first microinjecting transcription activator-like effector nuclease (TALEN) mRNAs into early embryos and then evaluating TALEN activity and mutation phenotypes.ResultsWe assembled a TALEN construct specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for distal limb development in invertebrates and vertebrates, and evaluated its activity in vitro by single-strand annealing assay. Then, we injected TALEN mRNAs into eggs within 1 hour post-ovulation. Injected embryos presented with defects in the second antenna and altered appendage development, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.ConclusionsWe succeeded, for the first time in D. pulex, in targeted mutagenesis by use of Platinum TALENs. This genome editing technique makes it possible to conduct reverse genetic analysis in D. pulex, making this species an even more appropriate model organism for environmental, evolutionary, and developmental genomics.
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Progressive change in joint degeneration in patients with knee or hip osteoarthritis treated with fentanyl in a randomized trial.
Yonsei Med. J.
PUBLISHED: 07-23-2014
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Opioids improve pain from knee and hip osteoarthritis (OA) and decrease the functional impairment of patients. However, there is a possibility that opioids induce analgesia and suppress the physiological pain of OA in patients, thereby inducing the progression of OA changes in these patients. The purpose of the current study was to investigate the possibility of progressive changes in OA among patients using opioids.
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Transient receptor potential vanilloid 1-immunoreactive innervation increases in fractured rat femur.
Yonsei Med. J.
PUBLISHED: 06-28-2014
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Pain from vertebral or femoral neck fractures is a particularly important problem in clinical orthopaedics. Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel, and there are recent reports on an association between bone pain and TRPV1. However, an increase in TRPV1 activity has not been reported following femoral fracture.
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Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems.
Sci Rep
PUBLISHED: 06-27-2014
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Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.
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Drosophila Strip serves as a platform for early endosome organization during axon elongation.
Nat Commun
PUBLISHED: 06-18-2014
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Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. Here we show that Drosophila Strip is a key regulator for neuronal morphogenesis. Strip knockdown disturbs the early endosome clustering, and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip-mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that has important roles in neuronal morphogenesis.
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Differential expression of periostin in the nasal polyp may represent distinct histological features of chronic rhinosinusitis.
Auris Nasus Larynx
PUBLISHED: 06-18-2014
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Chronic rhinosinusitis (CRS) is thought to be a multifactorial disease, and it is classified into a number of subtypes according to clinicohistological features. Periostin, a 90-kDa secreted protein, was reported to exist in nasal polyps (NPs) associated with CRS. We compared the expression of periostin with the degree of eosinophilic infiltration as well as tissue remodeling.
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Immunohistochemical analysis of the expression of E-cadherin and ZEB1 in non-small cell lung cancer.
Pathol. Int.
PUBLISHED: 06-10-2014
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We performed an immunohistochemical analysis of the expression of zinc-finger E-box binding homeobox 1 (ZEB1), a master regulator of epithelial-mesenchymal transition (EMT), and determined its relationship with E-cadherin in 157 non-small cell lung carcinomas (93 adenocarcinomas, 36 squamous cell carcinomas, 18 large cell carcinomas, and 10 pleomorphic carcinomas). Although the expression of E-cadherin was low in the subset of adenocarcinomas (10%) and squamous cell carcinomas (11%), ZEB1 expression was only observed in one case of squamous cell carcinoma and none of the adenocarcinomas. In contrast, the low expression of E-cadherin (50% and 90%, respectively) and the positive expression of ZEB1 (11% and 50%, respectively) were more frequently observed in poorly differentiated carcinomas (large cell carcinomas and pleomorphic carcinomas). Overall, the expression of ZEB1 was inversely correlated with that of E-cadherin. Furthermore, the distribution of ZEB1-positive cancer cells was more restricted than in the area in which the expression of E-cadherin was lost, and the former was detected within the latter. We concluded that the expression of ZEB1 was not necessarily associated with the low expression of E-cadherin in lung adenocarcinomas and squamous cell carcinomas. The expression of ZEB1 correlated with an undifferentiated and/or sarcomatoid morphology that may occur in the late stage of EMT.
