Lentiviral Vector-Mediated Gradients of GDNF in the Injured Peripheral Nerve: Effects on Nerve Coil Formation,Schwann Cell Maturation and Myelination

Although the peripheral nerve is capable of regeneration, only a small minority of patients regain normal function after surgical reconstruction of a major peripheral nerve lesion, resulting in a severe and lasting negative impact on the quality of life. Glial cell-line derived neurotrophic factor (GDNF) has potent survival- and outgrowth-promoting effects on motoneurons, but locally elevated levels of GDNF cause trapping of regenerating axons and the formation of nerve coils. This phenomenon has been called the “candy store” effect. In this study we created gradients of GDNF in the sciatic nerve after a ventral root avulsion. This approach also allowed us to study the effect of increasing concentrations of GDNF on Schwann cell proliferation and morphology in the injured peripheral nerve. We demonstrate that lentiviral vectors can be used to create a 4 cm long GDNF gradient in the intact and lesioned rat sciatic nerve. Nerve coils were formed throughout the gradient and the number and size of the nerve coils increased with increasing GDNF levels in the nerve. In the nerve coils, Schwann cell density is increased, their morphology is disrupted and myelination of axons is severely impaired. The total number of regenerated and surviving motoneurons is not enhanced after the distal application of a GDNF gradient, but increased sprouting does result in higher number of motor axon in the distal segment of the sciatic nerve. These results show that lentiviral vector mediated overexpression of GDNF exerts multiple effects on both Schwann cells and axons and that nerve coil formation already occurs at relatively low concentrations of exogenous GDNF. Controlled expression of GDNF, by using a viral vector with regulatable GDNF expression, may be required to avoid motor axon trapping and to prevent the effects on Schwann cell proliferation and myelination.     

Long-Term Resilience of Late Holocene Coastal Subsistence System in Southeastern South America

Isotopic and molecular analysis on human, fauna and pottery remains can provide valuable new insights into the diets and subsistence practices of prehistoric populations. These are crucial to elucidate the resilience of social-ecological systems to cultural and environmental change. Bulk collagen carbon and nitrogen isotopic analysis of 82 human individuals from mid to late Holocene Brazilian archaeological sites (∼6,700 to ∼1,000 cal BP) reveal an adequate protein incorporation and, on the coast, the continuation in subsistence strategies based on the exploitation of aquatic resources despite the introduction of pottery and domesticated plant foods. These results are supported by carbon isotope analysis of single amino acid extracted from bone collagen. Chemical and isotopic analysis also shows that pottery technology was used to process marine foods and therefore assimilated into the existing subsistence strategy. Our multidisciplinary results demonstrate the resilient character of the coastal economy to cultural change during the late Holocene in southern Brazil.     

Inhibitors of HIV-1 attachment. Part 9: An assessment of oral prodrug approaches to improve the plasma exposure of a tetrazole-containing derivative

7-(2H-Tetrazol-5-yl)-1H-indole 3 was found to be a potent inhibitor of HIV-1 attachment but the compound lacked oral bioavailability in rats. The cause of the low exposure was believed to be poor absorption attributed to the acidic nature of the tetrazole moiety and, in an effort to address this liability, three more lipohilic tetrazole analogs, N-acetoxymethyl 4, N-pivaloyloxymethyl 5, and N-methyl 6, were evaluated as potential oral prodrugs in rats. Prodrug 5 was ineffective in improving the plasma concentration of 3 in vivo but compound 4 provided a 15-fold enhancement of the plasma concentration of 3. Most interestingly, oral dosing of analog 6 afforded a substantial increase in the plasma concentration of the parent in rats when compared to dosing of parent. This represents a novel example of a methyl tetrazole that acts as a prodrug for a free NH tetrazole-containing compound.     

Bioencapsulation of apomyoglobin in nanoporous organosilica sol–gel glasses: Influence of the siloxane network on the conformation and stability of a model protein

Nanoporous sol–gel glasses were used as host materials for the encapsulation of apomyoglobin, a model protein employed to probe in a rational manner the important factors that influence the protein conformation and stability in silica‐based materials. The transparent glasses were prepared from tetramethoxysilane (TMOS) and modified with a series of mono‐, di‐ and tri‐substituted alkoxysilanes, R_nSi(OCH₃)_4?n (R = methyl‐, n = 1; 2; 3) of different molar content (5, 10, 15%) to obtain the decrease of the siloxane linkage (? Si? O? Si? ). The conformation and thermal stability of apomyoglobin characterized by circular dichroism spectroscopy (CD) was related to the structure of the silica host matrix characterized by ²⁹Si MAS NMR and N₂ adsorption. We observed that the protein transits from an unfolded state in unmodified glass (TMOS) to a native‐like helical state in the organically modified glasses, but also that the secondary structure of the protein was enhanced by the decrease of the siloxane network with the methyl modification (n = 0 < n = 1 < n = 2 < n = 3; 0 < 5 < 10 < 15 mol %). In 15% trimethyl‐modified glass, the protein even reached a maximum molar helicity (?24,000 deg. cm² mol^?1) comparable to the stable folded heme‐bound holoprotein in solution. The protein conformation and stability induced by the change of its microlocal environment (surface hydration, crowding effects, microstructure of the host matrix) were discussed owing to this trend dependency. These results can have an important impact for the design of new efficient biomaterials (sensors or implanted devices) in which properly folded protein is necessary. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 895–906, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Protein adsorption onto organically modified silica glass leads to a different structure than sol-gel encapsulation

The secondary structures of two proteins were examined by circular dichroism spectroscopy after adsorption onto a series of organically modified silica glasses. The glasses were prepared by the sol-gel technique and were varied in hydrophobicity by incorporation of 5% methyl, propyl, trifluoropropyl, or n-hexyl silane. Both cytochrome c and apomyoglobin were found to lose secondary structure after adsorption onto the modified glasses. In the case of apomyoglobin, the α-helical content of the adsorbed protein ranged from 21% to 28%, well below the 62% helix found in solution. In contrast, these same glasses led to a striking increase in apomyoglobin structure when the protein was encapsulated within the pores during sol-gel processing: the helical content of apomyoglobin increased with increasing hydrophobicity from 18% in an unmodified glass to 67% in a 5% hexyl-modified glass. We propose that proteins preferentially adsorb onto unmodified regions of the silica surface, whereas encapsulated proteins are more susceptible to changes in surface hydration due to the proximity of the alkyl chain groups.     

Fatty acid-depleted albumin induces the formation of echovirus A particles

Picornavirus infection requires virus uncoating, associated with the production of 135S "A" particles and 80S empty particles from 160S mature virions, to release the RNA genome into the cell cytoplasm. Normal albumin inhibits this process. We now show that when depleted of fatty acids, albumin induces the formation of echovirus A particles.