Convenient Synthesis of (E)-5-Aryl(halo)methylenebicyclo- [2.2.2]oct-2-enes and -[2.2.1]hept-2-enes via Lewis Acid- Promoted Carbohalogenation of Cyclic 2,6-Enynols

Abstract: An efficient synthesis of (E)-5-aryl(halo)-methylenebicyclo[2.2.2]oct-2-enes is reported. Lewis acid-promoted carbohalogenation of 4-(3-arylprop-2-ynyl)-cyclohex-2-enols in dichloromethane proceeds rapidly to afford the exo-methylenebridged bicycles in good yields. This method also provides an easy access to (E)-5-aryl(halo)methyl-A C H T U N G T R E N N U N G enebicyclo[2.2.1]hept-2-enes from the five-membered ring 2,6-enynols. The reactions are procedurally simple and high yielding, producing the aryl(halo)methylene-bridged bicycles in minutes under air and mild conditions

Serotype and multilocus sequence typing of Streptococcus suis from diseased pigs in Taiwan

Abstract Streptococcus suis (S. suis) infection can cause clinically severe meningitis, arthritis, pneumonia and septicemia in pigs. To date, studies on the serotypes, genotypes and antimicrobial susceptibility of S. suis in affected pigs in Taiwan are rare. In this study, we comprehensively characterized 388 S. suis isolates from 355 diseased pigs in Taiwan. The most prevalent serotypes of S. suis were serotypes 3, 7 and 8. Multilocus sequence typing (MLST) revealed 22 novel sequence types (STs) including ST1831-1852 and one new clonal complex (CC), CC1832. The identified genotypes mainly belonged to ST27, ST94 and ST1831, and CC27 and CC1832 were the main clusters. These clinical isolates were highly susceptible to ceftiofur, cefazolin, trimethoprim/sulfamethoxazole and gentamicin. The bacteria were prone to be isolated from cerebrospinal fluid and synovial fluid in suckling pigs with the majority belonging to serotype 1 and ST1. In contrast, ST28 strains that corresponded to serotypes 2 and 1/2 were more likely to exist in the lungs of growing-finishing pigs, which posted a higher risk for food safety and public health. This study provided the genetic characterization, serotyping and the most current epidemiological features of S. suis in Taiwan, which should afford a better preventative and treatment strategy of S. suis infection in pigs of different production stages

Alleviation of Carbon-Tetrachloride-Induced Liver Injury and Fibrosis by Betaine Supplementation in Chickens

Betaine is a food component with well-reported hepatoprotection effects. However, the effects and mechanisms of betaine on liver fibrosis development are still insufficient. Because metabolic functions of chicken and human liver is similar, we established a chicken model with carbon Tetrachloride- (CCl4-) induced fibrosis for studying antifibrotic effect of betaine in vivo and in vitro. Two-week-old male chicks were supplemented with betaine (1%, w/v) in drinking water for 2 weeks prior to the initiation of CCl4 treatment (i.p.) until sacrifice. Primary chicken hepatocytes were treated with CCl4 and betaine to mimic the in vivo supplementation. The supplementation of betaine significantly alleviated liver fibrosis development along with the inhibition of lipid peroxidation, hepatic inflammation cytokine, and transforming growth factor-β1 expression levels. These inhibitive effects were also accompanied with the attenuation of hepatic stellate cell activation. Furthermore, our in vitro studies confirmed that betaine provides antioxidant capacity for attenuating the hepatocyte necrosis by CCl4. Altogether, our results highlight the antioxidant ability of betaine, which alleviates CCl4-induced fibrogenesis process along with the suppression of hepatic stellate cells activation. Since betaine is a natural compound without toxicity, we suggest betaine can be used as a potent nutritional or therapeutic factor for reducing liver fibrosis

Transdermal Composite Microneedle Composed of Mesoporous Iron Oxide Nanoraspberry and PVA for Androgenetic Alopecia Treatment

The transdermal delivery of therapeutic agents amplifying a local concentration of active molecules have received considerable attention in wide biomedical applications, especially in vaccine development and medical beauty. Unlike oral or subcutaneous injections, this approach can not only avoid the loss of efficacy of oral drugs due to the liver's first-pass effect but also reduce the risk of infection by subcutaneous injection. In this study, a magneto-responsive transdermal composite microneedle (MNs) with a mesoporous iron oxide nanoraspberry (MIO), that can improve the drug delivery efficiency, was fabricated by using a 3D printing-molding method. With loading of Minoxidil (Mx, a medication commonly used to slow the progression of hair loss and speed the process of hair regrowth), MNs can break the barrier of the stratum corneum through the puncture ability, and control the delivery dose for treating androgenetic alopecia (AGA). By 3D printing process, the sizes and morphologies of MNs is able to be, easily, architected. The MIOs were embedded into the tip of MNs which can deliver Mx as well as generate mild heating for hair growth, which is potentially attributed by the expansion of hair follicle and drug penetration. Compared to the mice without any treatments, the hair density of mice exhibited an 800% improvement after being treated by MNs with MF at 10-days post-treatment

Embryonic cholesterol esterification is regulated by a cyclic AMP-dependent pathway in yolk sac membrane-derived endodermal epithelial cells.

