3C assay of DNA looping.

<p>Hind III restriction enzyme spliced at -5,274 and -631 bp in the hiNOS promoter. The gel of the 3C assay shown is representative of three experiments.</p

In vitro and In vivo analysis of AP-1 binding sites in the hiNOS enhancer.

<p>(A) Mutagenesis analysis of AP-1 sites at -5.1 kb downstream (Pr8-1d) or -5.3 kb upstream (Pr8-1u) in the hiNOS promoter. Mutant construct for each site or double AP-1 mutant (1u+1d) were generated in the hiNOS Pr8 promoter luciferase reporter plasmid driven by pCMV promoter. Wild-type hiNOS promoter luciferase reporter plasmid served as control. * Indicates p <0.05 vs. p300. (B) ChIP analysis of AP-1 binding sites in the hiNOS enhancer with various antibodies. * Indicates p <0.05 vs. Ig G.</p

ChIP-Loop assay. Gel assay for AP-1, RNA pol II, and p300 binding.

<p>Schematic representation of AP-1 sites in the human iNOS promoter with relevant target sequences for Hind III restriction endonucleases and location of PCR primers. Ig G serves as negative control. Upper lane: without p300 siRNA treatment; Lower lane: with p300 siRNA treatment, p300 antibody with scrambled control siRNA serves as positive control. Gel assay shown is representative of three similar experiments.</p

Tumor-Penetrating Peptide-Functionalized Redox-Responsive Hyperbranched Poly(amido amine) Delivering siRNA for Lung Cancer Therapy

Biosafety and the targeting ability of gene delivery systems are critical aspects for gene therapy of cancer. In this study, we report the synthesis and use of redox-responsive poly­(amido amine) (PAA) with good biocompatibility and biodegradation as a gene carrier material. A tumor-specific tissue penetration peptide, internalizing-RGD (iRGD) was then conjugated to PAA with an amidation reaction. In experiments using H1299 cells, PAA-iRGD was found to have a lower cytotoxicity and higher cellular uptake efficiency compared to PAA. An siRNA, specific to epidermal growth factor receptor (EGFR) that is overexpressed on the lung cancer cell surface and often targeted in lung cancer treatment, was designed to silence EGFR (i.e., siEGFR) for delivery by the gene carrier PAA-iRGD. <i>EGFR</i> gene silencing, apoptosis, antiproliferation, and antitumor effects of PAA-iRGD/siEGFR were evaluated <i>in vitro</i> and <i>in vivo</i>. PAA-iRGD/siEGFR displayed a much higher gene silencing ability compared to PAA and polyethylenimine (25 kDa), significantly inhibited the proliferation and migration of H1299 cells, and elicited significant cell apoptosis. Moreover, intravenously injected PAA-iRGD/siEGFR inhibited lung tumor growth <i>in vivo</i>. These results suggest that PAA-iRGD with good biocompatibility, biodegradation, and targeting ability could be a promising gene delivery system for gene therapy of cancers

p300 mediated transactivation of the hiNOS or heterologous promoter.

<p>(A) The –7.2 kb wild-type (WT) human iNOS promoter construct (Pr8) or deleted -5 to -6 kb enhancer region (Pr8-Del), were co-transfected into A549 cells with p300 expression vector. Basal and stimulated luciferase activities were determined 6 hours after cytokine mix (CM) stimulation. Relative luciferase activities (RLA) values are the means ± sd of at least three separate experiments performed in triplicate. *Indicates <i>P</i> < 0.05 <i>vs</i>. basal, # indicates P < 0.05 vs. control (B) The minimal TK promoter construct with ligated hiNOS enhancer was co-transfected into A549 cells with p300 expression vector. Co-transfection with empty vector served as control. Basal and stimulated luciferase activity was determined 6 hr after cytokine stimulation. Values shown are the means ± sd of at least three separate experiments performed in triplicate. *Indicates P < 0.05 vs. basal, # indicates P < 0.05 vs. control.</p

Redox-Responsive Biodegradable Polycation Poly(amido amine) Used As Intranasal Vaccine Delivery Systems

Polycations such as polyethylenimine and chitosan have been widely used as mucosal vaccine delivery systems due to their permeation enhancement effect. Preferably, environmentally responding biodegradable polycations would be better carrier materials for mucosal vaccine delivery. Disulfide bond-based redox-sensitive polycations could respond to the higher intracellular glutathione concentration and degrade in the cytoplasm via the breakage of the disulfide bonds, which are particularly suitable for antigen delivery. In this work, we evaluated the potential of redox-sensitive, biodegradable polycation poly­(amido amine) (PAA) as mucosal vaccine carriers. From the primary studies with ovalbumin used as a model protein antigen, it is found that PAA could complex with and encapsulate protein antigen via electrostatic attraction, enhance the cellular uptake of antigen by dendritic cell line DC2.4, prolong antigen residence in nasal cavity, and promote antigen permeation into nasal submucosal layer. Further, Balb/c mice were intranasally immunized with PAA-delivered recombinant hemagglutinin (HA) antigen protein of H7N9 influenza virus. The PAA/HA formulations induced significantly more potent systemic IgG response and mucosal IgA response, higher splenocyte proliferation activity, higher secretion levels of cytokines IFN-γ and IL-4 by splenocytes, more memory CD4⁺ and CD8⁺ T cells, and more DCs expressing MHC II molecule. From the results, the redox-responsive polycation PAA as vaccine carriers helped elicit more potent cellular and humoral immune responses. Particularly, PAA induced much higher cellular immune response compared with previously reported carrier materials. The intelligent PAA could be developed as efficient mucosal vaccine delivery systems for clinical applications

