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The use of low-dimensional inorganic or organic nanomaterials has advantages for DNA and protein recognition due to their sensitivity, accuracy, and physical size matching. In this research, poly(3-methylthiophene) (P3MT) nanowires (NWs) are electrochemically prepared with dopant followed by functionalization with probe DNA (pDNA) sequence through electrostatic interaction. Various lengths of pDNA sequences (10-, 20- and 30-mer) are conjugated to the P3MT NWs respectively followed with hybridization with their complementary target DNA (tDNA) sequences. The nanoscale photoluminescence (PL) properties of the P3MT NWs are studied throughout the whole process at solid state. In addition, the correlation between the PL enhancement and the double helix DNA with various lengths is demonstrated., Competing Interests: The authors declare no conflict of interest.

The purpose of our study was to compare pulmonary artery (PA) enhancement according to venous routes of contrast media (CM) administration in patients who underwent CT pulmonary angiography (CTPA) in the emergency department (ED).This retrospective study reviewed the CTPAs of 24 patients who administered CM via leg veins (group A) and 72 patients via arm veins (group B) with age and gender matching at a ratio of 1:3. Clinical data, aorta attenuation (Aoatten), and PA attenuation (PAatten) were compared between group A and B. Each group was subcategorized into diagnostic and nondiagnostic CTPA subgroups, with a threshold of 250 HU at the PA. Then, clinical data, rates of pulmonary embolism (PE), and right ventricle (RV) strain were compared. In group A, the relationship between the narrowest suprahepatic IVC area (IVCarea) and the attenuation ratio of the RV to the intrahepatic IVC (RV/IVCatten) was evaluated.Aoatten (236.6 HU vs 293.1 HU, P < .001) and PAatten (266.7 HU vs 321.4 HU, P = .026) were significantly lower in group A than in group B. The proportion of nondiagnostic CTPA was significantly higher in group A than in group B (58.3% vs 19.4%, P = .001). In the subgroup analysis in of group A, patients with a nondiagnostic CTPA were significantly younger (55.3 years vs 68.6 years, P = .026) and showed a significantly lower incidence rate of PE (14% vs 70%, P = .01) than patients with a diagnostic CTPA. However, the radiological diagnostic rate of RV strain was comparable between patients with nondiagnostic and diagnostic CTPA. In group A, IVCarea and RV/IVCatten were positively correlated, with a correlation coefficient of 0.430 (P < .036).In conclusion, administration of CM through the leg veins increases the nondiagnostic CTPA rate, reducing the detection rate of PE. When CM is injected via the leg veins, the degree of PA enhancement is related with to the diameter of the suprahepatic IVC; therefore, adjustment of respiratory maneuvers may be needed to promote IVC flow into the right cardiac chamber, and to improve PA enhancement.

Purpose: Beyond the limited scope of non-specific polyclonal regulatory T cell (Treg)-based immunotherapy, which depends largely on serendipity, the present study explored a target Treg subset appropriate for the delivery of a novel epitope spreader Pep19 antigen as part of a sophisticated form of immunotherapy with defined antigen specificity that induces immune tolerance., Methods: Human polyclonal CD4 + CD25 + CD127 lo- Tregs (127-Tregs) and naïve CD4 + CD25 + CD45RA + Tregs (45RA-Tregs) were isolated and were stimulated with target peptide 19 (Pep19)-pulsed dendritic cells in a tolerogenic milieu followed by ex vivo expansion. Low-dose interleukin-2 (IL-2) and rapamycin were added to selectively exclude the outgrowth of contaminating effector T cells (Teffs). The following parameters were investigated in the expanded antigen-specific Tregs: the distinct expression of the immunosuppressive Treg marker Foxp3, epigenetic stability (demethylation in the Treg-specific demethylated region), the suppression of Teffs, expression of the homing receptors CD62L/CCR7, and CD95L-mediated apoptosis. The expanded Tregs were adoptively transferred into an NOD/scid/IL-2Rγ -/- mouse model of collagen-induced arthritis., Results: Epitope-spreader Pep19 targeting by 45RA-Tregs led to an outstanding in vitro suppressive T cell fate characterized by robust ex vivo expansion, the salient expression of Foxp3, high epigenetic stability, enhanced T cell suppression, modest expression of CD62L/CCR7, and higher resistance to CD95L-mediated apoptosis. After adoptive transfer, the distinct fate of these T cells demonstrated a potent in vivo immunotherapeutic capability, as indicated by the complete elimination of footpad swelling, prolonged survival, minimal histopathological changes, and preferential localization of CD4 + CD25 + Tregs at the articular joints in a mechanistic and orchestrated way., Conclusions: We propose human naïve CD4 + CD25 + CD45RA + Tregs and the epitope spreader Pep19 as cellular and molecular targets for a novel antigen-specific Treg-based vaccination against collagen-induced arthritis., Competing Interests: Conflict of Interest: No potential conflict of interest relevant to this article was reported.

Multiple sclerosis (MS) is a T-lymphocyte-mediated autoimmune disease that is characterized by inflammation in the central nervous system (CNS). Although many disease-modifying therapies (DMTs) are presumed effective in patients with MS, studies on the efficacy and safety of DMTs for preventing MS relapse are limited. Therefore, we tested the immunosuppressive anti-inflammatory effects of oral-formulated tacrolimus (FK506) on MS in a mouse model of experimental autoimmune encephalomyelitis (EAE). The mice were randomly divided into 3 experimental groups: an untreated EAE group, a low-dose tacrolimus-treated EAE group, and a high-dose tacrolimus-treated EAE group. After autoimmunization of the EAE mice with myelin oligodendrocyte glycoprotein, symptom severity scores, immunohistochemistry of the myelination of the spinal cord, and western blotting were used to evaluate the EAE mice. After the autoimmunization, the symptom scores of each EAE group significantly differed at times. The group treated with the larger tacrolimus dose had the lowest symptom scores. The tacrolimus-treated EAE groups exhibited less demyelination and inflammation and weak immunoreactivity for all of the immunization biomarkers. Our results revealed that oral-formulated tacrolimus inhibited the autoimmunization in MS pathogenesis by inactivating inflammatory cells., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2017 The Korean Academy of Medical Sciences.)

Objective: Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT)., Methods: Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR., Results: Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1 , Sox2 , and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid., Conclusion: Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

Based on the structural framework of a pyriproxyfen metabolite, nineteen oxime ester derivatives were synthesized via reaction of the carboxylic acids with 4-(2-(2-pyridinyloxy)ethoxy)benzaldehyde oxime. The corresponding structures were comprehensively characterized by ¹H-nuclear magnetic resonance (NMR), 13 C-NMR, and electrospray ionization high-resolution mass spectrometry (ESI-HRMS). All of the compounds were screened for their insecticidal activities against Plutella xylostella and Myzus persicae , and for their ovicidal activities against Helicoverpa armigera eggs. The results obtained show that most of the oxime ester derivatives displayed moderate to high insecticidal activities and ovicidal activities at a concentration of 600 ug/mL. In particular, the ovicidal activity of compounds 5j , 5o , 5p , 5q , and 5s was determined to be 100%. Importantly, some of the compounds presented even higher biological activities than the reference compound pyriproxyfen. For example, compound 5 j displayed an insecticidal activity value of 87.5% against Myzus persicae , whereas the activity value of pyriproxyfen was 68.3% at a concentration of 600 ug/mL. Among the synthesized compounds 5 j and 5 s exhibited broad biological activity spectra., Competing Interests: All authors declare no conflicts of interest.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P). Four AD Tg cell lines were established from Jeju black pig ear fibroblasts (JB-PEFs); the resultant JB-PEFAD cells harbored transgene integration, expressed transgene mRNAs, and had normal karyotypes. Tg line #2-1, which expressed high levels of the transgenes, was used for SCNT; cleavage and blastocyst rates of embryos derived from this line were lower than those of Non-Tg. These embryos yielded three piglets (Jeju National University AD-Tg pigs, JNUPIGs) revealed by microsatellite testing to be genetically identical to JB-PEFAD. Transgenes were expressed in multiple tissues, and at especially high levels in brain, and Aβ-40/42, total Tau, and GFAP levels were high in brains of the Tg animals. Five or more copies of transgenes were inserted into chromosome X. This is the first report of an AD Tg pig derived from a multi-cistronic vector.

