Signalling pathway of U46619-induced vascular smooth muscle contraction in mouse coronary artery.

BACKGROUND: Thromboxane A2 (TXA2 ) participates in many pathophysiological processes of coronary artery disease. However, its mechanism of TXA2 -induced contraction in the coronary artery remains to be clarified. A multi myograph system was used to measure the isometric tension of the mouse coronary arteries and identify the effect and pathway of TXA2 analogues U46619. Confocal laser scanning microscopy was used to measure the intracellular calcium concentration ([Ca2+ ]i ) in mouse coronary artery smooth muscle cells. Results from the experiment had shown that contraction in coronary artery was generated by U46619 in a concentration-dependent manner, which was completely abolished by a specific TXA2 receptor blocker, GR32191. PI-PLC inhibitors U73122 and D609 and Rho-Kinase inhibitor Y-27632 can block the U46619 elicited coronary artery contraction in a dose-dependent manner. Then, the vasoconstriction response to U46619 was obviously inhibited by two pan-PKC inhibitors chelerythrine or GÓ§6983, and a selective PKCδ inhibitor rottlerin, but was not blocked by a selective PKCζ inhibitor PKC-PS or a selective PKCß inhibitor hispidin. Meanwhile, the PKC activator PDBu-induced vasoconstriction was significantly inhibited by 1 µmol/L nifedipine, then mostly inhibited by 100 µmol/L 2-APB and 10 µmol/L Y27632. We further found that the response to U46619 was inhibited, respectively, by three calcium channel blockers nifedipine, SKF96356 or 2-APB in a concentration-dependent manner. Although Store-operated Ca2+ (SOC) channels generated the increase of [Ca2+ ]i in mouse coronary artery smooth muscle cells, SOC channels did not contribute to the vasoconstriction in mouse coronary arteries. Caffeine-induced sarcoplasmic reticulum (SR) Ca2+ release could obviously induce coronal vasoconstriction. In addition, NPPB, a cell membrane Ca2+ activated C1- channel blocker, could obviously inhibit the U46619-induced vasoconstriction. The U46619-induced mouse coronary artery contraction was involved in the increase in [Ca2+ ]i mediated by Cav1.2, TRPC channels and SR release through the activation of G-protein-coupled TP receptors and the kinases signalling pathway in TP downstream proteins, while SOC channels did not participate in the vasoconstriction.

Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300.

Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

Synthesis of Indoles through Domino Reactions of 2-Fluorotoluenes and Nitriles.

Indoles are essential heterocycles in medicinal chemistry, and therefore, novel and efficient approaches to their synthesis are in high demand. Among indoles, 2-aryl indoles have been described as privileged scaffolds. Advanced herein is a straightforward, practical, and transition-metal-free assembly of 2-aryl indoles. Simply combining readily available 2-fluorotoluenes, nitriles, LiN(SiMe3 )2 , and CsF enables the generation of a diverse array of indoles (38 examples, 48-92 % yield). A range of substituents can be introduced into each position of the indole backbone (C4 to C7, and aryl groups at C2), providing handles for further elaboration.

Morphology evolution and gelation mechanism of alkali induced konjac glucomannan hydrogel.

A molecular-level mechanism of alkali induced konjac glucomannan (KGM) hydrogel gelation processing is considered with the application of nuclear magnetic resonance (NMR) spectroscopy and atomic force microscopy (AFM) as complementary methods to diffusive wave spectroscopy (DWS) microrheology and thermoanalysis. It is shown that deacetylation of KGM chains occurs immediately upon mixing with Na2CO3, inducing self-packaging. Partial unfolding of the packed loose structure of dehydrated KGM is observed upon heating. The configuration transition from random coils to self-assembling filament networks takes place before KGM aggregating to form large irreversible bundles with a lower degree of cross-linking. The gelation is not fulfilled until the temperature is increased to above 70 °C when the significant agglomeration is initiated among transitional fibrils to form junction zones essentially composed of acetyl-free portions. This suggests the intermolecular aggregation of KGM chains not simply regarding to hydrogen bonds, but essentially relating to hydrophobic interactions.

