James Bradley Summers

Design of a nonviral vector for site-selective, efficient integration into the human genome

Gene therapy in eukaryotes has met many obstacles. Research into the design of suitable nonviral vectors has been slow. To our knowledge, no nonviral vector has been proposed that allows for the possibility of highly efficient, site‐selective integration into the genome of mammalian cells. On the basis of prior studies investigating the components necessary for transposon, retrovirus‐like retrotransposon, and retroviral integration, we propose a nonviral system that would potentially allow for site‐selective, efficient integration into the mammalian genome. Transposons have been developed that can transform a variety of cell lines. For example, the Sleeping Beauty transposon (SB) can transform a wide range of vertebrate cells from fish to human, and it mediates stable integration and long‐term transgene expression in mice. However, the efficiency of transposition varies significantly among cell lines, suggesting the possible involvement of host factors in SB transposition. Here, we propose the use of a chimeric transposase (i.e., transposase‐host DNA binding domain) to bypass the potential requirement of a host DNA‐directing factor (or factors) for efficient, site‐selective integration. We also discuss another potential method of docking the transposonbased vector adjacent to the host DNA, utilizing repetitive sequences for homologous recombination to promote efficient site‐selective integration, as well as other site‐selective nonviral approaches.—Kaminski, J. M., Huber, M. R., Summers, J. B., Ward, M. B. Design of a nonviral vector for site‐selective, efficient integration into the human genome. FASEB J. 16, 1242–1247 (2002)

Immunotherapy and prostate cancer.

Prostate cancer is the second leading cause of cancer death in the US, largely because of the limitations of our current therapeutic options, especially once the cancer has metastasized. Investigators have long sought new therapeutic modalities such as angiogenesis inhibitors, vaccines, and gene therapy, among others. It appears that a combination approach will be required to cure the majority of malignancies. Immunotherapy for prostate cancer appears feasible and a likely therapeutic modality in the armamentarium. Unfortunately, further research in basic immunology and the interaction of the immune system with other forms of therapy is needed. Many obstacles exist in immunotherapy, including vector design, tumouricidal specificity, and tumor evasion, which will have to be overcome in order to realize the maximum therapeutic benefit from this treatment modality.

TSP-1 secreted by bone marrow stromal cells contributes to retinal ganglion cell neurite outgrowth and survival.

Background Bone marrow stromal cells (BMSCs) are pluripotent and thereby a potential candidate for cell replacement therapy for central nervous system degenerative disorders and traumatic injury. However, the mechanism of their differentiation and effect on neural tissues has not been fully elucidated. This study evaluates the effect of BMSCs on neural cell growth and survival in a retinal ganglion cell (RGCs) model by assessing the effect of changes in the expression of a BMSC-secreted protein, thrombospondin-1 (TSP-1), as a putative mechanistic agent acting on RGCs. Methods and Findings The effect of co-culturing BMSCs and RGCs in vitro was evaluated by measuring the following parameters: neurite outgrowth, RGC survival, BMSC neural-like differentiation, and the effect of TSP-1 on both cell lines under basal secretion conditions and when TSP-1 expression was inhibited. Our data show that BMSCs improved RGC survival and neurite outgrowth. Synaptophysin, MAP-2, and TGF-β expression are up-regulated in RGCs co-cultured with BMSCs. Interestingly, the BMSCs progressively displayed neural-like morphology over the seven-day study period. Restriction display polymerase chain reaction (RD-PCR) was performed to screen for differentially expressed genes in BMSCs cultured alone or co-cultured with RGCs. TSP-1, a multifactorial extracellular matrix protein, is critically important in the formation of neural connections during development, so its function in our co-culture model was investigated by small interfering RNA (siRNA) transfection. When TSP-1 expression was decreased with siRNA silencing, BMSCs had no impact on RGC survival, but reduced neurite outgrowth and decreased expression of synaptophysin, MAP-2 and TGF-β in RGCs. Furthermore, the number of BMSCs with neural-like characteristics was significantly decreased by more than two-fold using siRNA silencing. Conclusions Our data suggest that the TSP-1 signaling pathway might have an important role in neural-like differentiation in BMSCs and neurite outgrowth in RGCs. This study provides new insights into the potential reparative mechanisms of neural cell repair.

