Human AdV-20-42-42, a promising novel adenoviral vector for gene therapy and vaccine product development

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination

Evidence for the Complexity of MicroRNA-Mediated Regulation in Ovarian Cancer: A Systems Approach

MicroRNAs (miRNAs) are short (∼22 nucleotides) regulatory RNAs that can modulate gene expression and are aberrantly expressed in many diseases including cancer. Previous studies have shown that miRNAs inhibit the translation and facilitate the degradation of their targeted messenger RNAs (mRNAs) making them attractive candidates for use in cancer therapy. However, the potential clinical utility of miRNAs in cancer therapy rests heavily upon our ability to understand and accurately predict the consequences of fluctuations in levels of miRNAs within the context of complex tumor cells. To evaluate the predictive power of current models, levels of miRNAs and their targeted mRNAs were measured in laser captured micro-dissected (LCM) ovarian cancer epithelial cells (CEPI) and compared with levels present in ovarian surface epithelial cells (OSE). We found that the predicted inverse correlation between changes in levels of miRNAs and levels of their mRNA targets held for only ∼11% of predicted target mRNAs. We demonstrate that this low inverse correlation between changes in levels of miRNAs and their target mRNAs in vivo is not merely an artifact of inaccurate miRNA target predictions but the likely consequence of indirect cellular processes that modulate the regulatory effects of miRNAs in vivo. Our findings underscore the complexities of miRNA-mediated regulation in vivo and the necessity of understanding the basis of these complexities in cancer cells before the therapeutic potential of miRNAs can be fully realized

Protandim, a Fundamentally New Antioxidant Approach in Chemoprevention Using Mouse Two-Stage Skin Carcinogenesis as a Model

Oxidative stress is an important contributor to cancer development. Consistent with that, antioxidant enzymes have been demonstrated to suppress tumorigenesis when being elevated both in vitro and in vivo, making induction of these enzymes a more potent approach for cancer prevention. Protandim, a well-defined combination of widely studied medicinal plants, has been shown to induce superoxide dismutase (SOD) and catalase activities and reduce superoxide generation and lipid peroxidation in healthy human subjects. To investigate whether Protandim can suppress tumor formation by a dietary approach, a two-stage mouse skin carcinogenesis study was performed. At the end of the study, the mice on a Protandim-containing basal diet had similar body weight compared with those on the basal diet, which indicated no overt toxicity by Protandim. After three weeks on the diets, there was a significant increase in the expression levels of SOD and catalase, in addition to the increases in SOD activities. Importantly, at the end of the carcinogenesis study, both skin tumor incidence and multiplicity were reduced in the mice on the Protandim diet by 33% and 57% respectively, compared with those on basal diet. Biochemical and histological studies revealed that the Protandim diet suppressed tumor promoter-induced oxidative stress (evidenced by reduction of protein carbonyl levels), cell proliferation (evidenced by reduction of skin hyperplasia and suppression of PKC/JNK/Jun pathway), and inflammation (evidenced by reduction of ICAM-1/VCAM-1 expression, NF-κB binding activity, and nuclear p65/p50 levels). Overall, induction of antioxidant enzymes by Protandim may serve as a practical and potent approach for cancer prevention

Imaging Single Retrovirus Entry through Alternative Receptor Isoforms and Intermediates of Virus-Endosome Fusion

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes

Optimized cross-layer forward error correction coding for H.264 AVC video transmission over wireless channels

Forward error correction (FEC) codes that can provide unequal error protection (UEP) have been used recently for video transmission over wireless channels. These video transmission schemes may also benefit from the use of FEC codes both at the application layer (AL) and the physical layer (PL). However, the interaction and optimal setup of UEP FEC codes at the AL and the PL have not been previously investigated. In this paper, we study the cross-layer design of FEC codes at both layers for H.264 video transmission over wireless channels. In our scheme, UEP Luby transform codes are employed at the AL and rate-compatible punctured convolutional codes at the PL. In the proposed scheme, video slices are first prioritized based on their contribution to video quality. Next, we investigate the four combinations of cross-layer FEC schemes at both layers and concurrently optimize their parameters to minimize the video distortion and maximize the peak signal-to-noise ratio. We evaluate the performance of these schemes on four test H.264 video streams and show the superiority of optimized cross-layer FEC design.Peer reviewedElectrical and Computer Engineerin