Axonal-Transport-Mediated Gene Transduction in the Interior of Rat Bone

BACKGROUND: Gene transduction has been considered advantageous for the sustained delivery of proteins to specific target tissues. However, in the case of hard tissues, such as bone, local gene delivery remains problematic owing to anatomical accessibility limitations of the target sites. METHODOLOGY/PRINCIPAL FINDINGS: Here, we evaluated the feasibility of exogenous gene transduction in the interior of bone via axonal transport following intramuscular administration of a nonviral vector. A high expression level of the transduced gene was achieved in the tibia ipsilateral to the injected tibialis anterior muscle, as well as in the ipsilateral sciatic nerve and dorsal root ganglia. In sciatic transection rats, the gene expression level was significantly lowered in bone. CONCLUSIONS/SIGNIFICANCE: These results suggest that axonal transport is critical for gene transduction. Our study may provide a basis for developing therapeutic methods for efficient gene delivery into hard tissues

Spintronics: Fundamentals and applications

Spintronics, or spin electronics, involves the study of active control and manipulation of spin degrees of freedom in solid-state systems. This article reviews the current status of this subject, including both recent advances and well-established results. The primary focus is on the basic physical principles underlying the generation of carrier spin polarization, spin dynamics, and spin-polarized transport in semiconductors and metals. Spin transport differs from charge transport in that spin is a nonconserved quantity in solids due to spin-orbit and hyperfine coupling. The authors discuss in detail spin decoherence mechanisms in metals and semiconductors. Various theories of spin injection and spin-polarized transport are applied to hybrid structures relevant to spin-based devices and fundamental studies of materials properties. Experimental work is reviewed with the emphasis on projected applications, in which external electric and magnetic fields and illumination by light will be used to control spin and charge dynamics to create new functionalities not feasible or ineffective with conventional electronics.Comment: invited review, 36 figures, 900+ references; minor stylistic changes from the published versio

Proteomic analysis of the lung in rats with hypobaric hypoxia-induced pulmonary hypertension

Experimental pulmonary hypertension that develops in hypobaric hypoxia is characterized by structural remodeling of the lung. Proteomics - which may be the most powerful way to uncover unknown remodeling proteins involved in enhancing cardiovascular performance - was used to study 150 male Wistar rats housed for up to 21 days in a chamber at the equivalent of 5500 m altitude level. After 14 days’ exposure to hypobaric hypoxia, pulmonary arterial pressure (PAP) was significantly increased. In lung tissue, about 140 matching protein spots were found among 8 groups (divided according to their hypobaric period) by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (pH4.5-pH6.5, 30 kDa-100 kDa). In hypobaric rats, three spots were increased two-fold or more (vs. control rats) in two-dimensional differential in-gel electrophoresis (2D-DIGE). The increased proteins were identified, by matrix-assisted laser desorption ionization time of flight (MALDI-TOF), as one isoform of heat shock protein 70 (HSP70) and two isoforms of protein disulfide isomerase associated 3. This result was confirmed by Western blotting analysis of 2D-PAGE. Conceivably, HSP70 and PDIA3 may play roles in modulating the lung structural remodeling that occurs due to pulmonary hypertension in hypobaric hypoxia

Therapeutic Effect of Exendin-4, a Long-Acting Analogue of Glucagon-Like Peptide-1 Receptor Agonist, on Nerve Regeneration after the Crush Nerve Injury

Glucagon-like peptide-1 (GLP-1) is glucose-dependent insulinotropic hormone secreted from enteroendocrine L cells. Its long-acting analogue, exendin-4, is equipotent to GLP-1 and is used to treat type 2 diabetes mellitus. In addition, exendin-4 has effects on the central and peripheral nervous system. In this study, we administered repeated intraperitoneal (i.p.) injections of exendin-4 to examine whether exendin-4 is able to facilitate the recovery after the crush nerve injury. Exendin-4 injection was started immediately after crush injury and was repeated every day for subsequent 14 days. Rats subjected to sciatic nerve crush exhibited marked functional loss, electrophysiological dysfunction, and atrophy of the tibialis anterior muscle (TA). All these changes, except for the atrophy of TA, were improved significantly by the administration of exendin-4. Functional, electrophysiological, and morphological parameters indicated significant enhancement of nerve regeneration 4 weeks after nerve crush. These results suggest that exendin-4 is feasible for clinical application to treat peripheral nerve injury

Gene expression in the interior of the bone by intramuscular administration of an HVJ-envelope-DNA complex.

<p>(A) Experimental design of gene transfer. A luciferase/HVJ-E vector (100 µl) containing 100 µg luciferase plasmid DNA was carefully injected percutaneously into the proximal one-third of the tibialis anterior muscle of the right hindlimb. The first gene administration was on day 0, and the second on day 7. Samples were harvested on days 1, 3, 5, 7, 10, and 12 after the first administration of a luciferase/HVJ-E vector (each group: n = 7). Luciferase activity was measured using a luciferase assay system. Pictures show tibia with tibialis anterior muscle (arrow) and tibia from which the periosteum was thoroughly stripped to avoid any contamination with muscles. (B) Relative luciferase activity (RLU/mg protein) in the ipsilateral tibia (without periosteum), and bone marrow of the ipsilateral tibia. Error bars: SEM. Each group: n = 7. The dotted lines indicate the average levels of control samples, which were harvested at each time point after PBS injection (n = 6). Error bars: SEM. *p<0.05. (C) Relative luciferase activity (RLU/mg protein) in the tibia (without periosteum) on day 3 after gene transfer. Black bar: the activity in the contralateral tibia (n = 7), gray bar: the activity in the ipsilateral tibia (n = 7), white bar: the activity in the tibia after injection of PBS as a control (n = 6). Error bars: SEM. *p<0.05. (D) Comparison of luciferase activities of the ipsilateral tibia between single and repeated gene transfers on days 10 and 12. When luciferase activity in the ipsilateral tibia without a second gene transfer is considered as 100%, its activity in the ipsilateral tibia with second gene transfer on day 7 is shown as a percentage. Black bars: rats with the second gene transfer on day 7, white bars: rats without a second gene transfer. Error bars: SEM. Each group: n = 7. *p<0.05.</p

Expression of luciferase mRNA in the bone.

<p>(A) RT-PCR was performed with a primer specific for firefly luciferase (product length, 261 bp). Luciferase mRNA was detected in the rat RNA extracted from the femur as well as the tibia. (B) Using four rats, RT-PCR for luciferase mRNA was performed 4 times. Quantification was performed using Image J software (n = 4). Error bars: SEM. (C) To ensure that there was no contamination of tibialis anterior muscle in any of the bone samples, RT-PCR was performed using all the above samples and a primer specific for MHC IIb (product length, 197 bp). MHC IIb mRNA was not detected in the ipsilateral femora, ipsilateral tibiae, or bone marrows from ipsilateral femora and tibiae (n = 4).</p

Possible mechanisms of gene transduction via axonal transport.

<p>If axons innervating both bone and muscle exist, gene expression could occur within the bone via axonal transport. The plasmid vector or its gene product could be transported retrogradely to the point of division of an axon. Next, we speculate two pathways of the plasmid vector or its gene product. In one pathway, a plasmid vector might be transported to DRG by fast retrograde axonal transport, and transcribed in DRG. Then, its gene product would be conveyed to peripheral sites by fast antegrade axonal transport. In the other pathway, a plasmid vector is conveyed to the other branch of the axon at its division point, and then reaches the bone. The right column shows the denervation of sciatic nerves after transection. Scissors show cut points. In this situation, no antegrade or retrograde axonal flow exists.</p