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

Purpose: Epitope spreading is a phenomenon in which distinct subdominant epitopes become major targets of the immune response. Heat shock protein (HSP) 60 from Porphyromonas gingivalis (PgHSP60) and peptide 19 from PgHSP60 (Pep19) are immunodominant epitopes in autoimmune disease patients, including those with periodontitis. It remains unclear whether Pep19 is a dominant epitope in subjects without periodontitis or autoimmune disease. The purpose of this study was to determine the epitope spreading pattern and verify Pep19 as an immunodominant epitope in healthy teenagers using dot immunoblot analysis. The patterns of epitope spreading in age-matched patients with type 1 diabetes mellitus (type 1 DM) and healthy 20- to 29-year old subjects were compared with those of healthy teenagers., Methods: Peptide from PgHSP60, Mycobacterium tuberculosis HSP60 (MtHSP60), and Chlamydia pneumoniae HSP60 (CpHSP60) was synthesized for comparative recognition by sera from healthy subjects and patients with autoimmune disease (type 1 DM). Dot immunoblot analysis against a panel of peptides of PgHSP60 and human HSP60 (HuHSP60) was performed to identify epitope spreading, and a densitometric image analysis was conducted., Results: Of the peptide from PgHSP60, MtHSP60, and CpHSP60, PgHSP60 was the predominant epitope and was most consistently recognized by the serum samples of healthy teenagers. Most sera from healthy subjects and patients with type 1 DM reacted more strongly with PgHSP60 and Pep19 than the other peptides. The relative intensity of antibody reactivity to Pep19 was higher in the type 1 DM group than in the healthy groups., Conclusions: Pep19 is an immunodominant epitope, not only in autoimmune disease patients, but also in healthy young subjects, as evidenced by their robust immunoreactivity. This result suggests that the Pep19-specific immune response may be an initiator that triggers autoimmune diseases., Competing Interests: Conflict of Interest: No potential conflict of interest relevant to this article was reported.

Aberrant nucleocytoplasmic localization of proteins has been implicated in many neurodegenerative diseases. Evidence suggests that cytoplasmic mislocalization of nuclear proteins such as transactive response DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS) may be associated with neurotoxicity in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. This study investigated the changes in nucleocytoplasmic distributions of the proteome and transcriptome in an in vitro model of ALS. After subcellular fractionation of motor neuron-like cell lines expressing wild-type or G93A mutant hSOD1, quantitative mass spectrometry and next-generation RNA sequencing (RNA-seq) were performed for the nuclear and cytoplasmic compartments. A subset of the results was validated via immunoblotting. A total of 1,925 proteins were identified in either the nuclear or cytoplasmic fractions, and 32% of these proteins were quantified in both fractions. The nucleocytoplasmic distribution of 37 proteins was significantly changed in mutant cells with nuclear and cytoplasmic shifts in 13 and 24 proteins, respectively (p<0.05). The proteins shifted towards the nucleus were enriched regarding pathways of RNA transport and processing (Dhx9, Fmr1, Srsf3, Srsf6, Tra2b), whereas protein folding (Cct5, Cct7, Cct8), aminoacyl-tRNA biosynthesis (Farsb, Nars, Txnrd1), synaptic vesicle cycle (Cltc, Nsf), Wnt signalling (Cltc, Plcb3, Plec, Psmd3, Ruvbl1) and Hippo signalling (Camk2d, Plcb3, Ruvbl1) pathways were over-represented in the proteins shifted to the cytoplasm. A weak correlation between the changes in protein and mRNA levels was found only in the nucleus, where mRNA was relatively abundant in mutant cells. This study provides a comprehensive dataset of the nucleocytoplasmic distribution of the proteome and transcriptome in an in vitro model of ALS. An integrated analysis of the nucleocytoplasmic distribution of the proteome and transcriptome demonstrated multiple candidate pathways including RNA processing/transport and protein synthesis and folding that may be relevant to the pathomechanism of ALS.

Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation. Canine serum progesterone concentrations during both proestrus and estrus were analyzed by RIA and ECLI to determine the ovulation day. Although both methods detected similar progesterone levels before ovulation, the mean progesterone concentration determined using ECLI was significantly higher than of RIA three days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of progesterone were used for determination of ovulation. A high percentage of mature oocytes was observed using ECLI when 6-15 ng/mL of progesterone was used for ovulation determination. To determine whether ECLI could be used for canine cloning, six canines were selected as oocyte donors, and two puppies were obtained after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.

Pilot-scale performances for ultrafiltration of Dongjiang river water without and with micro-coagulation and coagulation-adsorption-sedimentation pretreatments were conducted to investigate the effects of pretreatments on hydraulic irreversible fouling of ultrafiltration short processes. Scanning electron microscope (SEM) imaging, high performance size exclusion chromatography (HPSEC) and fluorescence excitation-emission matrix (EEM) were employed to identify foulants responsible for irreversible fouling during ultrafiltration with and without pretreatments. The results showed that the hydraulic irreversible fouling rate was 0.16 kPa·d -1 with micro-coagulation pretreatment at 10 L·(m 2 ·h) -1 , while the rate without pretreatment was 0.32 kPa·d -1 at the same flux. Meanwhile, the fouling rate with coagulation-adsorption-sedimentation pretreatment was almost zero at the same flux. Furthermore, when the flux increased to 17 L·(m 2 ·h) -1 , the irreversible fouling was not observable with coagulation-adsorption-sedimentation pretreatment (backwashing every 24 hours). Properties of foulants from external membrane and internal membrane after backwashing showed that the protein-like and fulvic-like substances were responsible for irreversible fouling in Dongjiang river water. Furthermore, structural alteration of cake layer resulted in less cake resistance and higher backwash efficiency after micro-coagulation pretreatment, leading to less irreversible fouling. Coagulation-adsorption-sedimentation pretreatment decreased irreversible fouling with higher removal efficiency of protein-like and fulvic substances, which were the foulants responsible for irreversible fouling during ultrafiltration. Both micro-coagulation and coagulation-adsorption-sedimentation pretreatments decreased irreversible fouling during ultrafiltration short process.

