Stereochemistry and enantiomeric purity of a novel anxiolytic agent, deramciclane fumarate.

The synthesis, stereostructure, and enantiomeric separation by chromatography of a new, chiral anxiolytic agent, deramciclane fumarate (2, (-)-[1R,2S,4R]-2-(2-dimethylaminoethoxy)-2-phenyl-1,7, 7-trimethylbicyclo[2.2.1]heptane fumarate, EGIS-3886), is described. The optical antipode and the racemate of compound 2 were also prepared. The structure was determined by single crystal X-ray diffraction analysis. The enantiomeric separation was accomplished by HPLC on Chiralcel OD (250 x 4.6 mm; 10 microm) and hexane-ethanol (99.5:0.5) as mobile phase at room temperature. The enantiomeric purity of the synthesized drug substance proved to be very high (>99. 9%). Some statements published earlier on the stereostructure of deramciclane fumarate are critically discussed.     

Codiversification of gastrointestinal microbiota and phylogeny in passerines is not explained by ecological divergence

Vertebrate gut microbiota (GM) is comprised of a taxonomically diverse consortium of symbiotic and commensal microorganisms that have a pronounced effect on host physiology, immune system function and health status. Despite much research on interactions between hosts and their GM, the factors affecting inter‐ and intraspecific GM variation in wild populations are still poorly known. We analysed data on faecal microbiota composition in 51 passerine species (319 individuals) using Illumina MiSeq sequencing of bacterial 16S rRNA (V3–V4 variable region). Despite pronounced interindividual variation, GM composition exhibited significant differences at the interspecific level, accounting for approximately 20%–30% of total GM variation. We also observed a significant correlation between GM composition divergence and host's phylogenetic divergence, with strength of correlation higher than that of GM vs. ecological or life history traits and geographic variation. The effect of host's phylogeny on GM composition was significant, even after statistical control for these confounding factors. Hence, our data do not support codiversification of GM and passerine phylogeny solely as a by‐product of their ecological divergence. Furthermore, our findings do not support that GM vs. host's phylogeny codiversification is driven primarily through trans‐generational GM transfer as the GM vs. phylogeny correlation does not increase with higher sequence similarity used when delimiting operational taxonomic units. Instead, we hypothesize that the GM vs. phylogeny correlation may arise as a consequence of interspecific divergence of genes that directly or indirectly modulate composition of GM.     

The ability of green fluorescent proteins for photoconversion under oxygen-free conditions is determined by the chromophore structure rather than its amino acid environment

Photoconversion of various green and cyan fluorescent proteins to the red fluorescent state under the oxygen-free conditions was studied. Such photoconversion has earlier been described for the EGFP green fluorescent protein. Phylogenetically distant fluorescent proteins that have a low identity of their amino acid sequences but contain chemically identical chromophores based on a Tyr residue were shown to be susceptible to this type of photoconversion. At the same time, the ECFP protein, which has 92% homology with EGFP but contains a chromophore based on tryptophan did not undergo the photoconversion. Thus, it is precisely the chromophore structure, rather than the amino acid environment that determines the ability of green fluorescent proteins to display photoconversion to the red fluorescent state under anaerobic conditions.     

A New Genetic Vaccine Platform Based on an Adeno-Associated Virus Isolated from a Rhesus Macaque