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Specific binding of the WASP N-terminal domain to Btk is critical for TLR2 signaling in macrophages.
Mol. Immunol.
PUBLISHED: 06-09-2014
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Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we revealed that WASP is involved in lipopolysaccharide-TLR4 signaling in macrophages by association of Bruton's tyrosine kinase (Btk) with the WASP N-terminal domain. Btk has been shown to play important roles in the signaling of several TLRs and to modulate the inflammatory response in macrophages. In this study, we evaluated the importance of the interaction between Btk and WASP in TLR2 signaling by using bone marrow-derived macrophage cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) or VL single-domain intrabodies. In this Tg bone marrow-derived macrophages, specific interaction between WASP and Btk were strongly inhibited by masking of the binding site in the WASP N-terminal domain. There was impairment of gene expression of TNF-?, IL-6, and IL-1? and phosphorylation of inhibitor of ?B ?/? (IKK?/?) and nuclear factor (NF)-?B upon stimulation with TLR2 ligands. Furthermore, tyrosine phosphorylation of WASP following TLR2-ligand stimulation was severely inhibited in the Tg bone marrow-derived macrophages, as shown by the impairment in WASP tyrosine phosphorylation following lipopolysaccharide stimulation. These results strongly suggest that the association between the WASP N-terminal domain and Btk plays an important role in the TLR2-signaling pathway in macrophages.
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Fluorescence-based endoscopic imaging of Thomsen-Friedenreich antigen to improve early detection of colorectal cancer.
Int. J. Cancer
PUBLISHED: 05-20-2014
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Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level.
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Development of olfactory projection neuron dendrites that contribute to wiring specificity of the Drosophila olfactory circuit.
Genes Genet. Syst.
PUBLISHED: 05-13-2014
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The antennal lobe (AL) of Drosophila is the first olfactory processing center in which olfactory input and output are spatially organized into distinct channels via glomeruli to form a discrete neural map. In each glomerulus, the axons of a single type of olfactory receptor neurons (ORNs) synapse with the dendrites of a single type of projection neurons (PNs). The AL is an ideal place to study how the wiring specificity between specific types of ORNs and PNs is established during development. During the past two decades, the involvement of diverse molecules in the specification and patterning of ORNs and PNs has been reported. Furthermore, local interneurons-another component of glomeruli-have been recently catalogued and their functions have been gradually dissected. Although there is accumulating knowledge about the involvement of these three cell types in the wiring specificity of the olfactory system, in this review, we focus especially on the development of PN dendrites.
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Temporary facial nerve dysfunction after parotidectomy correlates with tumor location.
Auris Nasus Larynx
PUBLISHED: 04-30-2014
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To investigate the clinical factors associated with temporary facial nerve dysfunction (TFND) following surgery for benign parotid gland tumors.
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Adverse drug events and medication errors in Japanese paediatric inpatients: a retrospective cohort study.
BMJ Qual Saf
PUBLISHED: 04-17-2014
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Knowledge about the epidemiology of adverse drug events (ADEs) and medication errors in paediatric inpatients is limited outside Western countries. To improve paediatric patient safety worldwide, assessing local epidemiology is essential.
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Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system.
Sci Rep
PUBLISHED: 04-04-2014
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CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications, not only for single gene targeting, but also for multiple targeted mutagenesis. To make the most of the multiplexity of CRISPR/Cas9, we established a system for constructing all-in-one expression vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette. We further demonstrated successful examples of multiple targeting including chromosomal deletions in human cells using the all-in-one CRISPR/Cas9 vectors constructed with our novel system. Our system provides an efficient targeting strategy for multiplex genome/epigenome editing, simultaneous activation/repression of multiple genes, and beyond.
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Incidence of nocturnal leg cramps in patients with lumbar spinal stenosis before and after conservative and surgical treatment.
Yonsei Med. J.