Unlike mouse results, cloning efficiency of nuclear transfer from porcine induced pluripotent stem cells (piPSCs) is very low. The present study was performed to investigate the effect of cell cycle inhibitors on the cell cycle synchronization of piPSCs. piPSCs were generated using combination of six human transcriptional factors under stem cell culture condition. To examine the efficiency of cell cycle synchronization, piPSCs were cultured on a matrigel coated plate with stem cell media and they were treated with staurosporine (STA, 20 nM), daidzein (DAI, 100 μM), roscovitine (ROSC, 10 μM), or olomoucine (OLO, 200 μM) for 12 h. Flow Cytometry (FACs) data showed that piPSCs in control were in G1 (37.5±0.2%), S (34.0±0.6%) and G2/M (28.5±0.4%). The proportion of cells at G1 in DAI group was significantly higher than that in control, while STA, ROSC and OLO treatments could not block the cell cycle of piPSCs. Both of viability and apoptosis were affected by STA and ROSC treatment, but there were no significantly differences between control and DAI groups. Real-Time qPCR and FACs results revealed that DAI treatment did not affect the expression of pluripotent gene, Oct4. In case of OLO, it did not affect both of viability and apoptosis, but Oct4 expression was significantly decreased. Our results suggest that DAI could be used for synchronizing piPSCs at G1 stage and has any deleterious effect on survival and pluripotency sustaining of piPSCs., Competing Interests: The authors declare that there is no financial conflict of interests to publish these results.

Indoleamine 2,3-dioxygenase (IDO) mediates immune tolerance, and suppressor of cytokine signaling 3 (SOCS3) negatively regulates the JAK/STAT signal transduction pathway. We determined previously that platelet-activating factor (PAF) protects mice against LPS-induced endotoxic shock, but its detailed mechanism of action was unknown. We performed survival experiments in IDO +/+ and IDO -/- mice using an LPS-induced endotoxemia model and rated organ injury (neutrophil infiltration and liver function). Using ELISA and Western blotting, we also investigated the mechanism of PAF-mediated endotoxin tolerance during endotoxemia. PAF-mediated endotoxin tolerance was dependent on IDO in vivo and in vitro and was not observed in IDO -/- mice. JAK/STAT signaling, crucial for SOCS3 expression, was also impaired in the absence of IDO. In an IDO- and STAT-dependent manner, PAF mediated a decrease in IL-12 and a dramatic increase in IL-10 and reduced mouse mortality. In addition, PAF attenuated LPS-mediated neutrophil infiltration into the lungs and interactions between neutrophil-like (THP-1) and endothelial cells (human umbilical vein endothelial cells). These results indicate that PAF-mediated endotoxin tolerance is initiated via IDO- and JAK/STAT-dependent expression of SOCS3. Our study has revealed a novel tolerogenic mechanism of IDO action and an important association between IDO and SOCS3 with respect to endotoxin tolerance., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cytoplasmic male sterility (CMS) in pepper is a better way to produce hybrid seeds compared to manual production. We used the two sequence characterized amplified region (SCAR) markers (CRF-SCAR and CMS-SCAR 130 ) in CMS pepper, to identify the genotype. We assembled two CMS yellow bud mutants (YBM; YBM12-A and YBM12-B). This mutation in leaf color is controlled by a single dominant nuclear gene. The aim was to create a new hybrid seed production method that reduces the costs and increases F 1 hybrid seed purity. The results suggest that the CRF-SCAR and CMS-SCAR 130 markers can be used together in multiple generations to screen for restorer or maintainer genes. We found the marker linked to the restorer gene (Rf) in the C-line and F 1 hybrids, as well as partially in the F 2 generation, whereas it was not found in the sterile YBM12-A or the maintainer line YBM12-B. In the F 2 population, sterility and fertility segregated at a 3:1 ratio based on the CRF-SCAR marker. A 130 bp fragment was produced in the YBM12-A, F 1 , and F 2 populations, suggesting that these lines contained sterile cytoplasm. A 140 bp fragment present in the YBM12-B and C-line indicated that these lines contained normal cytoplasm. In addition, we identified some morphological characters distinguishing sterile and fertile buds and flowers that may be linked to the sterility gene. If more restorer lines are identified, CMS expressing the YBM trait can be used in hybrid seed production.

Objectives: The aim of our study was to assess the value of diffusion-weighted imaging with reverse phase-encoding polarity (R-DWI) in addition to conventional DWI using forward phase-encoding polarity (F-DWI) in differentiating acute brainstem infarctions from hyperintense artefacts., Methods: Seventy-six patients with 38 hyperintense brainstem artefacts and 38 acute brainstem infarctions that had undergone F-DWI and R-DWI were retrospectively selected based on the clinicoradiological diagnosis. Four radiologists independently rated their confidence in diagnosing acute infarctions and ruling out brainstem artefacts in a blind manner, and then compared the diagnostic performance and confidence between F-DWI alone and F-DWI with R-DWI., Results: The areas under the curve determined for F-DWI with R-DWI in diagnosing infarctions were significantly higher than F-DWI alone for all readers (resident 1, 0.908 vs 0.776; resident 2, 0.908 vs 0.789; neuroradiologist, 0.961 vs 0.868; emergency radiologist, 0.934 vs 0.855, all p < 0.05). All readers were more confident using F-DWI with R-DWI than F-DWI alone (all p < 0.05) for diagnosing acute brainstem infarction, and three readers (readers except the neuroradiologist) were more confident using F-DWI with R-DWI for ruling out brainstem artefacts (p ≤ 0.001)., Conclusion: The addition of R-DWI to F-DWI is a valuable method for differentiating acute brainstem infarctions from hyperintense artefacts., Key Points: • Hyperintense brainstem artefacts can be confused with acute infarctions on DWI. • Additional R-DWI to F-DWI reduces inter-reader variability in diagnosing brainstem infarctions. • Additional R-DWI improves performance and confidence for discriminating infarctions from artefacts.

Background: Although periodontal pathogens show a strong association with development of atherosclerosis, little is known about how a microorganism contributes to disease onset and progression. Oxidation of low-density lipoprotein (LDL) is a major risk factor of atherogenesis. The principal objective of this study is to evaluate the ability of peptide 19 (Pep19) of Porphyromonas gingivalis (Pg) heat shock protein (HSP) as a potent inducer of LDL oxidation, and a secondary objective is to compare this ability with that of Pep19 from different bacteria., Methods: HSP60, Pep14, and Pep19 from Pg and THP-1 monocytes were cultured, and the extent of LDL oxidation induced by each peptide was evaluated by an assay for thiobarbituric acid-reactive substances (TBARS). Pep19 and HSP60 from Chlamydia pneumoniae and Mycobacterium tuberculosis were also cultured with THP-1 monocytes and evaluated by the TBARS assay. After incubation of macrophages with LDL and peptides from Pg, Oil Red O staining was performed for examination of foam cells, macrophages that took up the oxidized LDL., Results: Monocyte-mediated native-LDL oxidation under the influence of Pep19 or HSP60 from Pg was significantly stronger than oxidation induced by the counterpart Pep19 or HSP60 from C. pneumoniae or M. tuberculosis. Pep19 from Pg HSP60 showed a stronger ability to induce LDL oxidation than did Pep14 from Pg HSP60., Conclusion: These results suggest Pep19 from Pg HSP60 has a distinct ability to induce native-LDL oxidation as a plausible mechanism by which this peptide may drive epitope spreading to the neoantigen, i.e., oxidized LDL, in the pathogenesis of atherosclerosis.

Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT -(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+ ] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues., Competing Interests: The authors declare no conflict of interest.