We created a hybrid adeno-associated virus (AAV) from two related rhesus macaque isolates, called AAVrh32.33, and evaluated it as a vaccine carrier for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus antigens. The goal was to overcome the limitations of vaccines based on other AAVs, which generate dysfunctional T-cell responses and are inhibited by antibodies found in human sera. Injection of a Gag-expressing AAVrh32.33 vector into mice resulted in a high-quality CD8⁺ T-cell response. The resulting Gag-specific T cells express multiple cytokines at high levels, including interleukin-2, with many having memory phenotypes; a subsequent boost with an adenovirus vector yielded a brisk expansion of Gag-specific T cells. A priming dose of AAVrh32.33 led to high levels of Gag antibodies, which exceed levels found after injection of adenovirus vectors. Importantly, passive transfer of pooled human immunoglobulin into mice does not interfere with the efficacy of AAVrh32.33 expressing nucleoproteins from influenza virus, as measured by protection to a lethal dose of influenza virus, which is consistent with the very low seroprevalence to this virus in humans. Studies of macaques with vectors expressing gp140 from HIV-1 (i.e., with AAVrh32.33 as the prime and simian adenovirus type 24 as the boost) demonstrated results similar to those for mice with high-level and high-quality CD8⁺ T-cell responses to gp140 and high-titered neutralizing antibodies to homologous HIV-1. The biology of this novel AAV hybrid suggests that it should be a preferred genetic vaccine carrier, capable of generating robust T- and B-cell responses.The initial interest in vectors based on adeno-associated viruses (AAV) was for applications in gene therapy. Most of the initial work was with vectors derived from AAV serotype 2 (AAV2), which is one of the six initial isolates. In the first in vivo studies, several groups showed stable expression of the transgene Escherichia coli β-galactosidase following intramuscular (i.m.) injection of AAV2-LacZ without immune responses to the transgene (23, 44). The apparent tolerance of the host to AAV-encoded antigens to a variety of transgene products has been demonstrated in mice and some large animals (1, 35, 39). Several mechanisms have been proposed to explain the lack of T-cell responses following in vivo gene transfer with AAV, including ignorance (inadequate presentation of antigen), anergy, and suppression (1, 5, 18, 37).As applications of AAV vectors for in vivo gene transfer expanded, it became clear that the apparent immune privilege of AAV transgene products was not absolute. A number of examples emerged in which the host mounted vibrant T-cell responses to AAV-encoded transgene products. Several key parameters appeared to influence immunogenicity of the transgene. For example, Sarukhan et al. suggest that the subcellular localization of the protein influences the magnitude of the ensuing T-cell response after AAV gene transfer (37). The dose and route of administration of the AAV vector also contribute significantly to B- and T-cell responses to the transgene (3, 13). Wang et al. showed that inflammation at the site of AAV administration promotes antigen-specific immune responses to the transgene (47). A consistent observation has been that B-cell responses to AAV-encoded transgenes are much more intense and more consistently generated than CD8⁺ T-cell responses (8, 46, 51). A number of investigators have begun to explore AAV vectors as genetic vaccines against a variety of infectious and noninfectious diseases, based on the notion that it can be developed to stimulate transgene immune responses (14, 22, 26, 28, 48-50).The discovery of an expanded family of AAV capsids from human and nonhuman primates has provided an opportunity to evaluate the effects of capsid structure on vector performance. Most of this work has focused on the use of novel AAV serotypes for achieving higher levels of transgene expression for applications in gene therapy (7, 12, 36). Xin et al. recently evaluated, in mice, vectors as vaccines for human immunodeficiency virus type 1 (HIV-1) based on the original AAV isolates AAV1 to AAV6 and two novel AAVs we recently discovered, AAV7 and AAV8 (48). They showed significant capsid-dependent effects on T- and B-cell responses to HIV-1 gp160. We recently confirmed these observations and more thoroughly evaluated the quality of the CD8⁺ T-cell responses (26). AAV vectors of multiple serotypes encoding HIV-1 Gag were injected i.m. into mice, which all showed some level of CD8⁺ T-cell responses based on tetramer staining and peptide-induced gamma interferon (IFN-γ) expression. However, the quality of AAV-induced, Gag-specific T cells was substantially lower than that obtained with adenoviral vectors, based on several criteria. A majority of the tetramer-positive (Tet⁺) T cells were nonresponsive to antigen, and those that did respond to antigen produced low levels of IFN-γ and no interleukin-2 (IL-2). Very few memory T cells were generated, and animals primed with AAV vectors were not responsive to a boost with an adenoviral vector. However, all AAV serotypes studied did generate very high levels of antibodies to the Gag transgene product.A final issue to consider in the use of AAV as a genetic vaccine for HIV-1 is the presence of neutralizing antibodies (NAbs) to the vector due to prior AAV infections. We recently conducted an extensive screening of human populations from several continents and found high prevalence and high titers of NAbs to AAV1 and AAV2 and moderate levels of NAbs to AAV7 and -8 (4). In vivo gene transfer experiments indicate that AAV NAbs will likely impinge on vector efficacy (9, 33, 38).This study describes the creation of a novel AAV from rhesus macaque isolates, called AAVrh32.33, and its characterization as a genetic vaccine for HIV-1. AAVrh32.33 has properties unlike those of any others we have studied. We showed that vectors based on this novel capsid elicit strong CD8⁺ T-cell responses to reporter transgene products that are dependent on CD4⁺ T-cell help and dependent on signaling through CD40L and CD28 (L. E. Mays and J. M. Wilson, submitted for publication). Important to the use of this vector in the clinic is a very low incidence of NAbs to it in human populations. This study describes the development of vectors based on AAVrh32.33 as genetic vaccines.     

