Copper-Catalyzed [2 + 2 + 3] Annulation of 1,6-Enynes with α‑Bromo-1,3-Dicarbonyl Compounds: Synthesis of Dihydrooxepines

Copper-catalyzed [2 + 2 + 3] annulation of 1,6-enynes with α-bromo-1,3-dicarbonyl compounds is described. This reaction provides facile access to seven-membered dihydrooxepines for epidithiodiketopiperazines with two newly formed C–C bonds and one C–O bond

Voltage-Dependent Anion Channel 1(VDAC1) Participates the Apoptosis of the Mitochondrial Dysfunction in Desminopathy

<div><p>Desminopathies caused by the mutation in the gene coding for desmin are genetically protein aggregation myopathies. Mitochondrial dysfunction is one of pathological changes in the desminopathies at the earliest stage. The molecular mechanisms of mitochondria dysfunction in desminopathies remain exclusive. VDAC1 regulates mitochondrial uptake across the outer membrane and mitochondrial outer membrane permeabilization (MOMP). Relationships between desminopathies and Voltage-dependent anion channel 1 (VDAC1) remain unclear. Here we successfully constructed the desminopathy rat model, evaluated with conventional stains, containing hematoxylin and eosin (HE), Gomori Trichrome (MGT), (PAS), red oil (ORO), NADH-TR, SDH staining and immunohistochemistry. Immunofluorescence results showed that VDAC1 was accumulated in the desmin highly stained area of muscle fibers of desminopathy patients or desminopathy rat model compared to the normal ones. Meanwhile apoptosis related proteins bax and ATF2 were involved in desminopathy patients and desminopathy rat model, but not bcl-2, bcl-xl or HK2.VDAC1 and desmin are closely relevant in the tissue splices of deminopathies patients and rats with desminopathy at protein lever. Moreover, apoptotic proteins are also involved in the desminopathies, like bax, ATF2, but not bcl-2, bcl-xl or HK2. This pathological analysis presents the correlation between VDAC1 and desmin, and apoptosis related proteins are correlated in the desminopathy. Furthermore, we provide a rat model of desminopathy for the investigation of desmin related myopathy.</p></div

Analyze of the purified hpMSCs population.

<p>A. The phenotype characteristics of purified MSCs were verified by flow cytometry analysis, which showed that these expanded and plate-adhering populations of cells (at doublings 3 and 5) were positive for CD29 (>95%), CD44 (>95%), CD73 (>99%), CD90 (>95%), CD166 (>95%) and CD105 (>95%) surface marker expression and negative for CD34 (<3%) and CD45 (<3%). The solid line denotes unstained control cells and dotted line denotes isolated mesenchymal stem cells. B. Osteogenic and adipogenic differentiation determination in isolated mesenchymal stem cells. Left panel indicated positive calcium deposit (arrows), right panel indicated lipid vacuoles (arrows). C. Osteogenic and adipogenic differentiation determination in isolated mesenchymal stem cells after transfected with adenovirus. 48 h after transfection, cells were washed with PBS and cultured in OsteoDiff or Adipodiff induction medium for 3 weeks, then Oil-Red-O and Alizarin Red S were utilized to visualize calcium deposits and fat droplets separately. Lipid vacuoles and calcium deposits (arrows) could be visualized (×100).</p

VDAC1 is involved in the desminopathy patients.

<p>The skeletal muscle fibers of desminopathy patients were treated with HE, MGT, immunohistochemical, NADH, SDH or PAS stain <b>(A)</b>; individual atypical RRF and individual lipid droplets by MGT and ORO stains were shown in <b>(B)</b>; desmin in the muscle fibers of patients were detected by immunohistochemistry as shown in <b>(C)</b>, the muscle fiber of normal person was set as a control <b>(C)</b>; VDAC1 and desmin in the muscle fibers of desminopathy patients were analyzed by immunofluorescence as shown in <b>(D)</b>. Scale bars, 50μm.</p

Migratory ability of transduced hpMSCs <i>in vitro</i> and <i>in vivo</i>.

<p>A. Migration of hpMSCs/hpMSCs-Ad-Endo cells through an 8-um Millicell membrane. HpMSCs were cultured in 24-well plate onto 8-um pore-sized membranes, and A2780s or 293 cells were cultured in the lower chamber. The cells migrated to the lower side of the membrane were labeled with methyl violet. Original magnification×400. B. Induction of hpMSCs/hpMSCs-Ad-Endo migration stimulated by A2780s or 293 cells. Compared with 293 cells, A2780s cells promoted migration of hpMSCs//hpMSCs-Ad-Endo significantly. Data shown as means ± SD. C. To verify the specific homing property of transducted mesenchymal to tumor site, we transducted hpMSCs with adenovirus carrying GFP and injected i.p. to nude mice with established peritoneal ovarian tumor. After 3 days and 2 weeks of i.p. injection, mice were sacrificed and tumor nodules and organs were collected. Left panel: 3 days after i.p. injection, GFP signal (arrow) were detected at tumor periphery. Right panel: 2 weeks after i.p. injection, positive GFP signal were detected in the tumor parenchymal (arrow) which indicated hpMSCs persist in the xenograft and can integrate into tumor focus. There is no positive GFP signal detected in the organs.</p

VDAC1 is involved in the desminopathy rat model.