Glycogen synthase kinase-3 β (GSK-3 β ) inhibitors have been suggested as a core regulator of apoptosis and have been investigated as therapeutic agents for neurodegenerative diseases, including amyotrophic lateral sclerosis. However, GSK-3 β has an interesting paradoxical effect of being proapoptotic during mitochondrial-mediated intrinsic apoptosis but antiapoptotic during death receptor-mediated extrinsic apoptosis. We assessed the effect of low to high doses of a GSK-3 β inhibitor on survival and apoptosis of the NSC-34 motor neuron-like cell line after serum withdrawal. Then, we identified changes in extrinsic apoptosis markers, including Fas, Fas ligand, cleaved caspase-8, p38 α , and the Fas-Daxx interaction. The GSK-3 β inhibitor had an antiapoptotic effect at the low dose but was proapoptotic at the high dose. Proapoptotic effect at the high dose can be explained by increased signals in cleaved caspase-8 and the motor neuron-specific p38 α and Fas-Daxx interaction. Our results suggest that GSK-3 β inhibitor dose may determine the summation effect of the intrinsic and extrinsic apoptosis pathways. The extrinsic apoptosis pathway might be another therapeutic target for developing a potential GSK-3 β inhibitor.

In computed tomographic colonography (CTC), a patient is commonly scanned twice including supine and prone scans to improve the sensitivity of polyp detection. Typically, a radiologist must manually match the corresponding areas in the supine and prone CT scans, which is a difficult and time-consuming task, even for experienced scan readers. In this paper, we propose a method of supine-prone registration utilizing band-height images, which are directly constructed from the CT scans using a ray-casting algorithm containing neighboring shape information. In our method, we first identify anatomical feature points and establish initial correspondences using local extreme points on centerlines. We then correct correspondences using band-height images that contain neighboring shape information. We use geometrical and image-based information to match positions between the supine and prone centerlines. Finally, our algorithm searches the correspondence of user input points using the matched anatomical feature point pairs as key points and band-height images. The proposed method achieved accurate matching and relatively faster processing time than other previously proposed methods. The mean error of the matching between the supine and prone points for uniformly sampled positions was 18.41±22.07mm in 20 CTC datasets. The average pre-processing time was 62.9±8.6s, and the interactive matching was performed in nearly real-time. Our supine-prone registration method is expected to be helpful for the accurate and fast diagnosis of polyps., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background/aims: Radiofrequency ablation (RFA) is one of the most frequently applied curative treatments in patients with a single small hepatocellular carcinoma (HCC). However, the clinical significance of and risk factors for early massive recurrence after RFA-a dreadful event limiting further curative treatment-have not been fully evaluated., Methods: In total, 438 patients with a single HCC of size ≤3 cm who underwent percutaneous RFA as an initial treatment between 2006 and 2009 were included. Baseline patient characteristics, overall survival, predictive factors, and recurrence after RFA were evaluated. In addition, the incidence, impact on survival, and predictive factors of early massive recurrence, and initial recurrence beyond the Milan criteria within 2 years were also investigated., Results: During the median follow-up of 68.4 months, recurrent HCC was confirmed in 302 (68.9%) patients, with early massive recurrence in 27 patients (6.2%). The 1-, 3-, and 5-year overall survival rates were 95.4%, 84.7%, and 81.8%, respectively, in patients with no recurrence, 99.6%, 86.4%, and 70.1% in patients with recurrence within the Milan criteria or late recurrence, and 92.6%, 46.5%, and 0.05% in patients with early massive recurrence. Multivariable analysis identified older age, Child-Pugh score B or C, and early massive recurrence as predictive of poor overall survival. A tumor size of ≥2 cm and tumor location adjacent to the colon were independent risk factors predictive of early massive recurrence., Conclusion: Early massive recurrence is independently predictive of poor overall survival after RFA in patients with a single small HCC. Tumors sized ≥2 cm and located adjacent to the colon appear to be independent risk factors for early massive recurrence., Competing Interests: The authors have no conflicts to disclose.

Amyotrophic lateral sclerosis (ALS) is a degenerative disorder that involves the death of motor neurons in the cortex, brain stem, and spinal cord. Adipose-derived stem cells (ADSCs) are considered as a perspective remedy for therapy of neurodegenerative diseases including ALS. Stem cells secrete various factors which can modulate a hostile environment, called paracrine effect. Exosomes are small extracellular vesicles containing cell derived factors and mediate paracrine effect of cells. Thus, exosomes from ADSCs (ADSC-exo) can be a potential candidate of therapeutic effects of stem cells. To investigate the effect of ADSC-exo on the cellular phenotypes of ALS, we used neuronal stem cells (NSCs), which can be differentiated into neuronal cells, isolated from wild type or G93A ALS mice model. ADSC-exo was treated to neuronal cells from G93A ALS mice model. Immunocytochemistry and dot-blot assay result showed that ADSC-exo alleviated aggregation of superoxide dismutase 1 (SOD1). Reduction of cytosolic SOD1 level by ADSC-exo was also confirmed by western blot. Mitochondria display various abnormalities in ALS and the decrease of phospho-CREB and PGC-1α were observed in the G93A cells. ADSC-exo treatment showed normalization of phospho-CREB/CREB ratio and PGC-1α expression level. Our results suggest that ADSC-exo modulates cellular phenotypes of ALS including SOD-1 aggregation and mitochondrial dysfunction, and can be a therapeutic candidate for ALS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Effective immunosuppression strategies and genetically modified animals have been used to prevent hyperacute and acute xenograft rejection; however, the underlying mechanisms remain unknown. In this study, we evaluated the expression of a comprehensive set of immune system-related genes (89 genes, including five housekeeping genes) in the blood of cynomolgus monkeys (~5 yr old) used as graft recipients, before and after the xenografting of the islets and heart from single and double α-1,3-galactosyltransferase (GalT) knockout (KO) pigs (<6 weeks old). The immunosuppressive regimen included administration of cobra venom factor, anti-thymocyte globulin, rituximab, and anti-CD154 monoclonal antibodies to recipients before and after grafting. Islets were xenografted into the portal vein in type 1 diabetic monkeys, and the heart was xenografted by heterotopic abdominal heart transplantation. Genes from recipient blood were analyzed using RT(2) profiler PCR arrays and the web-based RT(2) profiler PCR array software v.3.5. Recipients treated with immunosuppressive agents without grafting showed significant downregulation of CCL5, CCR4, CCR6, CD4, CD40LG, CXCR3, FASLG, CXCR3, FOXP3, GATA3, IGNG, L10, IL23A, TRAF6, MAPK8, MIF, STAT4, TBX21, TLR3, TLR7, and TYK2 and upregulation of IFNGR1; thus, genes involved in protection against viral and bacterial infection were downregulated, confirming the risk of infection. Notably, C3-level control resulted in xenograft failure within 2 days because of a 7- to 11-fold increase in all xenotransplanted models. Islet grafting using single GalT-KO pigs resulted in upregulation of CXCL10 and MX1, early inflammation, and acute rejection-associated signals at 2 days after xenografting. We observed at least 5-fold upregulation in recipients transplanted with islets grafts from single (MX1) or double (C3, CCR8, IL6, IL13, IRF6, CXCL10, and MX1) GalT-KO pigs after 77 days; single GalT-KO incurred early losses owing to immune attacks. Our results suggest that this novel, simple, non-invasive, and time-efficient procedure (requiring only 1.5 ml blood) for evaluating graft success, minimizing immune rejection, and blocking infection., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: To evaluate thread sign of biliary intraductal papillary mucinous neoplasm (B-IPMN) on magnetic resonance imaging (MRI)., Methods: Thread sign was defined as intraductal linear or curvilinear hypointense striations. Two radiologists independently evaluated the presence and location of thread sign on MR cholangiography (thin-slice, thick-slab and 3D MRC) and axial MR images (T2 TSE, T2 HASTE and DWI) in patients with B-IPMN (n = 38) and in matched control groups with benign (n = 36) or malignant (n = 35) biliary diseases. Sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of thread sign for diagnosing B-IPMN were evaluated., Results: Thread sign was observed only in patients with B-IPMN on MRC (44.7-52.6 % [17/38-20/38], P < 0.001) and axial MR images (31.6 % [12/38], P < 0.001), except in one patient with recurrent pyogenic cholangitis on MRC (2.8 %, 1/36). The sensitivity, specificity, accuracy, PPV and NPV of thread sign for diagnosing B-IPMN on MRC were 0.53, 0.99, 0.83, 0.95 and 0.80, respectively (reader 1) and 0.45, 1.0, 0.81, 1.0 and 0.77, respectively (reader 2). Thread sign was detected mainly at the extrahepatic bile duct (52.6 %, 20/38)., Conclusion: B-IPMN can manifest thread sign, a novel specific MR finding, mainly at the extrahepatic bile duct on MRI, especially on MRC., Key Points: • Some B-IPMNs manifest thread sign within the bile ducts on MRI. • Thread sign is a highly specific finding for B-IPMN on MRI. • MRC is superior to axial T2WI and DWI for detecting thread sign.