Enatiomeric determination of tramadol and O-desmethyltramadol in human urine by gas chromatography–mass spectrometry

A GC–MS assay for stereoselective determination of tramadol and its pharmacologically active phase I metabolite O-desmethyltramadol in human urine was developed. Nefopam was used as internal standard. The method involves a simple solid phase extraction with chiral analysis by gas chromatography–electron ionization mass spectrometry using m/z 263; 58, 249; 58, and 179; 58 for the determination of concentration of tramadol, O-desmethyltramadol and internal standard, respectively. Chromatography was performed on a Rt-βDEXcst column containing alkylated beta-cyclodextrins as a chiral selector. The calibration curves were linear in the concentration range 0.1–20 μg/mL (R² ≥ 0.998). Intra-day accuracies ranged between 97.2–104.9%, 96.1–103.2%, and 97.3–102.8% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged between 95.2–105.7%, 99.1–105.2%, and 96.5–101.2% at the lower, intermediate, and high concentration for all analytes, respectively. This method was successfully used to determine the concentration of enantiomers of T and ODT in a pharmacogenetic study.     

Occurrence and chemical diversity of the genus Bryoria in the Sučí Potok Valley, Veporské Vrchy Mountains (Central Slovakia)

Four species of the genus, Bryoria were found in the Sučí Potok Valley: B. capilaris (Ach.) Brodo & D. Hawksw., B. fuscescens (Gyeln.) Brodo & D. Hawksw., B. implexa (Hoffm.) Brodo & D. Hawksw., B. nadvornikiana (Gyeln.) Brodo & D. Hawksw. The most common species in the valley was B. implexa. Four chemotypes of this lichen were recognized.     

Measurement of the optical parameters of purple membrane and plant light-harvesting complex films with optical waveguide lightmode spectroscopy

Purple membrane (bacteriorhodopsin) and plant light-harvesting complexes (LHCII) were dried on the optical waveguide sensor with varying thicknesses in a wide range (from 20 to several hundreds of nanometers) and the optical parameters were studied with optical waveguide lightmode spectroscopy. It was found that applying the approximate 4-layer mode equations for the measured effective refractive indices resulted in unacceptable results for the optical parameters: with increasing thickness the refractive index decreased monotonously from 1.5 to 1.1. Therefore an inverse waveguide numerical method was developed and used to obtain reliable results from the experiments. The inverse method yielded an approximately constant (1.53) refractive index independently of the thickness for the purple membrane and LHCII films. Light-induced changes in the optical parameters of the purple membrane and LHCII films were also studied. For purple membrane films the most significant effect is the change in refractive index and absorption. For LHCII films prolonged illumination induced irreversible structural changes, most probably of thermo-optic origin.     

AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development

The exocyst is a hetero-oligomeric protein complex involved in exocytosis and has been extensively studied in yeast and animal cells. Evidence is now accumulating that the exocyst is also present in plants. Bioinformatic analysis of genes encoding plant homologs of the exocyst subunit, Exo70, revealed that three Exo70 subgroups are evolutionarily conserved among angiosperms, lycophytes and mosses. Arabidopsis and rice contain 22 and approximately 39 EXO70 genes, respectively, which can be classified into nine clusters considered to be ancient in angiosperms (one has been lost in Arabidopsis). We characterized two independent T-DNA insertional mutants of the AtEXO70A1 gene (exo70A1-1 and exo70A1-2). Heterozygous EXO70A1/exo70A1 plants appear to be normal and segregate in a 1:2:1 ratio, suggesting that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, both exo70A1-1 and exo70A1-2 homozygotes exhibit an array of phenotypic defects. The polar growth of root hairs and stigmatic papillae is disturbed. Organs are generally smaller, plants show a loss of apical dominance and indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that the putative exocyst subunit EXO70A1 is involved in cell and organ morphogenesis.     

Increasing incidence of Geomyces destructans fungus in bats from the Czech Republic and Slovakia

Background

White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown.

Methodology/Principal Findings

We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval.

Conclusions/Significance

G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme.