A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome^1,2 or hyperoxic injuries of the neonatal lung³. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)⁴, genetic variants of surfactant deficiencies⁵ and α1-antitrypsin deficiency⁶.Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual''s immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies⁷.In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep⁸, and even in a clinical setting⁹, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection^10-12, (ultrasound-guided) intrapulmonary injection^13,14 and intravenous administration into the yolk sac vessels^15,16 or umbilical vein¹⁷. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques¹⁸.     

Simulations of a protein crystal: explicit treatment of crystallization conditions links theory and experiment in the streptavidin-biotin complex

A 250 ns molecular dynamics simulation of the biotin-liganded streptavidin crystal lattice, including cryoprotectant molecules and crystallization salts, is compared to a 250 ns simulation of the lattice solvated with pure water. The simulation using detailed crystallization conditions preserves the initial X-ray structure better than the simulation using pure water, even though the protein molecules display comparable mobility in either simulation. Atomic fluctuations computed from the simulation with crystallization conditions closely reproduce fluctuations derived from experimental temperature factors (correlation coefficient of 0.88, omitting two N-terminal residues with very high experimental B-factors). In contrast, fluctuations calculated from the simulation with pure water were less accurate, particularly for two of the streptavidin loops exposed to solvent in the crystal lattice. Finally, we obtain good agreement between the water and cryoprotectant densities obtained from the simulated crystallization conditions and the electron density due to solvent molecules in the X-ray structure. Our results suggest that detailed lattice simulations with realistic crystallization conditions can be used to assess potential function parameters, validate simulation protocols, and obtain valuable insights that solution-phase simulations do not easily provide. We anticipate that this will prove to be a powerful strategy for molecular dynamics simulations of biomolecules.     

FK506 binding protein 12 differentially accelerates fibril formation of wild type alpha-synuclein and its clinical mutants A30P or A53T

Aggregation of alpha-synuclein (α-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of α-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P α-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants α-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.     

Synthesis and antiviral properties of some polyphenols related to Salvia genus

An efficient synthesis of the acid part of salvianolic acid E 2 is described. Compound 2 was obtained from vanillin in 10 steps and 21% overall yield. During the synthesis of 2 an unexpected 5-oxo-4b,9b-dihydroindano[1,2-b]benzofuran rac-12 was isolated. Both compounds together with the acid part of salvianolic acid D were active as HIV-1 integrase inhibitors at the submicromolar level. But they did not inhibit the replication of the virus on MT-4 cells.     

Rehabilitation of Degraded Areas of Central Amazonia Using Direct Sowing of Forest Tree Seeds

Deforestation in the Amazon Basin is still increasing, and the rehabilitation of these lands continues to be a challenge. Autoecological studies of most Amazonian species are rare, and efficient techniques for restoration of forested habitats have yet to be developed. The aim of this study was to test direct sowing as a rehabilitation technique for sites with different degrees of disturbance: bare soil, pasture, and secondary and mature forests in Central Amazonia, Brazil. At each site, we sowed seeds of 11 native tree species. Throughout the following year we evaluated germination and seedling survival. The germination differed according to the study site and species. Seedling survival in degraded sites was higher than in other areas. After 1 year in the bare soil site, 33% of the sown seeds of eight species developed seedlings; in the pasture the establishment was 23%, in secondary forest 15%, and in mature forest 12% of only four species. The only widespread survivor with more than 45% emergence in all perturbed sites was Caryocar villosum. No pioneer seedlings remained after 1 year. There was a positive correlation between seed size and survival. Large‐seeded non‐pioneer species seem to be more suitable for direct sowing than small‐seeded species. We recommend a combination of direct sowing and planting of seedlings as an appropriate means to accelerate the rehabilitation of degraded areas in Central Amazonia.     

