A high-efficiency Cre/loxP-based system for construction of adenoviral vectors

Adenovirus (Ad) vectors provide a highly efficient means of mammalian gene transfer and are widely used for high-level protein expression in mammalian cells, as recombinant vaccines and for gene therapy. A commonly used method for constructing Ad vectors relies on in vivo homologous recombination between two Ad DNA-containing bacterial plasmids cotransfected into 293 cells. While the utility of this two-plasmid approach is well established, its efficiency is low owing to the inefficiency of homologous recombination. To address this, we have developed an improved method for Ad vector construction based on Cre-mediated site-specific recombination between two bacterial plasmids, each bearing a loxP site. Ad vectors are generated as a result of Cre-mediated site-specific recombination between the two plasmids after their cotransfection into 293 cells expressing Cre recombinase. The frequency of Ad vector rescue by Cre-mediated site-specific recombination is significantly higher (approximately 30-fold) than by in vivo homologous recombination. The efficiency and reliability of this method should greatly simplify and expedite the construction of recombinant Ad vectors for mammalian gene transfer. Ad vectors are commonly constructed by homologous recombination between two plasmids cotransfected into 293 cells. This method has numerous advantages but results in low numbers of plaques owing to inefficient recombination. We have developed an improved method based on Cre-mediated site-specific recombination, which results in vector rescue at frequencies approximately 30-fold higher than by homologous recombination. This method should greatly simplify and expedite the construction of recombinant Ad vectors for mammalian gene transfer.     

Synthesis and Characterization of Solution Processable Poly(pyrazolines)

Solution‐processable poly(dibenzalacetone) and poly(dibenzalcyclohexanone) are prepared by the condensation of 1,4‐dialkoxy‐bisbenzaldehyde and acetone/cyclohexanone using classic Claisen–Schmidt condensation. The prepared polymers are readily soluble in common organic solvents due to the presence of alkoxy chains present on the aromatic ring. The availability of α,β‐unsaturated ketone on the backbone is readily functionalized with hydrazine hydrate to obtain poly(pyrazoline acetate), a nonconjugated polymer on the backbone, which shows enhanced emission characteristics on comparison with the pristine polymer and small molecule analogues. The highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO) of levels of poly(pyrazolines) has a band gap of 1.54 eV as calculated from cyclic voltammetry and UV–vis studies. Uniform thin film is obtained by spin casting as 1% solution on chlorobenzene and annealing under vacuum. Atomic force microscopy analysis shows fine morphology of the thin film. I–V characterization of the film shows low turn‐on voltage of 5.3 V. HOMO–LUMO is calculated by density functional theory and the result suggests that these molecules have great potential toward polymer organic light‐emitting diode applications.     

Copper nanoparticles synthesized by polyol process used to control hematophagous parasites

The present study was based on assessments of the anti-parasitic activities of the hematophagous (blood feeding) larvae of malaria vector, Anopheles subpictus Grassi, filariasis vector, Culex quinquefasciatus, Say (Diptera: Culicidae), and the larvae of cattle tick Rhipicephalus (Boophilus) microplus, Canestrini (Acari: Ixodidae). The metallic copper nanoparticles (Cu NPs) synthesized by polyol process from copper acetate as precursor and Tween 80 were used as both the medium and the stabilizing reagent. The efficacy of synthesized Cu NPs was tested against the larvae of blood-sucking parasites. UV-vis spectra characterization was performed, and peak was observed at 575 nm, which is the characteristic to the surface plasmon bond of Cu NPs. The strong surface plasmon absorption band observed at 575 nm may be due to the formation of non-oxidized Cu NPs. X-ray diffraction (XRD) spectral data showed concentric rings corresponding to the 26.79 (111), 34.52 (200), and 70.40 (220) reflections. XRD spectrum of the copper nanoparticles exhibited 2θ values corresponding to the copper nanocrystal. No peaks of impurities are observed in XRD data. The scanning electron micrograph (SEM) showed structures of irregular polygonal, cylindrical shape, and the size range was found to be 35–80 nm. The size of the Cu NPs was measured by atomic force microscope (AFM) in non-contact mode. For imaging by AFM, the sample was suspended in acetone and spins coated on a silicon wafer. The line profile image was drawn by the XEI software and the horizontal line at 6 μm on a 2D AFM image. Research has demonstrated that metallic nanoparticles produce toxicity in aquatic organisms that is due largely to effects of particulates as opposed to release of dissolved ions. Copper acetate solution tested against the parasite larvae exposed to varying concentrations and the larval mortality was observed for 24 h. The larval percent mortality observed in synthesized Cu NPs were 36, 49, 75, 93,100; 32, 53, 63, 73, and 100 and 36, 47, 69, 88, 100 at 0.5, 1.0, 2.0, 4.0, and 8.0 mg/L against A. subpictus, C. quinquefasciatus and R. microplus, respectively. The larval percent mortality shown in copper acetate solution were 16, 45, 57, 66 and 100, 37, 58, 83, 87, and 100 and 41, 59, 79, 100, and 100 at 10, 20, 30, 40, and 50 mg/L against A. subpictus, C. quinquefasciatus, and R. microplus, respectively. The maximum efficacy was observed in Cu NPs and copper acetate solution against the larvae of A. subpictus, C. quinquefasciatus, and R. microplus with LC₅₀ and r ² values of 0.95 and 23.47, 1.01 and 15.24, and 1.06 and 14.14 mg/L with r ² = 0.766; 0.957 and 0.908; 0.946; and 0.816 and 0.945, respectively. The control (distilled water) showed nil mortality in the concurrent assay. The chi-square value was significant at p ≤ 0.05 level. This is the first report on anti-parasitic activity of the synthesized Cu NPs and copper acetate solution.     