During avian embryonic development, endodermal epithelial cells (EECs) absorb yolk through the yolk sac membrane. Sterol O-acyltransferase (SOAT) is important for esterification and yolk lipid utilization during development. Because the major enzyme for yolk sac membrane cholesteryl ester synthesis is SOAT1, we cloned the avian SOAT1 promoter and elucidated the cellular functions of SOAT1. Treatments with either glucagon, isobutylmethylxanthine (IBMX), an adenylate cyclase activator (forskolin), a cAMP analog (dibutyryl-cAMP), or a low glucose concentration all increased SOAT1 mRNA accumulation in EECs from Japanese quail, suggesting that SOAT1 is regulated by nutrients and hormones through a cAMP-dependent pathway. Activity of protein kinase A (PKA) was increased by IBMX, whereas co-treatment with the PKA inhibitor, H89 negated the increase in PKA activity. Cyclic AMP-induced EECs had greater cholesterol esterification than untreated EECs. By promoter deletion and point-mutation, the cAMP-response element (-349 to -341 bp) was identified as critical in mediating transcription of SOAT1. In conclusion, expression of SOAT1 was regulated by a cAMP-dependent pathway and factors that increase PKA will increase SOAT1 to improve the utilization of lipids in the EECs and potentially modify embryonic growth

Positive regulation of transcription and enzymatic function of <i>SOAT1</i>.

<p>(A-D) Effects of forskolin and dibutyryl-cyclic AMP (db-cAMP) on <i>SOAT1</i> mRNA accumulations in EECs and hepatocytes. EECs and hepatocytes were treated with different concentrations of forskolin or db-cAMP for 24 hours. <i>SOAT1</i> mRNA accumulation was measured and normalized to <i>β-actin</i> mRNA. (n = 4) (E-F) H89 inhibited IBMX-induced <i>SOAT1</i> mRNA accumulations in EECs and hepatocytes. <i>SOAT1</i> mRNA accumulation was increased by 0.5 mM IBMX treatment for 24 hours, whereas the PKA inhibitor, H89 (10 μM) abolished the IBMX-mediated increase of <i>SOAT1</i>. (n = 6) (G-H) The IBMX treatment increased PKA activity in EECs and hepatocytes. The cells were treated with 0.5 mM IBMX with or without 10 μM H89 for 24 hours. The cell lysates were assayed for PKA activity using the PepTag assay. Typical gel patterns are represented (three independent experiments) for EECs and hepatocytes with statistical analysis of the densitometric analysis represented by the graphs (quantified by ImageQuant software). (I) Fluorescence examination of <i>SOAT1</i> activity by applying NBD-cholesterol as the substrate. EECs were incubated with 10 μg/mL of NBD-cholesterol at 37°C for 30 mins or 1 hour. Green indicated the cholesterol ester inside of EECs and blue indicated cell nucleus stained with DAPI. (J-K) The fluorescent intensity was quantified by ImageJ software. EECs were treated with or without db-cAMP for 24 hours. EECs were then incubated with 10 μg/mL of NBD-cholesterol for 30 min or 1 hour and examined by confocal microscopy. (J) Fluorescent intensity of total area. K: Fluorescent intensity per area of lipid droplets in EECs. (n = 4) Statistical significance was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Control value was set as 1. Different letters indicate a significant difference (P≤0.05). (L) The <i>SOAT1</i> protein levels after db-cAMP or IBMX treatments. EECs were treated with 0.5 or 1 mM db-cAMP or 0.5 mM IBMX for 48 hours and protein levels were examined by Western blotting. The <i>β-actin</i> was used as internal control. Statistical significance was determined by one-way analysis of variance. Dunnett's multiple comparisons test was used to evaluate differences between means. Data were expressed as means ± S.E.M. (n = 8) and different letters indicate the significant difference (P≤0.05). (M-N) The effects of H89 with or without db-cAMP and IBMX treatments on NBD-cholesterol uptake and storage. (M) EECs were pre-treated with or without H89 (10 μM) for 2 hours, and added db-cAMP (1 mM) or IBMX (0.5 mM) for 24 hours. Cells were then incubated with 10 μg/mL NBD-cholesterol for one hour, fixed with 4% paraformaldehyde and finally examined by confocal microscopy. Green indicated the cholesterol ester inside of EECs and blue indicated cell nucleus stained with DAPI. Scale bar = 50 μm. (N) The quantitation of fluorescent intensity on NBD-cholesterol uptake and storage by ImageJ software in cells treated with H89 with or without IBMX or db-cAMP. Data were expressed as means ± S.E.M. (n = 4) Statistical significance among more than three different experimental groups was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Different letters indicate a significant difference (P≤0.05).</p

The chicken primers for real-time PCR quantification.

<p>The chicken primers for real-time PCR quantification.</p

Conditions and primers for serial promoter region deletion.

<p>Conditions and primers for serial promoter region deletion.</p