<p>In the desminopathy rat model, normal (control) or mutant desmin(rAd5-DES) transferred muscle fibers were analyzed by immunohistochemistry <b>(A)</b>; desminopathy muscle fibers were detected by HE, MGT, NSE, immunohistochemical, PAS, ORO, NADH or SDH stain <b>(B);</b> VDAC1 and desmin in the normal or desminopathy muscle fibers were detected by immunofluorescence as shown in <b>(C)</b>. Scale bars, 50μm.</p

Apoptosis related proteins are involved in the deminopathy rat model.

<p>Apoptosis related proteins bax <b>(A)</b>, bcl-2 <b>(B)</b>, bcl-xl <b>(C)</b>, ATF2 <b>(D)</b> or HK2 <b>(E)</b> was analyzed by immunofluorescence in the normal <b>(control)</b> or desminopathy muscle fibers. Scale bars, 50μm.</p

Evaluation of anti-angiogenesis effect by alginate assay and CD31 immunohistochemistry.

<p><b>A</b>. Anti-angiogenesis of hpMSCs + Ad-hEndo versus hpMSCs, hpMSCs + Ad-null, and normal saline (NS). Alginate beads containing A2780s tumor cells were implanted subcutaneously into nude mice. The hpMSCs-Ad-hEndo (1×10⁹pfu/100 μl) was injected i.p. on day 3, 6, 9, 12 after bead implantation, with hpMSCs-Ad-null, hpMSCs or saline as control. At day 14, image of the alginate implants was taken and indicated decreased vascularization in beads treated with hpMSCs-Ad-hEndo. B. Analysis of angiogenesis among the groups via CD31 staining. CD31-positive cells were plentiful in the NS, hpMSCs, and hpMSCs + Ad-null groups; however, few positive cells were found in the hpMSCs + Ad-hEndo group. C. FITC-dextran uptake decrease showed the reduction of vascularization in beads treated with hpMSCs-Ad-hEndo. (*, P<0.05) D. Vessel density was determined by counting the number of microvessels per high-power field (×400) and showed hpMSCs-Ad-hEndo significantly reduced CD31 positive microvessels compared with controls. Values are presented as means ± SD. (*, P<0.05)</p

Fluorescence images of transduced hpMSCs and the expression of endostatin protein determined by ELISA and Western blots.

<p>A. GFP fluorescence was assessed in the FITC channel. The hpMSCs transfected with GFP-labeled adenovirus showed green. Original magnification×400. B. Cells transfected with Ad-hEndo and control adenovirus (Ad-null) at an MOI of 2000 were analyzed by Western blot to detect the expression of the endostatin protein. hpMSCs transfected with Ad-hEndo showed obvious expression of endostatin protein when compared with control. C. To verify the expression level of endostatin secreted by engineered hpMSCs <i>in vitro</i>, ELISA assay was performed to evaluate endostatin in the supernatants of cells transfected with adenovirus carrying endostatin, hpMSCs and hpMSCs transfected with Ad-null were applied as control. At 48 h, 72 h and 96 h post transfection, cell culture supernatants were collected and endostatin concentration were determined. The concentration of endostatin in the cell culture supernatants was significantly higher than the control groups and the level of endotatin also increased with time and remained at a high level 96 h after transfection. D. Flow cytometry was applied to evaluate the transfection efficiency of Ad-hEndo-GFP. The transfection rate at MOI 1000, 2000 AND 3000 was 60.3%, 82.3% and 79% respectively. Compared with the control, infection at an MOI of 2000 resulted in higher transfection efficiency. The red frame represents positive zone on each plot.</p

One Novel Multiple-Target Plasmid Reference Molecule Targeting Eight Genetically Modified Canola Events for Genetically Modified Canola Detection

Multiple-target plasmid DNA reference materials have been generated and utilized as good substitutes of matrix-based reference materials in the analysis of genetically modified organisms (GMOs). Herein, we report the construction of one multiple-target plasmid reference molecule, pCAN, which harbors eight GM canola event-specific sequences (RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45) and a partial sequence of the canola endogenous reference gene <i>PEP</i>. The applicability of this plasmid reference material in qualitative and quantitative PCR assays of the eight GM canola events was evaluated, including the analysis of specificity, limit of detection (LOD), limit of quantification (LOQ), and performance of pCAN in the analysis of various canola samples, etc. The LODs are 15 copies for RF2, MS1, and RT73 assays using pCAN as the calibrator and 10 genome copies for the other events. The LOQ in each event-specific real-time PCR assay is 20 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and <i>PEP</i> assays are between 91% and 97%, and the squared regression coefficients (<i>R</i>²) are all higher than 0.99. The quantification bias values varied from 0.47% to 20.68% with relative standard deviation (RSD) from 1.06% to 24.61% in the quantification of simulated samples. Furthermore, 10 practical canola samples sampled from imported shipments in the port of Shanghai, China, were analyzed employing pCAN as the calibrator, and the results were comparable with those assays using commercial certified materials as the calibrator. Concluding from these results, we believe that this newly developed pCAN plasmid is one good candidate for being a plasmid DNA reference material in the detection and quantification of the eight GM canola events in routine analysis