PUBLISHED: 04-01-2014
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To examine the effects of conservative and surgical treatments for nocturnal leg cramps in patients with lumbar spinal stenosis (LSS). Nocturnal leg cramps is frequently observed in patients with peripheral neuropathy. However, there have been few reports on the relationship between nocturnal leg cramps and LSS, and it remains unknown whether conservative or surgical intervention has an impact on leg cramps in patients with LSS.
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Concurrent chemoradiotherapy with docetaxel, cisplatin and 5-fluorouracil (TPF) in patients with locally advanced squamous cell carcinoma of the head and neck.
Jpn. J. Clin. Oncol.
PUBLISHED: 03-30-2014
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Compared with radiotherapy alone, concurrent chemoradiotherapy significantly improves survival rates for patients with squamous cell carcinoma of the head and neck. The aim of this study was to retrospectively evaluate the efficacy, toxicity and long-term prognosis of concurrent chemoradiotherapy with docetaxel, cisplatin and 5-fluorouracil chemotherapy.
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Experimental pain in the gingiva and its impact on prefrontal cortical hemodynamics: a functional near-infrared spectroscopy study.
Neurosci. Lett.
PUBLISHED: 03-26-2014
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Evaluating alterations in brain activity in response to pain stimulus can help understand the mechanisms underlying pain perception. We measured oxygenated hemoglobin (oxy-Hb) levels using functional near-infrared spectroscopy (fNIRS) in order to assess prefrontal cortex activation after inducing a pain stimulus to the gingiva. Twenty-three right-handed, healthy male subjects (mean age: 29.3±3.6 years) were subjected to a mild pain stimulus to the tissue around the right maxillary central incisor. The periodontal pain stimulus (PPS) was elicited from a pocket probe, and a multi-channel fNIRS system with its accompanying 22-channel probes was used for measuring oxy-Hb levels. Mean oxy-Hb levels for each channel were calculated on the basis of values obtained at rest and during the PPS load, for 1min each. The change in oxy-Hb level was calculated by subtracting oxy-Hb at rest from oxy-Hb levels during PPS load. Oxy-Hb levels in each channel during both conditions were then compared using the paired t-test and Bonferroni correction. Pain stimulation caused oxy-Hb levels to decrease in virtually all areas of the prefrontal cortex, particularly, in the superior frontal gyrus, the middle frontal gyrus, and the orbital part of the superior, middle, and inferior frontal gyrus, on the brain side contralateral to the pain load. This measurement could prove beneficial as an index for objective pain evaluation.
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Increases in light and intermittent smoking among Asian Americans and non-Hispanic Whites.
Nicotine Tob. Res.
PUBLISHED: 03-17-2014
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Asian Americans are the fastest growing immigrant group in the United States and are more likely to be light and intermittent smokers (LITS) compared with non-Hispanic Whites (NHWs). LITS experience adverse health effects related to smoking. Previous research has aggregated Asian American ethnic groups, masking important differences between groups. We sought to compare LITS rates among Asian American subgroups before and after the 1998 Master Settlement Agreement (MSA) with NHWs in California utilizing data from the California Tobacco Surveys (CTS).
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Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases.
Genesis
PUBLISHED: 03-05-2014
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Targeted mutagenesis of genes-of-interest, or gene-knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis.
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Simple knockout by electroporation of engineered endonucleases into intact rat embryos.
Sci Rep
PUBLISHED: 02-24-2014
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Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, the microinjection of endonucleases into embryos requires a high skill level, is time consuming, and may cause damage to embryos. Here, we demonstrate that the electroporation of endonuclease mRNAs into intact embryos can induce editing at targeted loci and efficiently produce knockout rats. It is noteworthy that the electroporation of ZFNs resulted in an embryonic survival rate (91%) and a genome-editing rate (73%) that were more than 2-fold higher than the corresponding rates from conventional microinjection. Electroporation technology provides a simple and effective method to produce knockout animals.
