37 research outputs found

    Characterization of Morphology and Structure of Wax Crystals in Waxy Crude Oils by Terahertz Time-Domain Spectroscopy

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    The content, morphology, and structure of precipitated wax crystals are major factors affecting crude oil rheology. In this paper, model oils obtained by dissolving a realistic mixture of long-chain <i>n</i>-octacosane in diesel fuels were studied using terahertz time-domain spectroscopy (THz-TDS) and microscopy to gain insight into clusters composed of asphaltene and wax with increasing wax content. The fractal dimension was used for quantitative characterization of the morphology and structure of clusters in the model oils. From the measured absorption and extinction coefficients in the THz region, dynamic processes of the clusters in the model oils were analyzed and identified. The extinction coefficient in the THz region strongly depended on the dispersed and aggregated states of the asphaltene and wax crystals. These observations suggest that the aggregation state of the particles in model oils can be monitored with THz-TDS. In the future, THz-TDS technology may be used to effectively analyze particle dispersion or the aggregation state in crude oil and may thus be useful for rapid assessment of the effect of pour-point depressant on wax crystal aggregates

    The Schizophrenia-Related Protein Dysbindin-1A Is Degraded and Facilitates NF-Kappa B Activity in the Nucleus

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    <div><p>Dystrobrevin-binding protein 1 (<i>DTNBP1</i>), a gene encoding dysbindin-1, has been identified as a susceptibility gene for schizophrenia. Functioning with partners in synapses or the cytoplasm, this gene regulates neurite outgrowth and neurotransmitter release. Loss of dysbindin-1 affects schizophrenia pathology. Dysbindin-1 is also found in the nucleus, however, the characteristics of dysbindin in the nucleus are not fully understood. Here, we found that dysbindin-1A is degraded in the nucleus via the ubiquitin-proteasome system and that amino acids 2-41 at the N-terminus are required for this process. By interacting with p65, dysbindin-1A promotes the transcriptional activity of NF-kappa B in the nucleus and positively regulates MMP-9 expression. Taken together, the data obtained in this study demonstrate that dysbindin-1A protein levels are highly regulated in the nucleus and that dysbindin-1A regulates transcription factor NF-kappa B activity to promote the expression of MMP-9 and TNF-α.</p></div

    EGFP-NLS-dysbindin-1A del 2–41 is stable in nucleus.

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    <p>(A) The degradation of EGFP-dysbindin-1A del 2–41 or EGFP-NLS-dysbindin-1A del 2–41. (B) The quantitative analysis of (A). The values shown represent the means ± S.E. of three independent experiments. (C) The C-terminus of dysbindin-1A was not ubiquitinated in nucleus. The deletion mutants of dysbindin were transfected into cells. The ubiquitination of deletion mutants were examined.</p

    Dysbindin-1A is degraded via the ubiquitin-proteasome pathway in the nucleus.

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    <p>(A) Nuclear dysbindin-1A was degraded via the ubiquitin-proteasome pathway. Twenty-four hours after transfection with dysbindin-1A-EGFP, dysbindin-1A-NLS-EGFP and dysbindin-1A-NES mutant-NLS-EGFP, cells were treated with DMSO or MG132 (10 μM) for 12 hours, respectively. (B and C) Dysbindin-1A-NLS-EGFP and dysbindin-1A-NES mutant-NLS-EGFP were transfected into HEK293 cells for 24 hours. The cells were then treated with MG132 (10 μM) for 12 hours. Cell lysates were immunoprecipitated with GFP antibody and immunoblotted with ubiquitin antibody.</p

    Dysbindin-1A is degraded in the nucleus.