Blood Compatibility Evaluations of Fluorescent Carbon Dots

Because of their unique advantages, fluorescent carbon dots are gaining popularity in various biomedical applications. For these applications, good biosafety is a prerequisite for their use in vivo. Studies have reported the preliminary biocompatibility evaluations of fluorescent carbon dots (mainly cytotoxicity); however, to date, little information is available about their hemocompatibility, which could impede their development from laboratory to bedside. In this work, we evaluated the hemocompatibility of fluorescent carbon dots, which we prepared by hydrothermal carbonization of α-cyclodextrin. The effects of the carbon dots on the structure and function of key blood components were investigated at cellular and molecular levels. In particular, we considered the morphology and lysis of human red blood cells, the structure and conformation of the plasma protein fibrinogen, the complement activation, platelet activation, and in vitro and in vivo blood coagulation. We found that the carbon dots have obvious concentration-dependent effects on the blood components. Overall, concentrations of the fluorescent carbon dots at ≤0.1 mg/mL had few adverse effects on the blood components, but at higher doses, the carbon dots impair the structure and function of the blood components, causing morphological disruptions and lysis of red blood cells, interference in the local microenvironments of fibrinogen, activation of the complement system, and disturbances in the plasma and whole blood coagulation function in vitro. However, the carbon dots tend to activate platelets only at low concentrations. Intravenous administration of the carbon dots at doses up to 50 mg/kg did not impair the blood coagulation function. These results provide valuable information for the clinical application of fluorescent carbon dots

Polyethylenimine-Modified Fluorescent Carbon Dots As Vaccine Delivery System for Intranasal Immunization

Fluorescent carbon dots (CDs) as a luminescent nanomaterial have obtained much attention in the biomedical field. To make good use of their luminescent property and nanoscaled size, we developed CDs as a vaccine delivery system for intranasal immunization in this work. To this aim, polyethylenimine-modified CDs were prepared via a simple microwave method. Intranasal immunization was performed by using the CDs as an antigen carrier to deliver model protein antigen ovalbumin. The results showed that the CDs as an intranasal vaccine delivery system enhanced the immunization efficacy by significantly increasing IgG titer, IgA induction in the local and distant mucous membrane sites, splenocyte proliferation, cytokine IFN-γ secretion by splenocytes, and memory T cells. From the results, the CDs could be used as vaccine delivery systems with the advantage of tracing the antigen transportation from administration site to the lymph organs

Recombinant HOPS can support vacuole fusion.

<p>Vacuoles were purified from each of the two tester strain (<i>pep4</i>∆ and <i>pho8</i>∆) that each carry the <i>vps11-1</i> mutation (Stroupe [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081534#B29" target="_blank">29</a>]and Wickner, 2006). Fusion reactions (30 µl total) contained 3 µg of each vacuole type in fusion salt (0.5 mM MgCl₂, 125 mM KCl, 20 mM PIPES/KOH, pH 6.8, 200 mM sorbitol) and an ATP-regenerating system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081534#B14" target="_blank">14</a>]. (A) The indicated volumes of each fraction of the HOPS purification were titrated into the fusion reaction. Reactions were incubated for 90 min at 26°C, and then assayed for Pho8 activity by addition of p-Nitrophenolphosphate [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081534#B14" target="_blank">14</a>]. As a control, purified yeast HOPS complex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081534#B16" target="_blank">16</a>] was added at the indicated concentrations. (B) Titration of fraction 9 at higher volumes into the fusion reaction. The analysis was conducted as in (A). Control indicates the addition of volumes of purification buffer into the reaction. </p

<i>In</i><i>vitro</i> assembly of subcomplexes of HOPS and CORVET complexes.

<p>(A) An example of subcomplex containing Vps11, Vps16, GFP-Vps18 and Vps33 co-expressed in LTE extract and isolated on the anti-GFP matrix. (B) Graphic summary of subcomplexes isolated in this study. (C) Fluorescence scan of the SDS-PAGE gel loaded with <i>in </i><i>vitro</i> reconstituted and affinity purified HOPS complex. The positions of the individual subunits are indicated on the right hand side. (D) Coomassie stained SDS-PAGE loaded with the HOPS containing fraction eluted from the gel filtration column. (E) Identification of the complex forming domains in the Vps subunits of HOPS and CORVET complex. GFP-tagged Vps11 was co-expressed with the truncated variants of Vps39 subunit. The samples were processed as in A. </p