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Murine leukemia virus uses TREX components for efficient nuclear export of unspliced viral transcripts.
Viruses
PUBLISHED: 02-21-2014
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Previously we reported that nuclear export of both unspliced and spliced murine leukemia virus (MLV) transcripts depends on the nuclear export factor (NXF1) pathway. Although the mRNA export complex TREX, which contains Aly/REF, UAP56, and the THO complex, is involved in the NXF1-mediated nuclear export of cellular mRNAs, its contribution to the export of MLV mRNA transcripts remains poorly understood. Here, we studied the involvement of TREX components in the export of MLV transcripts. Depletion of UAP56, but not Aly/REF, reduced the level of both unspliced and spliced viral transcripts in the cytoplasm. Interestingly, depletion of THO components, including THOC5 and THOC7, affected only unspliced viral transcripts in the cytoplasm. Moreover, the RNA immunoprecipitation assay showed that only the unspliced viral transcript interacted with THOC5. These results imply that MLV requires UAP56, THOC5 and THOC7, in addition to NXF1, for nuclear export of viral transcripts. Given that naturally intronless mRNAs, but not bulk mRNAs, require THOC5 for nuclear export, it is plausible that THOC5 plays a key role in the export of unspliced MLV transcripts.
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Injection of bupivacaine into disc space to detect painful nonunion after anterior lumbar interbody fusion (ALIF) surgery in patients with discogenic low back pain.
Yonsei Med. J.
PUBLISHED: 02-18-2014
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Bupivacaine is commonly used for the treatment of back pain and the diagnosis of its origin. Nonunion is sometimes observed after spinal fusion surgery; however, whether the nonunion causes pain is controversial. In the current study, we aimed to detect painful nonunion by injecting bupivacaine into the disc space of patients with nonunion after anterior lumbar interbody fusion (ALIF) surgery for discogenic low back pain.
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Screening methods to identify TALEN-mediated knockout mice.
Exp. Anim.
PUBLISHED: 02-14-2014
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Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms.
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Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts.
J. Virol.
PUBLISHED: 01-29-2014
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Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts, and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/TAP in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses, including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named the CAE (cytoplasmic accumulation element). The CAE enhanced the cytoplasmic accumulation of viral RNA transcripts and the expression of viral proteins without significantly affecting the stability, splicing, or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found that the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in an NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and nonspliced viral RNA transcripts.
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Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay.
Sci Rep
PUBLISHED: 01-28-2014
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Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated "affinity" silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.
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FAST-id system for enrichment of cells with TALEN-induced mutations and large deletions.
Genes Cells
PUBLISHED: 01-22-2014
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Transcription activator-like effector nuclease (TALEN)-mediated genome editing is a powerful technique for analyzing gene functions in various cells and organisms. At target loci, TALENs can not only introduce short insertions and deletions, but also yield large deletions through the use of two TALEN pairs. Here, we report easy and efficient methods for enrichment of cells with TALEN-induced mutations and large deletions. First, we established the fluorescence-activated sorting of TALEN-induced deletions (FAST-id) system that enabled fluorescence-activated cell sorting-mediated enrichment of cells with TALEN-induced mutations. In the FAST-id system, either EGFP or mCherry and TALENs were co-expressed. Using dual fluorescence selection, both left and right TALEN-expressing cells were easily concentrated, resulting in enrichment of TALEN-mediated mutated cells. Next, to apply the FAST-id system to enrichment of cells with large deletions, we developed the fast unification of separate endonucleases (FUSE) method for assembly of two TALENs into a single expression vector. Using the FUSE method, we easily obtained a TALEN pair-expressing plasmid driven by a single promoter. By combining the FAST-id system and FUSE method, cells with large deletions were efficiently enriched. To the best of our knowledge, this is the first report of enrichment of cells with TALEN-induced large deletions.
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A new in vitro system for evaluation of passive intestinal drug absorption: Establishment of a double artificial membrane permeation assay.