How Outdoor Trees Affect Indoor Particulate Matter Dispersion: CFD Simulations in a Naturally Ventilated Auditorium.

This study used computational fluid dynamics (CFD) models, coupling with a standard k-ε model based on the Reynolds-averaged Navier-Stokes (RANS) approach and a revised generalized drift flux model, to investigate effects of outdoor trees on indoor PM1.0, PM2.5, and PM10 dispersion in a naturally ventilated auditorium. Crown volume coverage (CVC) was introduced to quantify outdoor trees. Simulations were performed on various CVCs, oncoming wind velocities and window opening sizes (wall porosities were 3.5 and 7.0%, respectively, for half and fully opened windows). The results were as follows: (1) A vortex formed inside the auditorium in the baseline scenario, and the airflow recirculation created a well-mixed zone with little variation in particle concentrations. There was a noticeable decrease in indoor PM10 with the increasing distance from the inlet boundary due to turbulent diffusion. (2) Assuming that pollution sources were diluted through the inlet, average indoor particle concentrations rose exponentially with increasing oncoming wind speed. PM10 changed most significantly due to turbulent diffusion and surface deposition reduction intensified by the increased wind velocity. (3) Increasing the window opening improved indoor cross-ventilation, thus reducing indoor particle concentrations. (4) When 2.87 m³/m² ≤ CVC ≤ 4.73 m³/m², indoor PM2.5 could meet requirements of the World Health Organization's air quality guidelines (IT-3) for 24-hour mean concentrations; and (5) average indoor particle concentrations had positive correlations with natural ventilation rates (R² = 0.9085, 0.961, 0.9683 for PM1.0, PM2.5, and PM10, respectively, when the wall porosity was 3.5%; R² = 0.9158, 0.9734, 0.976 for PM1.0, PM2.5, and PM10, respectively, when the wall porosity was 7.0%).

Construction and characterization of HIV type 1 CRF07_BC infectious molecular clone from men who have sex with men.

This study aimed to investigate the biological characterization of HIV type 1 (HIV-1) CRF07_BC infection among men who have sex with men (MSM). From November 2011 to November 2013, a total of 66 blood samples were collected from MSM with acute HIV-1 infection with CRF07_BC subgroup strains. Deletion in the gag p6 region was detected by sequence alignment and comparative analysis. Peripheral blood mononuclear cells (PBMCs) of HNXX1301-1307 samples were separated by density gradient centrifugation. Nested polymerase chain reaction (nPCR) was used to amplify the viral DNA. The near full-length HIV-1 DNA products were ligated to the long terminal repeat (LTR) vector plasmid (07BCLTR) to construct a full-length HIV clone. The molecular clone was transfected into HEK-293T cells, TZM-b1 cells and patients' PBMCs. The pregenome of an infectious molecular clone of HIV-1 (pNL4-3) was amplified, and a subclone with CRF07_BC was developed to construct the full-length chimeric molecular clone pNL4-3/07BCLTR. Detection of p24 antigen and luciferase activity was used to measure the in vitro infectivity of pNL4-3/07BCLTR. Among the 66 MSM patients infected with CRF07_BC strains, deletion mutations of the Gag P6 proteins were found in 7 of 18CRF07_BC strains; deletion mutations of 2-13 amino acids in different regions were discovered in 6 strains; and the remaining 42 strains did not show deletions. Seven strains with amino acids deficiency in the P6 protein accounted for 27% of all strains and 75% of all deletion genotype strains. A total of 186 full-length molecular clones of CRF07_BC were constructed. There were 5, 9, 10 and 11 clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 that resulted in p24-positive supernatant when transfected into HEK-293T cells. Full-length clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 showed slight infection in the transfected TZM-b1 cells, as judged by the fluorescence values of TZM-b1 cells 48h post-transfection. However, we were unable to transfect the patients' PMBCs with the above four clones. The phylogenetic tree of the C2V3 segment of the Env gene showed that a significant gene cluster was formed by all of the chimeric full-length HNXX1306 clones, and the bootstrap value for this cluster was 97.5%. Patients' PBMCs could be infected by 1306N6, 1306N13 and 1306N22 chimeric full-length clones. The CRF07_BC subtype (6889-7407 nucleotide residues of HXB2) is one of the most prevalent epidemic HIV-1 virus strains among the MSM population. The full-length chimeric molecular clone pNL4-3/07BCLTR may significantly improve the in vitro infectivity of the CRF07_BC strain.