Objective: To investigate the significance of long non-coding RNA MALAT1 expression in osteosarcoma, and the potential mechanism by which MALAT1 promotes tumor metastasis., Methods: Twenty cases of osteosarcoma in the First Affiliated Hospital of Xinxiang Medical University and Ping Ding Shan First People's Hospital were collected from January 2014 to December 2015. The expression of MALAT1 in osteosarcoma tissue and paired adjacent noncancerous tissue were analyzed by qRT-PCR. Correlation of MALAT1 expression in osteosarcoma with clinical pathologic features was performed by the Mann-Whitney U test. U-2OS cells were transfected with lenti-virus carrying MALAT1-shRNA and nonspecific shRNA (LV-vector). The expression of MALAT1 was detected by qRT-PCR. The cell activity was evaluated by MTT asssy. The impact of MALAT1-shRNA on invasion in U-2OS cells were determined by transwell migration assay. The expression of Wnt/β-catenin signal pathway related proteins were detected by Immunofluorescence stain and Western blot., Results: The expression level of MALAT1 in osteosarcoma tissue was higher than that in paired adjacent noncancerous tissue and correlated significantly with nodal and pulmonary metastasis(P<0.01). MTT assay showed that knockdown of MALAT1 with lenti virus-MALAT1 shRNA inhibited the growth of U-2OS cells, along with marked decrease of invasive ability of U-2OS cells in the transwell migration assay. By immunofluorescence stain and Western blot assay, MALAT1 significantly reduced the expression of β-catenin, MMP7, and c-MYC in U-2OS cells., Conclusions: The expression of MALAT1 is high in osteosarcoma and correlates with tumor metastasis. MALAT1 promotes invasion and metastasis of osteosarcoma cells likely thought the Wnt/β-catenin signal pathway.

Objective: To investigate appendiceal location and misdiagnosis rate of appendicitis associated with deep pelvic cecum on multidetector computed tomography (MDCT)., Materials and Methods: Among 1107 appendicitis cases, 25 patients with deep pelvic cecum and 75 patients with conventional cecum on MDCT were retrospectively selected for analysis of appendiceal locations and preoperative misdiagnosis rate., Results: The major appendiceal direction in deep pelvic cecum group was ascending (84.0%). The misdiagnosis rates of appendicitis in deep pelvic and conventional cecum groups were 16% and 5.3%., Conclusion: A deep pelvic cecum may cause misdiagnosis of appendicitis. The appendix associated with deep pelvic cecum mainly demonstrates right ascending direction., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disease. The precise pathogenic mechanisms of the disease remain uncertain, and as of yet, there is no effective cure. Human adipose stem cells (hASC) can be easily obtained during operative procedures. hASC have a clinically feasible potential to treat neurodegenerative disorders, since cytosolic extract of hASC contain a number of essential neurotrophic factors. In this study, we investigated effects of hASC extract on the SOD1 G93A mouse model of ALS and in vitro test. Administration of hASC extract improved motor function and prolonged the time until symptom onset, rotarod failure, and death in ALS mice. In the hASC extracts group, choline acetyltransferase immunostaining in the ventral horn of the lumbar spinal cord showed a large number of motor neurons, suggesting normal morphology. The neuroprotective effect of hASC extract in ALS mice was also suggested by western blot analysis of spinal cord extract from ALS mice and in vitro test. hASC extract treatment significantly increased expression of p-Akt, p-CREB, and PGC-1α in SOD1 G93A mouse model and in vitro test. Our results indicated that hASC extract reduced apoptotic cell death and recovered mutant SOD1-induced mitochondrial dysfunction. Moreover, hASC extract reduced mitochondrial membrane potential. In conclusion, we have demonstrated, for the first time, that hASC extract exert a potential therapeutic action in the SOD1 G93A mouse model of ALS and in vitro test. These findings suggest that hASC hold promise as a novel therapeutic strategy for treating ALS.

Objectives: To determine the diagnostic superiority of parametric response mapping of apparent diffusion coefficient (ADCPR) for predicting glioblastoma treatment response, compared to single time point measurement., Methods: Fifty post-treatment glioblastoma patients were enrolled. ADCPR was calculated from serial apparent diffusion coefficient (ADC) maps acquired before and at the time of first detection of an enlarged contrast-enhancing lesion on voxel-by-voxel basis. The percentage-decrease in ADCPR and tenth percentile histogram cutoff value of ADC (ADC10) were compared at subsequent 3-month and 1-year follow-ups., Results: The percentage-decrease in ADCPR was significantly higher in the progression group (mean = 33.2-38.3 %) than in the stable-response group (mean = 9.7 %) at 3 months follow-up (corrected p < 0.001 for both readers). ADCPR significantly improved area under the receiver operating characteristic curve from 0.67 to 0.88 (corrected p = 0.037) and from 0.70 to 0.92 (corrected p = 0.020) for both readers, respectively, compared to ADC10 at 3-month follow-up, but did not significantly improve at 1-year follow-up. The inter-reader agreement was higher for ADCPR than ADC10 (intraclass correlation coefficient, 0.93 versus 0.86)., Conclusion: Voxel-based ADCPR appears to be a superior imaging biomarker than ADC, particularly for predicting early tumour progression in patients with glioblastoma., Key Points: • Treatment response pattern of glioblastoma was evaluated using voxel-based ADCPR and ADC10. • Voxel-based ADCPR was more accurate in predicting treatment response pattern than ADC10. • Inter-reader agreement was higher in ADCPR calculation than in ADC10 calculation. • Voxel-based ADCPR can be a predictor of early treatment response pattern for glioblastoma.

The National Aeronautics and Space Administration Animal Enclosure Module (AEM) was developed as a self-contained rodent habitat for shuttle flight missions that provides inhabitants with living space, food, water, ventilation, and lighting, and this study reports whether, after minimal hardware modification, the AEM could support an extended term up to 35 days for Sprague-Dawley rats and C57BL/6 female mice for use on the International Space Station. Success was evaluated based on comparison of AEM housed animals to that of vivarium housed and to normal biological ranges through various measures of animal health and well-being, including animal health evaluations, animal growth and body masses, organ masses, rodent food bar consumption, water consumption, and analysis of blood contents. The results of this study confirmed that the AEMs could support 12 adult female C57BL/6 mice for up to 35 days with self-contained RFB and water, and the AEMs could also support 5 adult male Sprague-Dawley rats for 35 days with external replenishment of diet and water. This study has demonstrated the capability and flexibility of the AEM to operate for up to 35 days with minor hardware modification. Therefore, with modifications, it is possible to utilize this hardware on the International Space Station or other operational platforms to extend the space life science research use of mice and rats., Competing Interests: The authors declare no conflict of interest.

