Probing a polar cluster in the retinal binding pocket of bacteriorhodopsin by a chemical design approach

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C(13) of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.     

Rapid throughput analysis of filamentous fungal growth using turbidimetric measurements with the Bioscreen C: a tool for screening antifungal compounds

A rapid method for screening antifungal compounds and performing ecophysiological studies with filamentous fungi has been developed by the use of specific semi-solid media and spectrophotometric/turbidimetric measurements using the Bioscreen C with 2×100 microtitre well plates. The medium composition and preparation, inoculum size, medium volumes, and incubation parameters for measuring initial germination and growth dynamics have been optimised. These have been applied to assess the effectiveness of 18 concentrations of propyl propane thiosulfinate (PTS) against Aspergillus flavus in YES medium under different environmental regimes. Minimum inhibitory concentrations (MIC), and non-inhibitory concentration (NIC) values, and a newly developed MIC(50), have been calculated for the efficacy of these concentrations of PTS under four different environmental conditions (0.995 and 0.95 water activity (a(w)); 20 and 25°C) over 7d periods with automated measurements every 20 min. Data were modelled using the Lambert-Pearson Model, a mathematical modelling approach previously used for bacterial inhibition. Results have been compared with traditional growth rate data and Lethal Dose(50) (LD(50)) values. This approach could have major implications for rapid screening assays for growth and secondary metabolite production by filamentous organisms and has major advantages in terms of time reduction, culture medium volumes required, measurement and the ability to integrate and model key parameters for comparing efficacy.     

Ring1a/b polycomb proteins regulate the mesenchymal stem cell niche in continuously growing incisors

Rodent incisors are capable of growing continuously and the renewal of dental epithelium giving rise to enamel-forming ameloblasts and dental mesenchyme giving rise to dentin-forming odontoblasts and pulp cells is achieved by stem cells residing at their proximal ends. Although the dental epithelial stem cell niche (cervical loop) is well characterized, little is known about the dental mesenchymal stem cell niche. Ring1a/b are the core Polycomb repressive complex1 (PRC1) components that have recently also been found in a protein complex with BcoR (Bcl-6 interacting corepressor) and Fbxl10. During mouse incisor development, we found that genes encoding members of the PRC1 complex are strongly expressed in the incisor apical mesenchyme in an area that contains the cells with the highest proliferation rate in the tooth pulp, consistent with a location for transit amplifying cells. Analysis of Ring1a(-/-);Ring1b(cko/cko) mice showed that loss of Ring1a/b postnatally results in defective cervical loops and disturbances of enamel and dentin formation in continuously growing incisors. To further characterize the defect found in Ring1a(-/-);Ring1b(cko/cko) mice, we demonstrated that cell proliferation is dramatically reduced in the apical mesenchyme and cervical loop epithelium of Ring1a(-/-);Ring1b(cko/cko) incisors in comparison to Ring1a(-/-);Ring1b(fl/fl)cre- incisors. Fgf signaling and downstream targets that have been previously shown to be important in the maintenance of the dental epithelial stem cell compartment in the cervical loop are downregulated in Ring1a(-/-);Ring1b(cko/cko) incisors. In addition, expression of other genes of the PRC1 complex is also altered. We also identified an essential postnatal requirement for Ring1 proteins in molar root formation. These results show that the PRC1 complex regulates the transit amplifying cell compartment of the dental mesenchymal stem cell niche and cell differentiation in developing mouse incisors and is required for molar root formation.     

Antiviral activity and synthesis of quaternized promazine derivatives against HSV-1

N-(4-chlorobenzyl)triflupromazinium chloride, a known antitubercular agent, has been found to also be active against HSV-1. A preliminary structure-activity relation has been explored to determine which groups are crucial to viral inhibition. Antiviral assessments such as GFP reduction, plaque reduction, treatment timing and wash-out studies have also been probed to determine a mode of action for QPD-1. Based on this preliminary data, it appears that QPD-1 is a reversible inhibitor, suspected to inhibit early stages of viral replication of HSV-1 at 50μM, equipotent to acyclovir.     

Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses

Background

The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein.

Methods

We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation.

Results

The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections.

Conclusion

These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.     

Phylogenetic relatedness as a tool in restoration ecology: a meta-analysis

Biotic interactions assembling plant communities can be positive (facilitation) or negative (competition) and operate simultaneously. Facilitative interactions and posterior competition are among the mechanisms triggering succession, thus representing a good scenario for ecological restoration. As distantly related species tend to have different phenotypes, and therefore different ecological requirements, they can coexist, maximizing facilitation and minimizing competition. We suggest including phylogenetic relatedness together with phenotypic information as a predictor for the net effects of the balance between facilitation and competition in nurse-based restoration experiments. We quantify, by means of a Bayesian meta-analysis of nurse-based restoration experiments performed worldwide, the importance of phylogenetic relatedness and life-form disparity in the survival, growth and density of facilitated plants. We find that the more similar the life forms of neighbouring plants are the greater the positive effect of phylogenetic distance is on survival and density. This result suggests that other characteristics beyond life form are also contained in the phylogeny, and the larger the phylogenetic distance, the less is the niche overlap, and therefore the less is the competition. As a general rule, we can maximize the success of the nurse-based practices by increasing life-form disparity and phylogenetic distances between the neighbour and the facilitated plant.     

