TLR3 and TLR9 Agonists Improve Postexposure Vaccination Efficacy of Live Smallpox Vaccines

Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV), a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C) even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs) were less protective than the TLR3 agonist poly(I:C). We show that activation of type 1 IFN by poly(I:C) and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination.     

Direct and indirect effects of climate on decomposition in native ecosystems from central Argentina

Abstract Climate affects litter decomposition directly through temperature and moisture, determining the ecosystem potential decomposition, and indirectly through its effect on plant community composition and litter quality, determining litter potential decomposition. It would be expected that both the direct and indirect effects of climate on decomposition act in the same direction along gradients of actual evapotranspiration (AET). However, studies from semiarid ecosystems challenge this idea, suggesting that the climatic conditions that favour decomposition activity, and the consequent ecosystem potential decomposition, do not necessarily lead to litter being easier to decompose. We explored the decomposition patterns of four arid to subhumid native ecosystems with different AET in central‐western Argentina and we analysed if ecosystem potential decomposition (climatic direct effect), nutrient availability and leaf litter potential decomposition (climatic indirect effect) all increased with AET. In general, the direct effect of climate (AET) on decomposition (i.e. ecosystem potential decomposition), showed a similar pattern to nutrient availability in soils (higher for xerophytic and mountain woodlands and lower for the other ecosystems), but different from the pattern of leaf litter potential decomposition. However, the range of variation in the ecosystem potential decomposition was much higher than the range of variation in litter potential decomposition, indicating that the direct effect of climate on decomposition was far stronger than the indirect effect through litter quality. Our results provide additional experimental evidence supporting the direct control of climate over decomposition, and therefore nutrient cycling. For the ecosystems considered, those with the highest AET are the ecosystems with the highest potential decomposition. But what is more interesting is that our results suggest that the indirect control of climate over decomposition through vegetation characteristics and decomposability does not follow the same trend as the direct effect of climate. This finding has important implications in the prediction of the effects of climate change on semiarid ecosystems.     

Effect of some heterocyclic herbicides on CO2 assimilation by spinach chloroplasts

We have studied the inhibitory effect of heterocyclic herbicides simazine, paraquat, pyrazon and amitrole on photosynthetic CO₂ fixation and on the level of intermediates of the CO₂ assimilation cycle in isolated spinach (Spinacia oleracea) chloroplasts, as well as their in vitro activities on ribulose-1,5-bisphosphate carboxylase and fructose-1,6-bisphosphatase. The half inhibitory concentrations (I₅₀) of CO₂ assimilation were about 1 μM for simazine and paraquat, and 10 μM for pyrazon. Amitrole, with an I₅₀ 100 μM, gave only a weak inhibition. In the presence of simazine or pyrazon the triose-phosphates/phosphoglycerate ratio diminished because of a decrease of the triose-phosphates percentage from 47% to 19%, which means an inhibition of the phosphoglycerate reduction step by a low NADPH synthesis. However, there was not a parallel increase of phosphoglycerate, because of collateral pathways leading to phospho-enolpyruvate, amino acids and other non-identified compounds. Paraquat did not give such a decreased ratio, which could be explained as an inhibition of some step of the Calvin cycle later than triose-phosphates by the H₂O₂ formed in a Mehler reaction. Amitrole did not show any effect on the pattern of intermediates. Simazine and pyrazon at 10 μM concentration promote a 20–30% activation of ribulose-1,5-bisphosphate carboxylase activity, whereas paraquat, pyrazon and simazine showed an I₅₀ about 100 μM for the inhibition of the photosynthetic fructose-1,6-bisphosphatase.     

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3–11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events – large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.     