In the insect immune system, nodules are known to be a product of the cellular response against microorganisms and may be a preferential target for melanization. However, the mechanism of nodule-preferential melanization remains to be explored. In this study, we identified several mechanisms of nodule-preferential melanization by analyzing congregation and the activation of several factors involved in the prophenoloxidase (proPO)-activating system in the silkworm, Bombyx mori. Microorganism-binding assays revealed that B. mori larval plasma have an effective invading microorganism-surveillance network consisting of at least six pattern-recognition receptors (PRRs). We also found that a hemolymph serine proteinase, BmHP14, can bind to Saccharomyces cerevisiae. Pull-down assays showed that PRR C-type lectins form protein complexes with serine proteinase homologs, BmSPH1 and BmSPH2, which leads to the activated forms of BmSPH1 and BmSPH2 being gathered on microorganisms and trapped in nodules. Immunostaining analysis revealed that most factors in the proPO-activating system and some factors in the triggering system for antimicrobial peptide production exist in the granules of hemocytes which can gather in nodules. Western blot analysis showed that factors in the proPO-activating system are congregated in formed nodules by their concentration in plasma and aggregating hemocytes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The anti-obesity effect of Korean traditional food, Memilmuk, was examined through inhibition of differentiation of 3T3-L1 preadipocytes by buckwheat flour extract. Oil-Red O staining showed that lipid accumulation in adipocytes was reduced upon adding buckwheat flour extract, indicating effective inhibition of adipocyte differentiation. Buckwheat flour extract also inhibited the expression of adipogenic transcription factor, peroxisome proliferator-activated receptor γ (PPARγ), and AMP-activated protein kinase (AMPK), an intracellular regulator of energy balance. Overall, the anti-obesity effect of Korean Memilmuk might be mediated through down-regulation of PPARγ expression via AMPK activation by buckwheat flour.

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine protein kinase that is known to mediate cancer cell death. Here, we show that B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, is regulated by GSK-3β and that GSK-3β-mediated regulation of Bcl-2 is crucial for mitochondrial-dependent cell death in paclitaxel-stimulated cells. We demonstrate that MCF7 GSK-3β siRNA cells are more sensitive to cell death than MCF7 GFP control cells and that in the absence of GSK-3β, Bcl-2 levels are reduced, a result enhanced by paclitaxel. Paclitaxel-induced JNK (c-Jun N-terminal kinase) activation is critical for Bcl-2 modulation. In the absence of GSK-3β, Bcl-2 was unstable in an ubiquitination-dependent manner in both basal- and paclitaxeltreated cells. Furthermore, we demonstrate that GSK-3β-mediated regulation of Bcl-2 influences cytochrome C release and mitochondrial membrane potential. Taken together, our data suggest that GSK-3β-dependent regulation of Bcl-2 is crucial for mitochondria-dependent cell death in paclitaxel-mediated breast cancer therapy.

Background: The primary problems with porcine oocyte vitrification are their low viability and development; both need improvement., Objective: This study was designed to improve the survival and developmental rates in vitrified-warmed porcine oocytes., Materials and Methods: Porcine oocytes matured in vitro were vitrified-warmed with Cryotop. Then the oocytes were supplemented with Q10 during recovery culture., Results: The survival rates immediately after warming were 92.9% by morphological inspection and 39.3% by fluorescein diacetate (FDA) assay. The group of recovery culture with Q10 (VC+Q10) showed significantly higher viability compared to the group of recovery culture without Q10 (VC+) analyzed by morphology and the FDA. The VC+Q10 group showed a low Bax/Bcl-xl ratio and a high expression of MAP3K12 and TGFB3 compared to the VC+. The cleavage rate did not differ in both groups but, blastocyst yield was higher in VC+Q10 than the VC+ group., Conclusion: Supplementation of Q10 during recovery culture led to a higher blastocyst yield by increasing survival rates and regulating mRNA expressions.

Background: Vitrification with the Cryotop device is the most promising technique for oocyte cryopreservation, but the high post-warming morphological survival of bovine oocytes does not guarantee high developmental competence after in vitro fertilization (IVF)., Objective: This study was designed to examine achievement of normal fertilization in bovine oocytes vitrified-warmed with the Cryotop device., Materials and Methods: Oocytes were matured in vitro and vitrified-warmed after complete removal of the cumulus layers. Distribution of cortical granules (CGs) was assessed by Lens culinaris agglutinin (LCA) lectin staining. Ten hours after IVF, presumptive zygotes were analyzed for pronuclear formation. Day-8 blastocysts were harvested and stained with Hoechst-33342 for total cell counting., Results: Both yield and mean cell number of the blastocysts were impaired by Cryotop vitrification. Incidence of polyspermic fertilization was three-times higher in vitrified oocytes compared to fresh oocytes. No difference in CG distribution was found between vitrified and fresh oocytes., Conclusion: Polyspermic fertilization induced in vitrified-warmed bovine oocytes may be one of the possible causes responsible for their low developmental potential.

We describe here for the first time the low temperature superplasticity of nanostructured low carbon steel (microalloyed with V, N, Mn, Al, Si, and Ni). Low carbon nanograined/ultrafine-grained (NG/UFG) bulk steel was processed using a combination of cold-rolling and annealing of martensite. The complex microstructure of NG/UFG ferrite and 50-80 nm cementite exhibited high thermal stability at 500 °C with low temperature elongation exceeding 100% (at less than 0.5 of the absolute melting point) as compared to the conventional fine-grained (FG) counterpart. The low temperature superplasticity is adequate to form complex components. Moreover, the low strength during hot processing is favorable for decreasing the spring back and minimize die loss.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by selective degeneration of motor neurons. Mutant superoxide dismutase 1 (SOD1) is often found as aggregates in the cytoplasm in motor neurons of various mouse models and familial ALS patients. The interplay between motor neurons and astrocytes is crucial for disease outcome, but the mechanisms underlying this phenomenon remain unknown. In this study, we investigated whether transient transfection with wild-type and mutant-type SOD1 may lead to amplification of mutant SOD1-mediated toxicity in cortical neurons and astrocytes derived from wild-type and mutant-type (human G93A-SOD1) mice. In transgenic mice expressing either wild- or mutant-type SOD1, we found that green fluorescent protein (GFP)-wtSOD1 was present in the cytoplasm and nuclei of wild-type cortical neurons and astrocytes, whereas GFP-mutant SOD1 was mainly cytoplasmic in wild- and mutant-type cortical neurons and astrocytes. These findings indicate that intracellular propagation of misfolding of GFP-wt or mtSOD1 are possible mediators of toxic processes involved in initiating mislocalization and aggregation. Here, we provide evidence that cytoplasmic aggregates induce apoptosis in G93A-SOD1 mouse cortical neurons and astrocytes and that the toxicity of mutant SOD1 in astrocytes is similar to the pathological effects of ALS on neurons in vitro. Collectively, our results indicate that mtSOD1 probably interacts with wtSOD1 via an unknown mechanism to produce augmented toxicity and may influence aggregate formation and apoptosis.

Lichen sclerosus (LS) is a rare, chronic, inflammatory disease of the skin that primarily affects postmenopausal women but may occur in men and children as well. Approximately 7% to 15% of cases are believed to occur in children. The epidemiologic data for LS have been limited and treatment options are not well studied, particularly in children. We reviewed new developments available in the current literature on the epidemiology and management of LS for children., (© 2015 Wiley Periodicals, Inc.)