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    <p>(A) HEK293 cells that had been transfected with dysbindin-1A-EGFP were pre-treated with leptomycin B 20 ng/ml or equal volumes of ethanol for 1 hour, respectively. The cells were then treated with CHX for the indicated time, and cell extracts were subjected to immunoblot analysis. (B) The band intensity of dysbindin-1A-EGFP relative to GAPDH is shown. The values shown represent the means ± S.E. of three independent experiments. *, <i>P</i><0.05; **, <i>P</i><0.01; one-way ANOVA. (C) Subcellular localization of dysbindin-1A-EGFP and its variants that harbor a nuclear localization signal and/or a nuclear export signal mutant. HEK293 cells were transfected with the indicated plasmids, and the nuclei were stained with DAPI; The bar represents 10 μm. (D) The half-life of dysbindin-1A-NLS or the NES mutant was shorter than that of wild type dysbindin-1A. Dysbindin-1A-EGFP, dysbindin-1A-NLS-EGFP and dysbindin-1A-NES mutant-NLS-EGFP were transfected into HEK293 cells for 24 hours; the cells were then treated with CHX (100 μg/ml) for the indicated time. (E) The data from three independent experiments of (D) were quantified. The values shown represent means ± S.E. (F) The HEK293 cells were pre-treated with leptomycin B 20 ng/ml or equal volumes of ethanol for 1 hour, respectively. The cells were then treated with CHX for the indicated time, and cell extracts were subjected to immunoblot analysis. (G) The quantified analysis from three independent experiments of (F). The values shown represent the means ± S.E. *, <i>P</i><0.05; one-way ANOVA.</p

    Data_Sheet_1.XLSX

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    <p>Avian-like H5N1 canine influenza virus (CIV) causes severe respiratory infections in dogs. However, the mechanism underlying H5N1 CIV infection in dogs is unknown. The present study aimed to identify differentially expressed miRNAs and mRNAs in the lungs and trachea in H5N1 CIV-infected dogs through a next-generation sequencing-based method. Eighteen 40-day-old beagles were inoculated intranasally with CIV, A/canine/01/Guangdong/2013 (H5N1) at a tissue culture infectious dose 50 (TCID<sub>50</sub>) of 10<sup>6</sup>, and lung and tracheal tissues were harvested at 3 and 7 d post-inoculation. The tissues were processed for miRNA and mRNA analysis. By means of miRNA-gene expression integrative negative analysis, we found miRNA–mRNA pairs. Lung and trachea tissues showed 138 and 135 negative miRNA–mRNA pairs, respectively. One hundred and twenty negative miRNA–mRNA pairs were found between the different tissues. In particular, pathways including the influenza A pathway, chemokine signaling pathways, and the PI3K-Akt signaling pathway were significantly enriched in all groups in responses to virus infection. Furthermore, dysregulation of miRNA and mRNA expression was observed in the respiratory tract of H5N1 CIV-infected dogs and notably, TLR4 (miR-146), NF-κB (miR-34c) and CCL5 (miR-335), CCL10 (miR-8908-5p), and GNGT2 (miR-122) were found to play important roles in regulating pathways that resist virus infection. To our knowledge, the present study is the first to analyze miRNA and mRNA expression in H5N1 CIV-infected dogs; furthermore, the present findings provide insights into the molecular mechanisms underlying influenza virus infection.</p

    Dysbindin-1A interacts with Rel A (p65) and promotes NF-kappa B activity.

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    <p>(A) A dual luciferase reporter assay shows that dysbindin-1A promotes NF-kappa B transcriptional activity. (B) Dysbindin-1A knockdown does not influence the protein levels of p65 in N2a cells. N2a cells were transfected with si-dys1# or si-dys2#. The resulting cell lysates were subjected to immunoblot analysis using antibodies to dysbindin-1A or p65. (C) p65 interacts with Flag-dysbindin-1A. In HEK293 cells that were co-transfected with Flag-dysbindin-1A and EGFP-p65, EGFP or EGFP-p65 was immunoprecipitated using an anti-GFP antibody. The immunoprecipitants were subjected to immunoblot analysis with the indicated antibodies. (D) Dysbindin-1A interacts with p65 in a GST-pulldown assay. (E) Endogenous dysbindin-1A interacts with endogenous p65 in SH-SY5Y cells. (F) EGFP-p65 interacts with Flag-dysbindin-1A in nucleus. The cytoplasm fraction or nuclear fraction of HEK293 cells transfected with EGFP/EGFP-p65 and Flag-dysbindin-1A were immunoprecipitated using an anti-GFP antibody. The immunoprecipitants were subjected to immunoblot analysis with the indicated antibodies. <i>IB</i>, immunoblot; <i>IP</i>, immunoprecipitation.</p