Designed synthesis of a highly conjugated hexaethynylbenzene-based host for supramolecular architectures.

The construction of efficient synthetic functional receptors with tunable cavities, and the self-organization of guest molecules within these cavities through noncovalent interactions can be challenging. Here we have prepared a double-cavity molecular cup based on hexaethynylbenzene that possesses a highly π-conjugated interior for the binding of electron-rich guests. X-ray crystallography, NMR spectroscopy, UV/Vis spectroscopy, fluorescent spectroscopy, cyclic voltammetry, and SEM were used to investigate the structures and the binding behaviors. The results indicated that the binding of a guest in one cavity would affect the binding of the same or another guest in the other cavity. The effect of electron transfer in this system suggests ample opportunities for tuning the optical and electronic properties of the molecular cup and the encapsulated guest. The encapsulation of different guests would also lead to different aggregate nanostructures, which is a new way to tune their supramolecular architectures.

Designed synthesis and supramolecular architectures of furan-substituted perylene diimide.

Novel furan-substituted perylene diimides are successfully synthesized and an efficient supramolecular architecture approach to construct zero/one-dimensional nano- and micro-structures by controlling solvents has been demonstrated. The aggregate structure conversion in different molecular structures can be controlled in the form of sphere-like, rod-like, and vesicle-like structures. As expected, these solid supramolecular rod-like architectures displayed interesting optical waveguide behavior, which indicates the aggregate structure materials of furan-substituted perylene diimides have the potential application as micro-scale photonic elements.

Synthesis and self-assembly of dihydroxyperylene bisimides for the tuning of photophysical properties.

The synthesis of 1,10-dihydroxyperylene bisimides by nucleophilic substitution of brominated perylene bisimide is described. 1,10-Dihydroxyperylene bisimides formed J aggregates in nonpolar solvents and showed a clearly redshifted absorption band. The solvent polarity also influenced the hydrogen bond with the hydroxyl group, and thus, the photophysical properties of perylene bisimide. The photophysical properties of these dihydroxyperylene perylene bisimides can also be tuned by changing charge transfer from the hydroxyl groups to the perylene core through the introduction of metal ions.

Selective and colorimetric fluoride anion chemosensor based on s-tetrazines.

s-Tetrazine and their derivatives are found to be a class of selective and colorimetric chemosensors for fluoride anions. The supramolecular interaction (anion-π and charge/electron transfer) between fluoride ion and π-electron deficient tetrazines receptor could facilitate the F(-)-tetrazines electron transfer (ET) event that generates tetrazine˙(-) radical anion. Accordingly, the color of its solution changed from red to green. Furthermore, the recognition of F(-) in DMSO is tolerant to water (DMSO-H(2)O = 9:1, v/v) and shows excellent selectivity towards other anions, especially H(2)PO(4)(-) and OAc(-).

The hypertension scale of the system of Quality of Life Instruments for Chronic Diseases, QLICD-HY: a development and validation study.