Two DNA/RNA binding proteins, TDP-43 and FUS/TLSU, are involved in RNA processing, and their aberrant mutations induce inherited amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions. Wild type TDP-43 and FUS (wtTDP-43 and wtFUS) are mainly localized in the nucleus and biochemically interact with the microRNA processing enzyme Drosha. In this study, we investigated Drosha stability in Neuro 2A cells by gain and loss of function studies of wtTDP-43 and wtFUS and cycloheximide mediated protein degradation assay. We also generated three different phosphomimetic mutants of TDP-43 (S379E, S403/404E and S409/410E) by using a site-directed mutagenesis method and examined Drosha stability to elucidate a correlation between the phosphorylated TDP-43 mutants and Drosha stability. Overexpression of wtTDP-43 and/or wtFUS increased Drosha stability in Neuro 2A cells and double knockdown of wtTDP-43 and wtFUS reduced its stability. However, knockdown of wtTDP-43 or wtFUS did not affect Drosha stability in Neuro 2A cells. Interestingly, a phosphomimetic mutant TDP-43 (S409/410E) significantly reduced Drosha stability via prevention of protein-protein interactions between wtFUS and Drosha, and induced cytotoxicity in Neuro 2A cells. Our findings suggest that TDP-43 and FUS controls Drosha stability in Neuro 2A cells and that a phosphomimetic mutant TDP-43 (S409/410E) which is associated with Drosha instability can induce neuronal toxicity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background/objectives: Athletes generally desire changes in body composition in order to enhance their athletic performance. Often, athletes will practice chronic energy restrictions to attain body composition changes, altering their energy needs. Prediction of resting metabolic rates (RMR) is important in helping to determine an athlete's energy expenditure. This study compared measured RMR of athletic and non-athletic adolescents with predicted RMR from commonly used prediction equations to identify the most accurate equation applicable for adolescent athletes., Subjects/methods: A total of 50 athletes (mean age of 16.6 ± 1.0 years, 30 males and 20 females) and 50 non-athletes (mean age of 16.5 ± 0.5 years, 30 males and 20 females) were enrolled in the study. The RMR of subjects was measured using indirect calorimetry. The accuracy of 11 RMR prediction equations was evaluated for bias, Pearson's correlation coefficient, and Bland-Altman analysis., Results: Until more accurate prediction equations are developed, our findings recommend using the formulas by Cunningham (-29.8 kcal/day, limits of agreement -318.7 and +259.1 kcal/day) and Park (-0.842 kcal/day, limits of agreement -198.9 and +196.9 kcal/day) for prediction of RMR when studying male adolescent athletes. Among the new prediction formulas reviewed, the formula included in the fat-free mass as a variable [RMR = 730.4 + 15 × fat-free mass] is paramount when examining athletes., Conclusions: The RMR prediction equation developed in this study is better in assessing the resting metabolic rate of Korean athletic adolescents.

Objective: We aimed to compare iohexol vs. diatrizoate as fecal/fluid tagging agents for computed tomography colonography (CTC) regarding examination quality., Methods: Forty prospective patients (M:F = 23:17; 63 ± 11.6 years) received CTC using 50 mL (350 mgI/mL) oral iohexol for tagging. Forty other indication-matched, age-matched, and sex-matched patients who underwent CTC using 100 mL diatrizoate for tagging and otherwise the same technique, were retrospectively identified. Two groups were compared regarding overall examination quality, per-patient and per-segment scores of colonic bubbles (0 [no bubbles] to 5 [the largest amount]), and the volume, attenuation, and homogeneity (untagged, layered, and homogeneous) of the residual colonic fluid., Results: The iohexol group demonstrated a greater amount of colonic bubbles than the diatrizoate group: mean per-patient scores ± SD of 1.2 ± 0.8 vs. 0.7 ± 0.6, respectively (p = 0.003); and rates of segments showing ≥ grade 3 bubbles of 12.9 % (85/659) vs. 1.6 % (11/695), respectively (p = 0.001). Residual colonic fluid amount standardized to the colonic volume did not significantly differ: 7.2 % ± 4.2 vs. 7.8 % ± 3.7, respectively (p = 0.544). Tagged fluid attenuation was mostly comparable between groups and the fluid was homogeneously tagged in 98.7 % (224/227) vs. 99.5 % (218/219) segments, respectively (p = 0.344). Iohexol caused more colonic bubbles when used during cathartic CTC. Otherwise, examination quality was similarly adequate with both iohexol and diatrizoate., Key Points: • When used for tagging, iohexol caused significantly more colonic bubbles than diatrizoate. • The residual colonic fluid amount did not significantly differ between iohexol and diatrizoate. • The quality of fluid tagging was similarly adequate in both iohexol and diatrizoate.

The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast cells originated from KNP were cultured and used as donor cell. After thawing, the donor cells were cultured for 1 hour with 15 μM roscovitine prior to the nuclear transfer. The numbers of reconstructed and parthenogenetic embryos transferred were 98 ± 35.2 and 145 ± 11.2, respectively. The pregnancy and delivery rate were 3/5 (60%) and 2/5 (40%). One healthy SLA homotype-defined SCNT KNP was successfully generated. The recipient-based individual cloning efficiency ranged from 0.65 to 1.08%. Taken together, it can be postulated that the methodological establishment of the production of SLA homotype-defined cloned KNP can be applied to the generation of transgenic cloned KNP as model animals for human disease and xenotransplantation researches.

We found that resveratrol enhances interferon (IFN)-γ-induced tryptophanyl-tRNA-synthetase (TTS) expression in bone marrow- derived dendritic cells (BMDCs). Resveratrol-induced TTS expression is associated with glycogen synthase kinase-3β (GSK-3β) activity. In addition, we found that resveratrol regulates naïve CD8+ T-cell polarization by modulating GSK-3β activity in IFN-γ-stimulated BMDCs, and that resveratol induces upregulation of TTS in CD8+ T-cells in the in vivo tumor environment. Taken together, resveratrol upregulates IFN-γ-induced TTS expression in a GSK-3β-dependent manner, and this TTS modulation is crucial for DC-mediated T-cell modulation.

Objective: The purpose of this study was to determine whether the occurrence of numerous colonic bubbles during CT colonography (CTC) performed with polyethylene glycol cleansing and oral iohexol fecal/fluid tagging could be prevented by use of simethicone., Subjects and Methods: Adults with suspected colonic neoplasia who had been randomly assigned to control and simethicone intervention groups underwent CTC after cleansing with 4 L of polyethylene glycol, tagging with 50 mL of 350 mg I/mL oral iohexol, and without (control) or with (intervention) oral administration of 200 mg of simethicone. Colonic segments in the control and intervention groups were evaluated for amount of colonic bubbles during CTC. A 6-point grading system was used in which 0 indicated no bubbles and 5 indicated that more than three fourths of the air-distended mucosa was covered with bubbles. The primary endpoint was a per-patient colonic bubble grade, derived as an average of the segmental grades., Results: Eighty adults with suspected colonic neoplasia were randomly assigned to the control (40 patients) and simethicone intervention (40 patients) groups. A total of 659 colonic segments in the control group and 689 segments in the intervention group were evaluated for amount of colonic bubbles during CTC. The per-patient colonic bubble score was significantly lower in the simethicone intervention group than in the control group. The mean score was 0.0±0.1 (SD) versus 1.2±0.8 (p<0.001; 95% CI for the mean difference, -1.4 to -1.0). In the intervention group, 673 (97.7%) segments were grade 0, and 16 (2.3%) were grade 1. In contrast, in the control group, 226 (34.3%) segments were grade 0; 173 (26.3%), grade 1; 175 (26.6%), grade 2; 45 (6.8%), grade 3; 23 (3.5%), grade 4; and 17 (2.6%), grade 5., Conclusion: The colonic bubbles associated with fecal/fluid tagging with iohexol can be successfully prevented by adding simethicone to the colonic preparation.