Eur J Pharm Biopharm
PUBLISHED: 01-16-2014
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The aim of this present study was to establish a new in vitro assay, double artificial membrane permeation assay (DAMPA), to evaluate the human intestinal permeability of drugs. A double artificial membrane with an intracellular compartment was constructed in side-by-side chambers by sandwiching a filter containing buffer solution with impregnated lipophilic filters with dodecane containing 2w/v% phosphatidylcholine. Permeation data of ionic compounds clearly indicated that not only the pH value of the apical solution but also that of the intracellular compartment affected the permeability across the double artificial membrane. DAMPA was performed with 20 compounds at physiological pH (apical; 6.5, intracellular and basal; 7.4). Paracellular and transcellular permeabilities of compounds in human epithelium were estimated based on the characteristics of the paracellular pathway using physicochemical properties of compounds with the Renkin function and the area factor i.e. the difference in the effective surface area between human epithelium and the double artificial membrane, respectively. The human intestinal permeability of each compound was predicted by the sum of estimated transcellular and paracellular permeabilities. Predicted human intestinal permeability was significantly correlated with the fraction of absorbed dose in humans, indicating that DAMPA has the potential to predict oral absorption of drugs in humans.
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Versatile strategy for isolating transcription activator-like effector nuclease-mediated knockout mutants in Caenorhabditis elegans.
Dev. Growth Differ.
PUBLISHED: 01-14-2014
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Targeted genome editing using transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems has recently emerged as a potentially powerful method for creating locus-specific mutations in Caenorhabditis elegans. Due to the low mutation frequencies, one of the crucial steps in using these technologies is screening animals that harbor a targeted mutation. In previous studies, identifying targeted mutations in C. elegans usually depended on observations of fluorescent markers such as a green fluorescent protein or visible phenotypes such as dumpy and uncoordinated phenotypes. However, this strategy is limited in practice because the phenotypes caused by targeted mutations such as defects in sensory behaviors are often apparently invisible. Here, we describe a versatile strategy for isolating C. elegans knockout mutants by TALEN-mediated genome editing and a heteroduplex mobility assay. We applied TALENs to engineer the locus of the neural gene glr-1, which is a C. elegans AMPA-type receptor orthologue that is known to have crucial roles in various sensory behaviors. Knockout mutations in the glr-1 locus, which caused defective mechanosensory behaviors, were efficiently identified by the heteroduplex mobility assay. Thus, we demonstrated the utility of a TALEN-based knockout strategy for creating C. elegans with mutations that cause invisible phenotypes.
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Nuclease-mediated genome editing: At the front-line of functional genomics technology.
Dev. Growth Differ.
PUBLISHED: 01-05-2014
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Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose.
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Role of glutamine-169 in the substrate recognition of human aminopeptidase B.
Biochim. Biophys. Acta
PUBLISHED: 01-02-2014
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Aminopeptidase B (EC 3.4.11.6, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB.
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Establishment of c-myc-immortalized Kupffer cell line from a C57BL/6 mouse strain.
Results Immunol
PUBLISHED: 01-01-2014
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We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells) in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7-10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5) was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4-5 days intervals for more than 5 months, with a population doubling time of 19 h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro.
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Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5?-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production.
PLoS ONE
PUBLISHED: 01-01-2014
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Old world monkey TRIM5? is a host factor that restricts human immunodeficiency virus type-1 (HIV-1) infection. Previously, we reported that rhesus macaque TRIM5? (RhTRIM5?) restricts HIV-1 production by inducing degradation of precursor Gag. Since suppressor of cytokine signaling 1 (SOCS1) is known to enhance HIV-1 production by rescuing Gag from lysosomal degradation, we examined if SOCS1 is involved in RhTRIM5?-mediated late restriction. Over-expression of SOCS1 restored HIV-1 production in the presence of RhTRIM5? to a level comparable to that in the absence of RhTRIM5? in terms of titer and viral protein expression. Co-immunoprecipitation studies revealed that SOCS1 physically interacted with RhTRIM5?. Over-expression of SOCS1 affected RhTRIM5? expression in a dose-dependent manner, which was not reversed by proteasome inhibitors. In addition, SOCS1 and RhTRIM5? were detected in virus-like particles. These results suggest that SOCS1 alleviates RhTRIM5?-mediated regulation in the late phase of HIV-1 life cycle probably due to the destabilization of RhTRIM5?.
