Modality-Specific Axonal Regeneration: Toward Selective Regenerative Neural Interfaces

Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control of robotic prosthetic devices. However, sensory and motor axons at the neural interface are of mixed sub-modality types, which difficult the specific recording from motor axons and the eliciting of precise sensory modalities through selective stimulation. Here we evaluated the possibility of using type specific neurotrophins to preferentially entice the regeneration of defined axonal populations from transected peripheral nerves into separate compartments. Segregation of mixed sensory fibers from dorsal root ganglion neurons was evaluated in vitro by compartmentalized diffusion delivery of nerve growth factor (NGF) and neurotrophin-3 (NT-3), to preferentially entice the growth of TrkA+ nociceptive and TrkC+ proprioceptive subsets of sensory neurons, respectively. The average axon length in the NGF channel increased 2.5-fold compared to that in saline or NT-3, whereas the number of branches increased threefold in the NT-3 channels. These results were confirmed using a 3D “Y”-shaped in vitro assay showing that the arm containing NGF was able to entice a fivefold increase in axonal length of unbranched fibers. To address if such segregation can be enticed in vivo, a “Y”-shaped tubing was used to allow regeneration of the transected adult rat sciatic nerve into separate compartments filled with either NFG or NT-3. A significant increase in the number of CGRP+ pain fibers were attracted toward the sural nerve, while N-52+ large-diameter axons were observed in the tibial and NT-3 compartments. This study demonstrates the guided enrichment of sensory axons in specific regenerative chambers, and supports the notion that neurotrophic factors can be used to segregate sensory and perhaps motor axons in separate peripheral interfaces

Bioluminescence Imaging for Assessment and Normalization in Transfected Cell Arrays

Transfected cell arrays (TCAs) represent a high-throughput technique to correlate gene expression with functional cell responses. Despite advances in TCAs, improvements are needed for the widespread application of this technology. We have developed a TCA that combines a two-plasmid system and dual-bioluminescence imaging to quantitatively normalize for variability in transfection and increase sensitivity. The two-plasmids consist of: (i) normalization plasmid present within each spot, and (ii) functional plasmid that varies between spots, responsible for the functional endpoint of the array. Bioluminescence imaging of dual-luciferase reporters (renilla, firefly luciferase) provides sensitive and quantitative detection of cellular response, with minimal post-transfection processing. The array was applied to quantify estrogen receptor α (ERα) activity in MCF-7 breast cancer cells. A plasmid containing an ERα-regulated promoter directing firefly luciferase expression was mixed with a normalization plasmid, complexed with cationic lipids and deposited into an array. ER induction mimicked results obtained through traditional assays methods, with estrogen inducing luciferase expression 10-fold over the antiestrogen fulvestrant or vehicle. Furthermore, the array captured a dose response to estrogen, demonstrating the sensitivity of bioluminescence quantification. This system provides a tool for basic science research, with potential application for the development of patient specific therapies

Bioluminescence Imaging for Assessment and Normalization in Transfected Cell Arrays

Transfected cell arrays (TCAs) represent a high-throughput technique to correlate gene expression with functional cell responses. Despite advances in TCAs, improvements are needed for the widespread application of this technology. We have developed a TCA that combines a two-plasmid system and dual-bioluminescence imaging to quantitatively normalize for variability in transfection and increase sensitivity. The two-plasmids consist of: (i) normalization plasmid present within each spot, and (ii) functional plasmid that varies between spots, responsible for the functional endpoint of the array. Bioluminescence imaging of dual-luciferase reporters (renilla, firefly luciferase) provides sensitive and quantitative detection of cellular response, with minimal post-transfection processing. The array was applied to quantify estrogen receptor α (ERα) activity in MCF-7 breast cancer cells. A plasmid containing an ERα-regulated promoter directing firefly luciferase expression was mixed with a normalization plasmid, complexed with cationic lipids and deposited into an array. ER induction mimicked results obtained through traditional assays methods, with estrogen inducing luciferase expression 10-fold over the antiestrogen fulvestrant or vehicle. Furthermore, the array captured a dose response to estrogen, demonstrating the sensitivity of bioluminescence quantification. This system provides a tool for basic science research, with potential application for the development of patient specific therapies