AIMS: This paper is aimed to develop and validate the hypertension scale of the system of Quality of Life Instruments for Chronic Diseases, QLICD-HY. METHODS: The QLICD-HY instrument was developed based on programmed decision procedures with multiple nominal and focus group discussions and pilot testing. A total of 157 inpatients with hypertension were used to provide the data measuring QOL three times before and after treatment. The psychometric properties of the scale were evaluated with respect to validity, reliability and responsiveness employing correlation and factor analyses, and t-tests. RESULTS: Correlation and factor analyses confirmed good construct validity and criterion-related validity when using Short Form (36) Health Survey (SF-36) as a criterion. Test-retest reliability coefficients (Pearson r and intra-class correlation (ICC)) for the overall instrument score and all domains except for the hypertension-specific domain (SPD) (0.75) were higher than 0.80 with a range of 0.75-0.91; the internal consistency α for all domains except for the hypertension-specific domain (0.66) was higher than 0.70. The overall score and scores for most facets within each domain except for the social domain (SOD) had statistically significant changes (t-tests) after treatment with moderate effect sizes. CONCLUSION: QLICD-HY has good validity, reliability, responsiveness and can be used as the quality-of-life instrument for patients with hypertension.

Development and validation of a quality of life instrument for patients with drug dependence: comparisons with SF-36 and WHOQOL-100.

AIM: Our goal was to develop a self-administered quality of life scale for patients with drug addiction/dependence (QOL-DA) and compare it with the SF-36 and the WHOQOL-100. METHODS: Employing theory and methodology of rating scale construction, a self-administered quality of life instrument for individuals with drug dependence QOL-DA was developed and evaluated utilizing responses from 212 drug-dependent subjects at the Kunming Municipal Mandatory Detoxification and Rehabilitation Center in China. Quality of life was measured using the SF-36, WHOQOL-100 and QOL-DA three times during the detoxification. RESULTS: Test-retest reliability in the domains of physical function, psychological function, social function and toxicity were 0.82, 0.64, 0.78, and 0.76, respectively. Cronbach's coefficient α for the 4 domains was 0.87, 0.89, 0.93 and 0.86, respectively. Correlations and factor analysis showed good construct validity. Criterion-related and convergent validity was confirmed by using the SF-36 and the WHOQOL-100 simultaneously. The instrument does show the change in QOL after two weeks of detoxification with higher standardized response mean higher than that of SF-36 and WHOQOL-100. CONCLUSION: The instrument developed has good validity, reliability and better responsiveness than instruments currently used, and can be employed effectively to measure the quality of life of individuals with drug dependence.

Non-invasive methods for identifying oocysts of Sarcocystis spp. from definitive hosts.

Because the excreted sporocysts and/or oocysts of various species of Sarcocystis may not be discriminated morphologically, we sought to validate a diagnostic technique based on variation in the 18S rDNA sequence. Oocysts and/or sporocysts from three taxa of Sarcocystis were collected from human, feline, and canine definitive hosts that had fed upon meats infected with the muscle cysts of Sarcocystis hominis, Sarcocystis fusiformis and a species of Sarcocystis from water buffalo that could not be distinguished from Sarcocystis cruzi. Using a new collection method employing filter paper, these excreted oocysts and sporocysts were subjected to DNA extraction, as were the corresponding muscle cysts. Methods employing PCR-RFLP and DNA sequencing of a partial 18S rDNA gene (ssrRNA) sequence were then used to successfully distinguish among the three taxa. The same, unique restriction digestion pattern characterizes the tissue cysts and oocysts and/or sporocysts of each parasite taxon. The technique makes possible amplification and identification of species specific gene sequences based on DNA extracted from as few as 7 excreted sporocysts (the equivalent of 3 and 1/2 oocysts) from freshly prepared material, or as few as 50 sporocysts from feces samples that had been stored in potassium dichromate (K2Cr2O7) for as long as 6 years. This represents the first report using molecular diagnostic procedures to diagnose oocysts of Sarcocystis in faecal samples, describing a valuable new tool for studying the epidemiology of various Sarcocystis species.