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Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona.
Development
PUBLISHED: 12-18-2013
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Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.
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TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-16-2013
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Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA) syndrome] is a rare autosomal recessive disorder characterized by constitutional aneuploidy and a high risk of childhood cancer. We previously reported monoallelic mutations in the BUB1B gene (encoding BUBR1) in seven Japanese families with the syndrome. No second mutation was found in the opposite allele of any of the families studied, although a conserved BUB1B haplotype and a decreased transcript were identified. To clarify the molecular pathology of the second allele, we extended our mutational search to a candidate region surrounding BUB1B. A unique single nucleotide substitution, G > A at ss802470619, was identified in an intergenic region 44 kb upstream of a BUB1B transcription start site, which cosegregated with the disorder. To examine whether this is the causal mutation, we designed a transcription activator-like effector nuclease-mediated two-step single-base pair editing strategy and biallelically introduced this substitution into cultured human cells. The cell clones showed reduced BUB1B transcripts, increased PCS frequency, and MVA, which are the hallmarks of the syndrome. We also encountered a case of a Japanese infant with PCS (MVA) syndrome carrying a homozygous single nucleotide substitution at ss802470619. These results suggested that the nucleotide substitution identified was the causal mutation of PCS (MVA) syndrome.
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Efficacy of massage treatment technique in masseter muscle hardness: robotic experimental approach.
Cranio
PUBLISHED: 12-07-2013
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The study aimed to clarify the masseter muscle hardness in patients with myofascial pain, to examine their changes after massage, and to analyze whether the hardness can be an index for massage treatment. Sixteen patients with myofascial pain (12 with unilateral and 4 with bilateral masseter muscle pain) and 24 healthy volunteers were enrolled in this study. The masseter hardness between patients and the healthy volunteers was compared. The changes in the hardness in patients after massage were examined. The relation of the hardness with massage regimens and efficacies was analyzed. There was a significant right-and-left difference of the hardness in patients, although there was no difference in the healthy volunteers. The hardness decreased after massage. The pretreatment asymmetry index of the hardness showed a significant correlation with the massage pressure. It was concluded that there was a significant difference between the right and left masseter hardness in patients with myofascial pain. After massage treatment, the masseter hardness and right-and-left difference decreased. The hardness may be an index for determining the massage pressure.
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Differences between tumor necrosis factor-? receptors types 1 and 2 in the modulation of spinal glial cell activation and mechanical allodynia in a rat sciatic nerve injury model.
Spine
PUBLISHED: 10-18-2013
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Immunohistological analysis of spinal glial cells and analysis of pain behavior in the rat neuropathic pain model were investigated to clarify the function of tumor necrosis factor (TNF)-? receptors p55 type 1 and p75 type 2.
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The HSP90 inhibitor 17-N-allylamino-17-demethoxy geldanamycin (17-AAG) synergizes with cisplatin and induces apoptosis in cisplatin-resistant esophageal squamous cell carcinoma cell lines via the Akt/XIAP pathway.
Oncol. Rep.