Controlled surface-associated delivery of genes and oligonucleotides

A system and methods for controlled gene delivery comprising condensed nucleic acids complexed with polylinkers, wherein the complexes are covalently and/or noncovalently bound to the surface of a substrate capable of supporting cell adhesion. The gene delivery system achieves temporal and spatial control of nucleic acid delivery to a target cell or cells through control of complex density on the surface of the support substrate, and reversibility of the attachment of the polylinker to the support substrate. The system and method of the invention can be used to create spatial patterns of gene expression, and in tissue engineering, high throughput screening, and gene therapy applications. What is claimed is: 1. A method for increasing transgene expression, comprising making a controlled nucleic acid delivery system, said system comprising forming nucleic acid polylinker complexes capable of being delivered to cells cultured on a support substrate, wherein said complexes are formed prior to being covalently or non-covalently immobilized to the surface of a support substrate, and wherein said method comprises: a) contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex, said complex being formed prior to attachment to a support substrate; and b) immobilizing the nucleic acid-polylinker complex to a support substrate; and wherein said cells are added to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate. 2. The method of claim 1, further comprising modification of the support substrate with serum prior to addition of the nucleic acid-polylinker complex, and wherein said modification allows for an increase in transgene expression. 3. The method of claim 1, wherein the extent of transgene expression is dependent upon substrate modification and complex formation. 4. The method of claim 1, wherein said nucleic acid polylinker complexes are polyplexes or lipoplexes. 5. The method of claim 1, wherein said support substrate is polystyrene, gold, hyaluronic acid collagen hydrogels or polylactide-co-glycolide (PLG). 6. The method of claim 2, wherein said substrate modification is made by treatment with serum. 7. The method of claim 1, wherein delivery of the nucleic acid-polylinker complexes to cells occurs from a polystyrene surface treated with serum, and wherein said delivery results in similar or greater percentage of transfected cells relative to bolus delivery. 8. The method of claim 1, said method further comprising release of the nucleic acid from the nucleic acid-polylinker complexes, wherein said release is maximized when the support substrate is treated with serum. 9. A method for increasing transgene expression, comprising making a controlled nucleic acid delivery system, said system comprising forming nucleic acid polylinker complexes capable of being delivered to cells cultured on a support substrate, wherein said complexes are covalently or non-covalently immobilized to the surface of a support substrate, and wherein said method comprises: a) contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; and c) adding cells to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein the release of nucleic acid from the nucleic acid-polylinker complexes is further enhanced when the support substrate containing the complexes is treated with serum or is incubated in conditioned medium. 10. The method of claim 2, wherein the delivery of the nucleic acid-polylinker complexes to cells from a serum-modified support substrate results in higher cellular association of the nucleic acid-polylinker complexes with the support substrate. 11. A method for increasing transgene expression, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex, wherein said complex is formed prior to addition to a support substrate; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate. 12. The method of claim 11, wherein said support substrate comprises a biodegradable or non-biodegradable material. 13. The method of either one of claim 1 or 11, wherein said complexes are formed prior to attachment to the solid support substrate. 14. The method of claim 12, wherein said biodegradable material is a hydrogel and said non-biodegradable material is polystyrene or gold. 15. The method of claim 12, wherein said hydrogel comprises a mixture of hyaluronic acid and collagen. 16. A method for increasing transgene expression, wherein said method promotes transfection of primary cells, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein the biodegradable material is a hydrogel and the non-biodegradable material is polystyrene or gold, and wherein the hvdrogel comprises a mixture of hyaluronic acid and collagen. 17. The method of either one of claim 1 or 11, wherein said nucleic acid polylinker complexes are immobilized to the support substrate using biotin and avidin, or an avidin derivative, or by non-specific adsorption. 18. The method of claim 17, wherein said avidin derivative is streptavidin or neutravidin. 19. The method of any one of claim 11–15, wherein the method further comprises controlling the size of the nucleic acid polylinker complex by regulating the salt content during complex formation. 20. The method of claim 19, wherein controlling the size of said complex formation is accomplished by the presence or absence of salt during the formation of the complexes, wherein the forming of large diameter complexes in the presence of salt results in increased transgene expression, and wherein the forming of small diameter complexes in the absence of salt results in a greater percentage of cells being transfected. 21. The method of claim 19, wherein the salt is sodium chloride. 22. A method for increasing transgene expression, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein said method further comprises release of the nucleic acid from the substrate, wherein the release is optimized by using conditioned medium. 23. The method of claim 11, wherein said method further comprises biotinylation of said complex to enhance release of said complex from said substrate. 24. The method of either of claim 1 or 11, wherein the nucleic acid is DNA, RNA or an oligonucleotide. 25. The method of claim 24, wherein said oligonucleotide is an antisense oligonucleotide or a catalytic RNA capable of interfering with the expression of a gene. 26. The controlled nucleic acid delivery system of either of claim 1 or 11, wherein the polylinker is a cationic polymer, cationic lipid, cationic protein, or cationic peptide

Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases

Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer

The Membrane Fusion Step of Vaccinia Virus Entry Is Cooperatively Mediated by Multiple Viral Proteins and Host Cell Components

For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry

A Kinome RNAi Screen Identified AMPK as Promoting Poxvirus Entry through the Control of Actin Dynamics

Poxviruses include medically important human pathogens, yet little is known about the specific cellular factors essential for their replication. To identify genes essential for poxvirus infection, we used high-throughput RNA interference to screen the Drosophila kinome for factors required for vaccinia infection. We identified seven genes including the three subunits of AMPK as promoting vaccinia infection. AMPK not only facilitated infection in insect cells, but also in mammalian cells. Moreover, we found that AMPK is required for macropinocytosis, a major endocytic entry pathway for vaccinia. Furthermore, we show that AMPK contributes to other virus-independent actin-dependent processes including lamellipodia formation and wound healing, independent of the known AMPK activators LKB1 and CaMKK. Therefore, AMPK plays a highly conserved role in poxvirus infection and actin dynamics independent of its role as an energy regulator

The Role of Macroeconomic Fundamentals in Malaysian Post Recession Growth

This study aims to find out the role of macroeconomic fundamentals in Malaysian post recession growth. The selected macroeconomic variables are exports, imports, price level, money supply, interest rate, exchange rate and government expenditure. The technique of cointegration was employed to assess the long run equilibrium relationships among the variables. Then, this study performs the Granger causality tests based on VECM to establish the short run causality among the variables. The long-run cointegrating relationship shown that an increase in exports, government expenditure or depreciation of exchange rate will promote long-term economic growth while increase in inflation, interest rate and imports will tamper the Malaysian economic growth. The results of short-run Granger-causality indicated that price level and government spending Granger-caused economic growth in the short-run. In conclusion, based on the results of long-run and short run analysis, the fiscal policy is probably the most appropriate tool in promoting economic growth in Malaysia during the post recession period