Global warming and arctic terns: Estimating climate change impacts on the world's longest migration

Climate change is one of the top three global threats to seabirds, particularly species that visit polar regions. Arctic terns migrate between both polar regions annually and rely on productive marine areas to forage, on sea ice for rest and foraging, and prevailing winds during flight. Here, we report 21st-century trends in environmental variables affecting arctic terns at key locations along their Atlantic/Indian Ocean migratory flyway during the non-breeding seasons, identified through tracking data. End-of-century climate change projections were derived from Earth System Models and multi-model means calculated in two Shared Socioeconomic Pathways: ‘middle-of-the-road’ and ‘fossil-fuelled development’ scenarios. Declines in North Atlantic primary production emerge as a major impact to arctic terns likely to affect their foraging during the 21st century under a ‘fossil-fuelled development’ scenario. Minimal changes are, however, projected at three other key regions visited by arctic terns (Benguela Upwelling, Subantarctic Indian Ocean and the Southern Ocean). Southern Ocean sea ice extent is likely to decline, but the magnitude of change and potential impacts on tern survival are uncertain. Small changes (<1 m s⁻¹) in winds are projected in both scenarios, but with minimal likely impacts on migration routes and duration. However, Southern Ocean westerlies are likely to strengthen and contract closer to the continent, which may require arctic terns to shift routes or flight strategies. Overall, we find minor effects of climate change on the migration of arctic terns, with the exception of poorer foraging in the North Atlantic. However, given that arctic terns travel over huge spatial scales and live for decades, they integrate minor changes in conditions along their migration routes such that the sum effect may be greater than the parts. Meeting carbon emission targets is vital to slow these end-of-century climatic changes and minimise extinction risk for a suite of polar species.     

Processing of the Borna Disease Virus Glycoprotein gp94 by the Subtilisin-Like Endoprotease Furin

Open reading frame IV (ORF-IV) of Borna disease virus (BDV) encodes a protein with a calculated molecular mass of ca. 57 kDa (p57), which increases after N glycosylation to 94 kDa (gp94). The unglycosylated and glycosylated proteins are proteolytically cleaved by the subtilisin-like protease furin. Furin most likely recognizes one of three potential cleavage sites, namely, an arginine at position 249 of the ORF-IV gene product. The furin inhibitor decRVKRcmk decreases the production of infectious BDV significantly, indicating that proteolytic cleavage of the gp94 precursor molecule is necessary for the full biological activity of the BDV glycoprotein.     

DNA sequence divergence in the Drosophila virilis group

DNA sequence divergence was analyzed in some sibling species of the Drosophila virilis group. Clones comprising about 0.1% of the genome DNA were selected at random from a D. virilis library for a comparative study on DNA from D. lummei, D. novamexicana, D. borealis, and D. lacicola. Blot hybridization experiments indicated that about 70% of DNA from D. lummei and D. novamexicana and less than 50% of DNA from D. borealis and D. lacicola share sequences that are homologous to DNA in D. virilis. This finding is in excellent agreement with the genealogical tree based on cytological studies (Throckmorton 1982). - Four plasmids with inserts which are present in one or a few copies per genome were hybridized in situ to polytene chromosomes. These experiments demonstrate that (1) homologous "unique" DNA sequences are localized exclusively in homologous bands and (2) homologous bands that appear to be identical in different species may contain different DNA sequences.     

l-Glutamic acid,a neuromodulator of dopaminergic transmission in the rat corpus striatum

A superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [³H]dopamine by rat striatal slices. Neither l-glutamate nor other aminoacids such as l-aspartate and d-glutamate (5 × 10^?5 M) modified the spontaneous release of exogenous [³H]dopamine from rat striatal slices. In contrast, these neuroexcitatory aminoacids did potentiate spontaneous release of striatal [³H]dopamine newly synthesized from [³H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only l-glutamate (5 × 10^?6-1 × 10^?4 M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. In addition, similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [³H]dopamine was studied. The effect of l-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca²⁺ in the superfusion medium and it was blocked by l-glutamate diethylester. Furthermore, this effect of l-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca²⁺-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and stereoselective towards l-glutamate stimulation. The findings reported have led us to propose that l-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.