PUBLISHED: 10-09-2013
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Although cisplatin (CDDP) is a key drug in the treatment of esophageal squamous cell carcinoma (ESCC), acquired chemoresistance remains a major problem. Combination therapy may represent one strategy to overcome this resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition by small molecules, either alone or in combination, has shown promise in the treatment of solid malignancies. In the present study, we evaluated the synergistic effects of combining CDDP with the HSP90 inhibitor 17-N-allylamino-17-demethoxy geldanamycin (17-AAG) on two CDDP-resistant human esophageal squamous cancer cell lines, KYSE30 and KYSE150. The results obtained demonstrated the synergistic inhibitory effects of CDDP and 17-AAG on the growth of KYSE30 and KYSE150 cells. Cell growth and cell number were more effectively reduced by the combined treatment with CDDP and 17-AAG than by the treatment with either CDDP or 17-AAG alone. Western blotting revealed that the combined action of CDDP and 17-AAG cleaved poly (ADP-ribose) polymerase (PARP) and caspase-3, which demonstrated that the reduction in both cell growth and cell number was mediated by apoptosis. Time-course experiments showed that reduction in X-linked inhibitor of apoptosis protein (XIAP) and phosphorylated Akt were concomitant with apoptosis. The results of the present study demonstrate that 17-AAG synergizes with CDDP and induces apoptosis in CDDP-resistant ESCC cell lines, and also that modulation of the Akt/XIAP pathway may underlie this synergistic effect. Combination therapy with CDDP and an HSP90 inhibitor may represent a promising strategy to overcome CDDP resistance in ESCC.
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Transcription activator-like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration.
Dev. Growth Differ.
PUBLISHED: 09-18-2013
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Regeneration of a lost tissue in an animal is an important issue. Although regenerative studies have a history of research spanning more than a century, the gene functions underlying regulation of the regeneration are mostly unclear. Analysis of knockout animals is a very powerful tool with which to elucidate gene function. Recently, transcription activator-like effector nucleases (TALENs) have been developed as an effective technique for genome editing. This technique enables gene targeting in amphibians such as newts that were previously impossible. Here we show that newts microinjected with TALEN mRNAs designed for targeting the tyrosinase gene in single-cell stage embryos revealed an albino phenotype. Sequence analysis revealed that the tyrosinase genes were effectively disrupted in these albino newts. Moreover, precise genome alteration was achieved using TALENs and single strand oligodeoxyribonucleotides. Our results suggest that TALENs are powerful tools for genome editing for regenerative research in newts.
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Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.
Dev. Growth Differ.
PUBLISHED: 09-15-2013
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Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.
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Targeted mutagenesis in sea urchin embryos using TALENs.
Dev. Growth Differ.
PUBLISHED: 09-13-2013
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Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos.
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Effect of low-dose aspirin on primary prevention of cardiovascular events in Japanese diabetic patients at high risk.
Circ. J.
PUBLISHED: 09-13-2013
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Background:?Benefit of low-dose aspirin for primary prevention of cardiovascular events in diabetes remains controversial. The American Diabetes Association (ADA), the American Heart Association (AHA), and the American College of Cardiology Foundation (ACCF) recommend aspirin for high-risk diabetic patients: older patients with additional cardiovascular risk factors. We evaluated aspirins benefit in Japanese diabetic patients stratified by cardiovascular risk. Methods and Results:?In the JPAD trial, we enrolled 2,539 Japanese patients with type 2 diabetes and no history of cardiovascular disease. We randomly assigned them to aspirin (81-100mg daily) or no aspirin groups. The median follow-up period was 4.4 years. We stratified the patients into high-risk or low-risk groups, according to the US recommendation: age (older; younger) and coexisting cardiovascular risk factors. The risk factors included smoking, hypertension, dyslipidemia, family history of coronary artery disease, and proteinuria. Most of the patients were classified into the high-risk group, consisting of older patients with risk factors (n=1,804). The incidence of cardiovascular events was higher in this group, but aspirin did not reduce cardiovascular events (hazard ratio [HR], 0.83; 95% confidence interval [CI]: 0.58-1.17). In the low-risk group, consisting of older patients without risk factors and younger patients (n=728), aspirin did not reduce cardiovascular events (HR, 0.55; 95% CI: 0.23-1.21). These results were unchanged after adjusting for potential confounding factors. Conclusions:?Low-dose aspirin is not beneficial in Japanese diabetic patients at high risk.??(Circ J?2013; 77: 3023-3028).