Tomato yellow leaf curl viruses: ménage à trois between the virus complex,the plant and the whitefly vector

Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops in tropical, subtropical and temperate regions of the world. Here, we focus on the interactions through recombination between the different begomovirus species causing TYLCD, provide an overview of the interactions with the cellular genes involved in viral replication, and highlight recent progress on the relationships between these viruses and their vector, the whitefly Bemisia tabaci. Taxonomy: The tomato yellow leaf curl virus‐like viruses (TYLCVs) are a complex of begomoviruses (family Geminiviridae, genus Begomovirus) including 10 accepted species: Tomato yellow leaf curl Axarquia virus (TYLCAxV), Tomato yellow leaf curl China virus (TYLCCNV), Tomato yellow leaf curl Guangdong virus (TYLCGuV), Tomato yellow leaf curl Indonesia virus (TYLCIDV), Tomato yellow leaf curl Kanchanaburi virus (TYLVKaV), Tomato yellow leaf curl Malaga virus (TYLCMalV), Tomato yellow leaf curl Mali virus (TYLCMLV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tomato yellow leaf curl Vietnam virus (TYLCVNV) and Tomato yellow leaf curl virus(TYLCV). We follow the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV), the most important of which is an 89% nucleotide identity threshold between full‐length DNA‐A component nucleotide sequences for begomovirus species. Strains of a species are defined by a 93% nucleotide identity threshold. Host range: The primary host of TYLCVs is tomato (Solanum lycopersicum), but they can also naturally infect other crops [common bean (Phaseolus vulgaris), sweet pepper (Capsicum annuum), chilli pepper (C. chinense) and tobacco (Nicotiana tabacum)], a number of ornamentals [petunia (Petunia×hybrida) and lisianthus (Eustoma grandiflora)], as well as common weeds (Solanum nigrum and Datura stramonium). TYLCVs also infect the experimental host Nicotiana benthamiana. Disease symptoms: Infected tomato plants are stunted or dwarfed, with leaflets rolled upwards and inwards; young leaves are slightly chlorotic; in recently infected plants, fruits might not be produced or, if produced, are small and unmarketable. In common bean, some TYLCVs produce the bean leaf crumple disease, with thickening, epinasty, crumpling, blade reduction and upward curling of leaves, as well as abnormal shoot proliferation and internode reduction; the very small leaves result in a bushy appearance.     

A multiscale, spatially distributed model of asthmatic airway hyper-responsiveness

We present a multiscale, spatially distributed model of lung and airway behaviour with the goal of furthering the understanding of airway hyper-responsiveness and asthma. The model provides an initial computational framework for linking events at the cellular and molecular levels, such as Ca²⁺ and crossbridge dynamics, to events at the level of the entire organ. At the organ level, parenchymal tissue is modelled using a continuum approach as a compressible, hyperelastic material in three dimensions, with expansion and recoil of lung tissue due to tidal breathing. The governing equations of finite elasticity deformation are solved using a finite element method. The airway tree is embedded in this tissue, where each airway is modelled with its own airway wall, smooth muscle and surrounding parenchyma. The tissue model is then linked to models of the crossbridge mechanics and their control by Ca²⁺ dynamics, thus providing a link to molecular and cellular mechanisms in airway smooth muscle cells. By incorporating and coupling the models at these scales, we obtain a detailed, computational multiscale model incorporating important physiological phenomena associated with asthma.     

A standard vector for the chromosomal integration and characterization of BioBrick™ parts in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

The chromosomal integration of biological parts in the host genome enables the engineering of plasmid-free stable strains with single-copy insertions of the desired gene networks. Although different integrative vectors were proposed, no standard pre-assembled genetic tool is available to carry out this task. Synthetic biology concepts can contribute to the development of standardized and user friendly solutions to easily produce engineered strains and to rapidly characterize the desired genetic parts in single-copy context.

Results

In this work we report the design of a novel integrative vector that allows the genomic integration of biological parts compatible with the RFC10, RFC23 and RFC12 BioBrick? standards in Escherichia coli. It can also be specialized by using BioBrick? parts to target the desired integration site in the host genome. The usefulness of this vector has been demonstrated by integrating a set of BioBrick? devices in two different loci of the E. coli chromosome and by characterizing their activity in single-copy. Construct stability has also been evaluated and compared with plasmid-borne solutions.

Conclusions

Physical modularity of biological parts has been successfully applied to construct a ready-to-engineer BioBrick? vector, suitable for a stable chromosomal insertion of standard parts via the desired recombination method, i.e. the bacteriophage integration mechanism or homologous recombination. In contrast with previously proposed solutions, it is a pre-assembled vector containing properly-placed restriction sites for the direct transfer of various formats of BioBrick? parts. This vector can facilitate the characterization of parts avoiding copy number artefacts and the construction of antibiotic resistance-free engineered microbes, suitable for industrial use.