Background: Barley straw (Hordeum vulgare L.) is an attractive lignocellulosic material and one of the most abundant renewable resources for fuel ethanol production. Although it has high cellulose and hemicellulose contents, there are several challenges and limitations in the process of converting it to fuel ethanol. High ash, silica and lignin contents in barley straw make it an inferior feedstock for enzymatic hydrolysis. Therefore pretreatment of barley straw could play an important role in inducing structural and compositional changes that increase the efficiency of enzymatic hydrolysis and make the whole process economically viable., Results: Saccharification was enhanced using various concentrations (0.0, 0.5, 1.0, 2.0 and 3.0% v/v) of a solution of sodium hypochlorite (NaClO) and hydrogen peroxide (H₂O₂) and various reaction times (15, 30 and 45 min) during pretreatment at 121 °C. The highest yield of glucose (447 mg g⁻¹) was achieved by pretreatment with 2.0% NaClO+H₂O₂ solution for 30 min, representing an increase of 65.99% compared with untreated barley straw (152 mg g⁻¹). During fermentation, the highest amount of ethanol (207 mg g⁻¹) was obtained under anaerobic plus 0.4 mmol L⁻¹ benzoic acid conditions, representing an increase of 57.49, 38.16 and 10.14% compared with untreated sample (88 mg g⁻¹), aerobic (128 mg g⁻¹) and anaerobic (186 mg g⁻¹) conditions respectively., Conclusion: The results suggest that pretreatment with 2.0% NaClO+H₂O₂ solution disrupted the recalcitrant structure of barley straw and enhanced the glucose yield and subsequent bioethanol production., (© 2014 Society of Chemical Industry.)

Cordycepin is an adenosine analog originally extracted from Cordyceps militaris that possesses many pharmacological effects including immune activation and antioxidant and antitumor effects. However, the underlying relationship between apoptosis and telomerase activity in response to cordycepin exposure has not been investigated. In this study, we found that cordycepin-induced apoptosis of human leukemia cells (H937 and THP-1 cells) was associated with inactivation of telomerase and downregulation of human telomerase reverse transcriptase (hTERT) as well as the transcription factors c-Myc and Sp1, which are required for basal transcription from the hTERT gene promoter. Cordycepin also attenuated the activation of phosphoinositide-3-kinase (PI3K)/Akt signaling, thereby reducing phosphorylation and nuclear translocation of hTERT. We further showed that the PI3K inhibitor LY29004 significantly decreased telomerase activity in cordycepin-treated cells and increased cordycepin-induced cell death. These findings demonstrate that cordycepin is cytotoxic to human leukemia cells and suppresses telomerase activity through transcriptional and post-translational suppression of hTERT by inactivating the PI3K/Akt signaling pathway., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Objective: To identify imaging characteristics that differentiate small (≤2 cm) HCCs from small (≤2 cm) benign nodules in cirrhotic liver on gadoxetic acid-enhanced and diffusion-weighted (DW) magnetic resonance (MR) images., Materials and Methods: On gadoxetic acid-enhanced and DW MR images, we analysed signal intensity of 222 small HCCs and 61 benign nodules (diameter, 0.5-2 cm) at each sequence and rim enhancement during portal or equilibrium phases. Univariate and multivariate logistic regression analyses identified predictors of HCC. Combinations of significant MR findings in multivariate analysis were compared with American Association for the Study of Liver Disease (AASLD) practice guidelines., Results: In multivariate analysis, arterial enhancement (adjusted odds ratio [aOR], 8.6), T2 hyperintensity (aOR, 5.8), and hyperintensity on DW images (aOR, 3.8) were significant for differentiating small HCCs from benign nodules (p ≤ 0.004). When two or all three findings were applied as diagnostic criteria for differentiating small HCCs from benign nodules, sensitivity and accuracy were significantly higher compared with AASLD practice guidelines (91% vs. 78% and 89% vs. 81%, respectively; each p < 0.0001)., Conclusion: On gadoxetic acid-enhanced MR imaging, arterial enhancement and hyperintensity on T2-weighted and DW MR images are helpful for differentiating small HCCs from benign nodules in liver cirrhosis.

Aim: To develop a novel non-sequencing method for the detection of hepatitis B virus (HBV) pre-S deletion mutants in HBV carriers., Methods: The entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction (PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis (CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 μm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of 114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma (HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis., Results: CGE allowed the separation of PCR products differing in size > 3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls (47.1% vs 28.1%, P < 0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC (95%CI: 1.723-5.122, P < 0.001), large deletion (> 99 bp) did not show any association with HCC (P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9% (8/9) had deletions 2-5 years prior to HCC, while only 44.4%4 (4/9) contained such deletions 6-9 years prior to HCC., Conclusion: CGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.

Purpose: The incidence of congenital hypothyroidism (CH) has increased in several countries. Lower cut-off in screening programs have led to an increase in the proportion of transient hypothyroidism (TH) cases diagnosed, leading to debate on the associated clinical and economic impact. This study aimed to identify factors that would allow discrimination between TH and permanent CH (PH) in patients with a eutopic thyroid gland., Methods: Sixty-six patients with CH from 3 different hospitals were studied: 26 cases of TH, and 40 cases of PH. Laboratory findings and clinical parameters were analysed in 56 patients with eutopic thyroid gland., Results: Initial serum thyroid stimulating hormone levels and L-thyroxine dose at 12 and 24 months of age were significantly higher in PH than TH patients with a eutopic thyroid gland. The area under the curve for the 12-month and 24-month dose for the prediction of TH in eutopic CH was 0.799 (95% confidence interval [CI], 0.678-0.919; P<0.001) and 0.925 (95% CI, 0.837-1.000; P<0.001), respectively. The optimum 12-month and 24-month dose in predicting TH is 3.25 µg/kg (12-month: sensitivity, 87.1%; specificity, 68.0%; 24-month: sensitivity 93.5%, specificity 88%)., Conclusion: Infants with CH requiring lower L-thyroxine doses (<3.25 µg/kg) are likely to have TH, and thus might be re-evaluated at 12 months or 24 months rather than 3 years of age.

Ellagic acid (EA) is a well- known phytochemical that modulates various cellular processes. It is present in a variety of foods, including grapes, strawberries, and nuts. However, the influence of EA on immunological responses is not well defined. Here, we investigated the effects of EA on the lipopolysaccharide (LPS)-induced bone marrow-derived dendritic cells (BMDCs). EA was not cytotoxic to DCs. EA suppressed LPS-induced expression of costimulatory molecules (CD80 and CD86), but it did not suppress the expression of major histocompatibility complex (MHC) class I and MHC class II in BMDCs. In particular, EA blocked LPS-induced c-Jun N-terminal kinase (JNK) activation. LPS-mediated expression of proinflammatory cytokines (IL-12 and IFN-γ) was diminished by EA. We also confirmed that levels of IL-12 and IFN-γ were not influenced by EA in the presence of a JNK inhibitor. Taken together, these data demonstrate that EA regulates the immune response through the modulation of LPS-induced DC maturation.