Lithium chloride protects retinal neurocytes from nutrient deprivation by promoting DNA non-homologous end-joining

Lithium chloride is a therapeutic agent for treatment of bipolar affective disorders. Increasing numbers of studies have indicated that lithium has neuroprotective effects. However, the molecular mechanisms underlying the actions of lithium have not been fully elucidated. This study aimed to investigate whether lithium chloride produces neuroprotective function by improving DNA repair pathway in retinal neurocyte. In vitro, the primary cultured retinal neurocytes (85.7% are MAP-2 positive cells) were treated with lithium chloride, then cultured with serum-free media to simulate the nutrient deprived state resulting from ischemic insult. The neurite outgrowth of the cultured cells increased significantly in a dose-dependent manner when exposed to different levels of lithium chloride. Genomic DNA electrophoresis demonstrated greater DNA integrity of retinal neurocytes when treated with lithium chloride as compared to the control. Moreover, mRNA and protein levels of Ligase IV (involved in DNA non-homologous end-joining (NHEJ) pathway) in retinal neurocytes increased with lithium chloride. The end joining activity assay was performed to determine the role of lithium on NHEJ in the presence of extract from retinal neurocytes. The rejoining levels in retinal neurocytes treated with lithium were significantly increased as compared to the control. Furthermore, XRCC4, the Ligase IV partner, and the transcriptional factor, CREB and CTCF, were up-regulated in retinal cells after treating with 1.0mM lithium chloride. Therefore, our data suggest that lithium chloride protects the retinal neural cells from nutrient deprivation in vitro, which may be similar to the mechanism of cell death in glaucoma. The improvement in DNA repair pathway involving in Ligase IV might have an important role in lithium neuroprotection. This study provides new insights into the neural protective mechanisms of lithium chloride.

Properties of Concrete Incorporating Ultrafine Fly Ash and Silica Fume

A laboratory study on the influence of the combination of ultrafine fly ash (UFFA) and silica fume (SF) on the properties of fresh and hardened concrete is described. Also compared are the performance of concrete incorporating UFFA and SF (ternary blend of cement), concrete incorporating UFFA or SF (binary blend of cement), and control portland cement concrete. The test results show that the incorporation of SF or UFFA in concrete resulted in higher strength and improved durability (resistance to chloride penetration). These benefits were found to be more pronounced in the SF concrete. However, the SF concrete demonstrated several limitations such as low slump and high early-age shrinkage. These limitations were not observed in the UFFA concrete; addition of UFFA increased the slump and decreased the early-age shrinkage. To minimize the shortcomings of SF without losing its strength and durability benefits, a concrete mixture incorporating both SF and UFFA was prepared. The test results show that the incorporation of both SF and UFFA produces a concrete mixture that demonstrates high early-age strength and improved durability similar to those properties in SF concrete. In addition, unlike SF concrete, the new concrete mixture demonstrates a higher level of slump and a lower level of free shrinkage.

1100. Site-Specific Transposon Integration

Purpose/Objective: We are developing a novel platform technology that utilizes a chimeric transposase to direct integration into specific sites in the host DNA. Thus, this technology minimizes non-specific integration that may result in insertional mutagenesis which can induce cancer and may disrupt genes that affect the fitness of the organism unrelated to cancer.

Comment: Publishing Ethics — Duplication Indiscretion

mation of prostanoids (i.e., prostaglandin E2, thromboxane A2) involved in many pathophysiologic processes.4 Prostaglandins inhibit the contraction of gastrointestinal smooth muscle, and Waldum’s hypothesis regarding the effect of COX-2 inhibitors should help explain the evidence of cholestasis and pancreatitis observed respectively in our case and in the case reported by Carrillo-Jimenez and Nurnberger after administration of celecoxib. However, today, only research, rather than clinical experience, seems to support such an effect of celecoxib on the smooth muscle of the gastrointestinal system.5