Analysis of a naturally-occurring deletion mutant of Spodoptera frugiperda multiple nucleopolyhedrovirus reveals sf58 as a new per os infectivity factor of lepidopteran-infecting baculoviruses

The Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, is structured as a mixture of nine genotypes (A-I). Occlusion bodies (OBs) of SfMNPV-C, -D and -G pure genotypes are incapable of oral transmission; a phenotype which in SfMNPV-C and -D is due to the absence of pif1 and pif2 genes. The complete sequence of the SfMNPV-G genome was determined to identify possible factors involved in this phenotype. Deletions of 4860 bp (22,366-27,225) and 60 bp (119,759-119,818) were observed in SfMNPV-G genome compared with that of the predominant complete genotype SfMNPV-B (132,954 bp). However no genes homologous to previously described per os infectivity factors were located within the deleted sequences. Significant differences were detected in the nucleotide sequence in sf58 gene (unknown function) that produced changes in the amino acid sequence and the predicted secondary structure of the corresponding protein. This gene is conserved only in lepidopteran baculoviruses (alpha- and betabaculoviruses). To determine the role of sf58 in peroral infectivity a deletion mutant was constructed using bacmid technology. OBs of the deletion mutant (Sf58null) were not orally infectious for S. frugiperda larvae, whereas Sf58null rescue virus OBs recovered oral infectivity. Sf58null DNA and occlusion derived virions (ODVs) were as infective as SfMNPV bacmid DNA and ODVs in intrahemocelically infected larvae or cell culture, indicating that defects in ODV or OB morphogenesis were not involved in the loss of peroral infectivity. Addition of optical brightener or the presence of the orally infectious SfMNPV-B OBs in mixtures with SfMNPV-G OBs did not recover Sf58null OB infectivity. According to these results sf58 is a new per os infectivity factor present only in lepidopteran baculoviruses.     

Mutualism with plants drives primate diversification

Understanding the origin of diversity is a fundamental problem in biology. Evolutionary diversification has been intensely explored during the last years due to the development of molecular tools and the comparative method. However, most studies are conducted using only information from extant species. This approach probably leads to misleading conclusions, especially because of inaccuracy in the estimation of extinction rates. It is critical to integrate the information generated by extant organisms with the information obtained from the fossil record. Unfortunately, this integrative approach has been seldom performed, and thus, our understanding of the factors fueling diversification is still deficient. Ecological interactions are a main factor shaping evolutionary diversification by influencing speciation and extinction rates. Most attention has focused on the effect of antagonistic interactions on evolutionary diversification. In contrast, the role of mutualistic interactions in shaping diversification has been much less explored. In this study, by combining phylogenetic, neontological, and paleontological information, we show that a facultative mutualistic plant-animal interaction emerging from frugivory and seed dispersal has most likely contributed to the diversification of our own lineage, the primates. We compiled diet and seed dispersal ability in 381 extant and 556 extinct primates. Using well-established molecular phylogenies, we demonstrated that mutualistic extant primates had higher speciation rates, lower extinction rates, and thereby higher diversification rates than nonmutualistic ones. Similarly, mutualistic fossil primates had higher geological durations and smaller per capita rates of extinction than nonmutualistic ones. As a mechanism underlying this pattern, we found that mutualistic extinct and extant primates have significantly larger geographic ranges, which promotes diversification by hampering extinction and increasing geographic speciation. All these outcomes together strongly suggest that the establishment of a facultative mutualism with plants has greatly benefited primate evolution and fueled its taxonomic diversification.     

Non-surgical deep intrauterine transfer of superfine open pulled straw (SOPS)-vitrified porcine embryos: Evaluation of critical steps of the procedure

Previous trials achieved extremely poor results when using the one-step warming method in a syringe in combination with non-surgical deep intrauterine transfer (NET) of superfine open pulled straw (SOPS)-vitrified embryos. This study aimed to assess the effect of the warming procedure on the in vitro and in vivo development of SOPS-vitrified embryos. The effect of the passage of the vitrified-warmed (VW) embryos through the NET catheter was also evaluated. Groups of 4 to 6 morulae and blastocysts, collected from weaned sows, were SOPS-vitrified in 1 μL of vitrification medium, warmed by the one-step warming method in a dish or in a 1-mL syringe and cultured in vitro for 48 h to evaluate the embryo survival (ES) and hatching rates (HR). Warming in syringe had a deleterious effect (P < 0.05) on the in vitro ES (60.5 ± 10.4%) and HR (39.6 ± 9.5%) of VW embryos in comparison with embryos warmed in a dish (85.4 ± 10.6% and 69.0 ± 8.4%, respectively). This decreased embryonic development was due to the increased time required between the removal of the straws from the liquid nitrogen and the contact of the embryos with the warming medium when the warming was performed in a syringe in comparison with that for the warming in a dish. After verifying that the passage of VW embryos through the NET catheter does not have a damaging effect on their further in vitro development, the negative effect of warming in a syringe was also confirmed after NET. Fifteen fresh and SOPS-vitrified embryos warmed in a syringe or in a dish were transferred to each recipient (n = 28) and recovered 24 h later to assess their developmental progression. All embryos from the syringe group were found to have degenerated at recovery. The in vivo ES and HR from the dish group (80.4 ± 3.4% and 14.2 ± 7.2%, respectively) were lower (P < 0.05) than those from the fresh group (94.0 ± 4.1% and 36.8 ± 7.8%, respectively). Combining the warming in a dish and the NET procedure, 35 VW embryos were transferred to each of 10 gilts. Five recipients farrowed an average of 10.4 ± 0.9 piglets. In conclusion, the method of one-step warming in a syringe has a negative effect on the in vitro and in vivo viability of SOPS-vitrified porcine embryos. In addition, NET of SOPS-vitrified embryos warmed by the one-step method in a dish showed promising reproductive performance of recipients. However, despite the great potential of this technology, further developments are required for large-scale commercial applications.