Synaptotagmin I and IV are differentially regulated in the brain by the recreational drug 3,4-methylenedioxymethamphetamine (MDMA)

3,4-Methylenedioxymethamphetamine (MDMA or Ecstasy) is a widely abused drug. In brains of mice exposed to MDMA, we recently detected altered expression of several cDNAs and genes by using the differential display polymerase chain reaction (PCR) method. Expression of one such cDNA, which exhibited 98% sequence homology with the synaptic vesicle protein synaptotagmin IV, decreased 2 h after MDMA treatment. Herein, the effect of MDMA on expression of both synaptotagmin I and IV was studied in detail, since the two proteins are functionally interrelated. PCR analyses (semi-quantitative and real-time) confirmed that upon treatment with MDMA, expression of synaptotagmin IV decreased both in the midbrain and frontal cortex of mice. Decreases in the protein levels of synaptotagmin IV were confirmed by Western immunoblotting with anti-synaptotagmin IV antibodies. In contrast, the same exposure to MDMA increased expression of synaptotagmin I in the midbrain, a region rich in serotonergic neurons, but not in the frontal cortex. This differential expression was confirmed at the protein level with anti-synaptotagmin I antibodies. MDMA did not induce down- or up-regulation of synaptotagmin IV and I, respectively, in serotonin transporter knockout mice (-/-) that are not sensitive to MDMA. Therefore, psychoactive drugs, such as MDMA, appear to modulate expression of synaptic vesicle proteins, and possibly vesicle trafficking, in the brain.     

Antiulcerogenic activity of hydroalcoholic fruit extract of Pithecellobium dulce in different experimental ulcer models in rats

Ethnopharmacological relevance

The ethnopharmacological importance of Pithecellobium dulce is evidenced by its traditional use for gastric complications. The aim of the study is to evaluate the gastroprotective activity and the mechanism of action of hydroalcoholic fruit extract of P. dulce (HAEPD) in rats by using chemical and stress induced ulcer models.

Materials and methods

Gastric ulcer was induced by administering alcohol (or) acetylsalicylic acid (or) hypothermic restraint stress to rats pretreated with HAEPD (200 mg/kg b wt for 30 day). Volume of gastric fluid, pH, acidity, activities of pepsin, H⁺, K⁺-ATPase, myeloperoxidase, mucin content, nucleic acids, glycoproteins and prostaglandin E₂ (PGE₂) levels were assessed in gastric tissues.

Results

Ulcer score was significantly minimized in HAEPD administered animals. pH and acidity of gastric fluid were significantly minimized and the mucin, PGE₂ levels were significantly maintained in drug pre administered animals. The activities of H⁺, K⁺- ATPase and myeloperoxidase were found to be significantly elevated in ulcer control animals and found to be decreased in drug pretreated animals. The cell proliferation was found to be enhanced in drug received animals. The total protein bound carbohydrate to total protein ratio was found to be significantly maintained by HAEPD. The effects were found to be comparable with that of standard drug omeprazole.

Conclusion

It is concluded that HAEPD possess a potent antiulcer activity probably by acting as cytoprotective and antiacid secretory agent.     

T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells

Immune tolerance against enteric commensal bacteria is important for preventing intestinal inflammation. Deletion of phosphoinositide-dependent protein kinase 1 (Pdk1) in T?cells via Cd4-Cre induced chronic inflammation of the intestine despite the importance of PDK1 in T?cell activation. Analysis of colonic intraepithelial lymphocytes of PDK1-deficient mice revealed markedly increased CD8α(+) T?cell receptor (TCR)γδ(+) T?cells, including an interleukin-17 (IL-17)-expressing population. TCRγδ(+) T?cells were responsible for the inflammatory colitis as shown by the fact that deletion of Tcrd abolished spontaneous colitis in the PDK1-deficient mice. This dysregulation of intestinal TCRγδ(+) T?cells was attributable to a reduction in the number and functional capacity of PDK1-deficient T regulatory (Treg) cells. Adoptive transfer of wild-type Treg cells abrogated the spontaneous activation and proliferation of intestinal TCRγδ(+) T?cells observed in PDK1-deficient mice and prevented the development of colitis. Therefore, suppression of intestinal TCRγδ(+) T?cells by Treg cells maintains enteric immune tolerance.     

Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons

Many organ systems are adversely affected by diabetes, including the brain, which undergoes changes that may increase the risk of cognitive decline. Although diabetes influences the hypothalamic-pituitary-adrenal axis, the role of this neuroendocrine system in diabetes-induced cognitive dysfunction remains unexplored. Here we demonstrate that, in both insulin-deficient rats and insulin-resistant mice, diabetes impairs hippocampus-dependent memory, perforant path synaptic plasticity and adult neurogenesis, and the adrenal steroid corticosterone contributes to these adverse effects. Rats treated with streptozocin have reduced insulin and show hyperglycemia, increased corticosterone, and impairments in hippocampal neurogenesis, synaptic plasticity and learning. Similar deficits are observed in db/db mice, which are characterized by insulin resistance, elevated corticosterone and obesity. Changes in hippocampal plasticity and function in both models are reversed when normal physiological levels of corticosterone are maintained, suggesting that cognitive impairment in diabetes may result from glucocorticoid-mediated deficits in neurogenesis and synaptic plasticity.