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Vertebral Compression Exacerbates Osteoporotic Pain in an Ovariectomy-Induced Osteoporosis Rat Model.
Spine
PUBLISHED: 09-13-2013
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Study Design. Basic pain study using osteoporotic rodent models.Objective. To examine alterations in distribution of pain-related neuropeptides following compressive force on osteoporotic vertebrae and their chronic pain-related properties.Summary of Background Data. We previously reported significantly increased production of calcitonin gene-related peptide (CGRP), a marker of inflammatory pain, in the dorsal root ganglia (DRG) of vertebrae in osteoporosis-model ovariectomized (OVX) rats. Here, we hypothesized that longitudinal compressive force on vertebrae can affect osteoporotic pain properties, which has not been examined yet.Methods. OVX rats were used as the osteoporosis model. Female Sprague Dawley rats were prepared and Fluoro-Gold (FG) neurotracer was applied to the periosteal surface of the Co5 vertebra. After FG-labeling, the animals were divided into 4 groups: Control, Control + compression, OVX, and OVX + compression. The Control groups were not ovariectomized. In the compression groups, K-wires were stabbed transversely through Co4 and Co6 with Co5 compressed longitudinally by rubber bands bridged between the two. One, 2, 4, and 8 weeks after surgery, bilateral S1 to S3 DRGs were excised for immunofluorescence assays. Expression of CGRP and activating transcription factor 3 (ATF-3), a marker of neuronal injury, were compared among the 4 groups.Results. Sustained upregulation of CGRP in DRG neurons was observed following compression of the Co5 vertebra, and Co5 compression caused significant increase in CGRP production in DRG neurons, while a greater level of ATF-3 upregulation was observed in DRGs in OVX rats following dynamic vertebral compression 8 weeks after surgery, implying potential neuropathic pain.Conclusion: There was sustained upregulation of CGRP and ATF3 in DRGs in osteoporotic model rats compared with controls, and levels were further enhanced by dynamic vertebral compression. These findings imply that dynamic compression stress on vertebrae can exacerbate osteoporotic pain by inducing both inflammatory and neuropathic pain mediators.
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TALEN-induced gene knock out in Drosophila.
Dev. Growth Differ.
PUBLISHED: 09-06-2013
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We report here a case study of TALEN-induced gene knock out of the trachealess gene of Drosophila. Two pairs of TALEN constructs caused targeted mutation in the germ line of 39% and 17% of injected animals, respectively. In the extreme case 100% of the progeny of TALEN-injected fly was mutated, suggesting that highly efficient biallelic germ line mutagenesis was achieved. The mutagenic efficiency of the TALEN pairs paralleled their activity of single strand annealing (SSA) assay in cultured cells. All mutations were deletion of 1 to 20 base pairs. Merit and demerit of TALEN-based gene knockout approach compared to other genome editing technologies is discussed.
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Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity.
Sci Rep
PUBLISHED: 09-05-2013
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Transcription activator-like effector (TALE) nuclease (TALEN) is a site-specific nuclease, which can be freely designed and easily constructed. Numerous methods of constructing TALENs harboring different TALE scaffolds and repeat variants have recently been reported. However, the functionalities of structurally different TALENs have not yet been compared. Here, we report on the functional differences among several types of TALENs targeting the same loci. Using HEK293T cell-based single-strand annealing and Cel-I nuclease assays, we found that TALENs with periodically-patterned repeat variants harboring non-repeat-variable di-residue (non-RVD) variations (Platinum TALENs) showed higher activities than TALENs without non-RVD variations. Furthermore, the efficiencies of gene disruption mediated by Platinum TALENs in frogs and rats were significantly higher than in previous reports. This study therefore demonstrated an efficient system for the construction of these highly active Platinum TALENs (Platinum Gate system), which could establish a new standard in TALEN engineering.
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Production of Sry knockout mouse using TALEN via oocyte injection.
Sci Rep
PUBLISHED: 08-14-2013
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Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome. In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.
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