    Data_Sheet_2.docx

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    <p>Avian-like H5N1 canine influenza virus (CIV) causes severe respiratory infections in dogs. However, the mechanism underlying H5N1 CIV infection in dogs is unknown. The present study aimed to identify differentially expressed miRNAs and mRNAs in the lungs and trachea in H5N1 CIV-infected dogs through a next-generation sequencing-based method. Eighteen 40-day-old beagles were inoculated intranasally with CIV, A/canine/01/Guangdong/2013 (H5N1) at a tissue culture infectious dose 50 (TCID<sub>50</sub>) of 10<sup>6</sup>, and lung and tracheal tissues were harvested at 3 and 7 d post-inoculation. The tissues were processed for miRNA and mRNA analysis. By means of miRNA-gene expression integrative negative analysis, we found miRNA–mRNA pairs. Lung and trachea tissues showed 138 and 135 negative miRNA–mRNA pairs, respectively. One hundred and twenty negative miRNA–mRNA pairs were found between the different tissues. In particular, pathways including the influenza A pathway, chemokine signaling pathways, and the PI3K-Akt signaling pathway were significantly enriched in all groups in responses to virus infection. Furthermore, dysregulation of miRNA and mRNA expression was observed in the respiratory tract of H5N1 CIV-infected dogs and notably, TLR4 (miR-146), NF-κB (miR-34c) and CCL5 (miR-335), CCL10 (miR-8908-5p), and GNGT2 (miR-122) were found to play important roles in regulating pathways that resist virus infection. To our knowledge, the present study is the first to analyze miRNA and mRNA expression in H5N1 CIV-infected dogs; furthermore, the present findings provide insights into the molecular mechanisms underlying influenza virus infection.</p

    The N-terminal 2–41 amino acids of dysbindin-1A are important for its nuclear degradation.

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    <p>(A) The N-terminal sequence of dysbindin-1A (amino acids 1–42). The sequence was scored using Ubpred and BDM-PUB, and potential ubiquitination sites are colored red. (B) The potential dysbindin-1A ubiquitination site, lysine 21, was mutated to arginine. The ubiquitination of K21R and WT forms of dysbindin-1A was examined. (C) Dysbindin-1A-EGFP, EGFP-dysbindin (residues 1–189) or EGFP-dysbindin-1A residue 2–41 deletion mutant were co-transfected with HA-Ub into HEK293 cells, respectively. After culturing for 24 hours, 10 μM MG132 was added, and the cells were then cultured for an additional 12 hours. After lysis, the proteins were immunoprecipitated using an anti-GFP antibody and immunoblotted with an HA antibody. (D) The EGFP-dysbindin-1A residue 2–41 deletion mutant was more stable in the nucleus. HEK293 cells expressing dysbindin-1A-EGFP or EGFP-dysbindin-1A residue 2–41 deletion mutant were treated with leptomycin B (20 ng/ml) for 1 hour. The cells were then treated with CHX for the indicated time. Finally, cell extracts were subjected to immunoblot analysis. (E) The band intensity of the dysbindin-1A-EGFP and EGFP-dysbindin-1A residue 2–41 deletion mutant is shown, relative to GAPDH. The values shown represent the means ± S.E. of three independent experiments. *, <i>P</i><0.05; one-way ANOVA. (F) The localization of EGFP-dysbindin-1A del 2–41 under treatment of Ethanol or LMB. The nuclei were stained with DAPI; The bar represents 10 μm.</p

    Light source comparison: indoors and outdoors.

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    <p>(A) The KS-Detect system during an experiment using sunlight. Lens must be manually aligned with the sun as the sun moves across the sky. (B) For experiments conducted indoors, the lens and microfluidics are removed from our portable kit, and are fixed in front of a 100 Watt LED array. (C) A typical solar temperature profile is more variable when compared to a typical LED array temperature profile, due to cloud coverage (as seen at about 35 min.) and intermittent realignment of the lens with the sun.</p
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