Mammalian Adaptation in the PB2 Gene of Avian H5N1 Influenza Virus

The substitution of glutamic acid (E) for lysine (K) at position 627 of the PB2 protein of avian H5N1 viruses has been identified as a virulence and host range determinant for infection of mammals. Here, we report that the E-to-K host-adaptive mutation in the PB2 gene appeared from day 4 and 5 along the respiratory tracts of mice and was complete by day 6 postinoculation. This mutation correlated with efficient replication of the virus in mice.     

Response of macroalgal assemblages from rockpools to climate change: effects of persistent increase in temperature and CO2

Anthropogenically induced global climate change has important implications for marine ecosystems with unprecedented ecological and economic consequences. Climate change will include the simultaneous increase of temperature and CO₂ concentration in oceans. However, experimental manipulations of these factors at the community scale are rare. In this study, we used an experimental approach in mesocosms to analyse the combined effects of elevated CO₂ and temperature on macroalgal assemblages from intertidal rock pools. Our model systems were synthetic assemblages of varying diversity and understory component and canopy species identity. We used assemblages invaded by the non‐indigenous canopy forming alga Sargassum muticum and assemblages with the native canopy species Cystoseira tamariscifolia. We examined the effects of both climate change factors on several ecosystem functioning variables (i.e. photosynthetic efficiency, productivity, respiration and biomass) and how these effects could be shaped by the diversity and species identity of assemblages. CO₂ alone or in combination with temperature affected the performance of macroalgae at both individual and assemblage level. In particular, high CO₂ and high temperature (20°C) drastically reduced the biomass of macroalgal assemblages and affected their productivity and respiration rates. The identity of canopy species also played an important role in shaping assemblage responses, whereas species richness did not seem to affect such responses. Species belonging to the same functional effect group responded differently to the same environmental conditions. Data suggested that assemblages invaded with S. muticum might be more resistant in a future scenario of climate change. Thus, in a future scenario of increasing temperature and CO₂ concentration, macroalgal assemblages invaded with canopy‐forming species sharing response traits similar to those of S. muticum could be favoured.     

Inhibitors of HIV-1 attachment. Part 7: Indole-7-carboxamides as potent and orally bioavailable antiviral agents

A series of substituted carboxamides at the indole C7 position of the previously described 4-fluoro-substituted indole HIV-1 attachment inhibitor 1 was synthesized and the SAR delineated. Heteroaryl carboxamide inhibitors that exhibited pM potency in the primary cell-based assay against a pseudotype virus expressing a JRFL envelope were identified. The simple methyl amide analog 4 displayed a promising in vitro profile, with its favorable HLM stability and membrane permeability translating into favorable pharmacokinetic properties in preclinical species.     

Bacterial species selective toxicity of two isomeric α/β-peptides: Role of membrane lipids

We have studied how membrane interactions of two synthetic cationic antimicrobial peptides with alternating α- and β-amino acid residues (“α/β-peptides”) impact toxicity to different prokaryotes. Electron microscopic examination of thin sections of Escherichia coli and of Bacillus subtilis exposed to these two α/β-peptides reveals different structural changes in the membranes of these bacteria. These two peptides also have very different effects on the morphology of liposomes composed of phosphatidylethanolamine and phosphatidylglycerol in a 2:1 molar ratio. Freeze fracture electron microscopy indicates that with this lipid mixture, α/β-peptide I induces the formation of a sponge phase. ³¹P NMR and X-ray diffraction are consistent with this conclusion. In contrast, with α/β-peptide II and this same lipid mixture, a lamellar phase is maintained, but with a drastically reduced d-spacing. α/β-Peptide II is more lytic to liposomes composed of these lipids than is I. These findings are consistent with the greater toxicity of α/β-peptide II, relative to α/β-peptide I, to E. coli, a bacterium having a high content of phosphatidylethanolamine. In contrast, both α/β-peptides display similar toxicity toward B. subtilis, in accord with the greater anionic lipid composition in its membrane. This work shows that variations in the selectivity of these peptidic antimicrobial peptides toward different strains of bacteria can be partly determined by the lipid composition of the bacterial cell membrane.     

Sensitivity to vinyl phenol derivatives produced by phenolic acid decarboxylase activity in Escherichia coli and several food-borne Gram-negative species

Ferulic, p-coumaric, and caffeic acids are phenolic acids present in soil, food, and gut, which have antimicrobial effects. Some Gram (+) bacteria metabolize these phenolic acids into vinyl derivatives due to phenolic acid decarboxylase activity (PAD) involved in the phenolic acid stress response (PASR). In this study, the antimicrobial activity of phenolic acids and their vinyl derivatives was tested on a panel of desirable and undesirable food-borne bacteria, especially Gram (?) species of Salmonella, Enterobacter, Klebsiella, and Pseudomonas, most of them without PAD activity. Native and engineered Escherichia coli strains either expressing or not PAD activity were included. Gram (?) bacteria of the panel were not significantly inhibited by phenolic acids at 3 mM, but were dramatically inhibited by the corresponding vinyl derivatives. On the contrary, Gram (+) bacteria displaying the PASR face the toxicity of phenolic acids by PAD activity and are not inhibited by vinyl phenols. In E. coli, the genes aaeB and marA, encoding efflux pumps for antimicrobial compounds, are upregulated by the addition of p-coumaric acid, but not by its derivative 4-vinyl phenol (p-hydroxystyrene). These results suggest that phenolic acids and their vinyl phenol derivatives produced by PAD (+) species could have a significant impact on undesirable or pathogenic food-borne Gram (?) bacteria in complex microbial ecosystems.     

Diversifying Incomes and Losing Landscape Complexity in Quilombola Shifting Cultivation Communities of the Atlantic Rainforest (Brazil)

Shifting cultivation systems have been blamed as the primary cause of tropical deforestation and are being transformed through various forms of conservation and development policies and through the emergence of new markets for cash crops. Here, we analyze the outcomes of different policies on land use/land cover change (LUCC) in a traditional, shifting cultivation landscape in the Atlantic Forest (Brazil), one of the world’s top biodiversity hotspots. We also investigate the impacts of those policies on the environment and local livelihoods in Quilombola communities, which are formed by descendants of former Maroon colonies. Our findings show that conservation and social policies have had mixed effects both on the conservation of the Atlantic Forest and on the livelihoods of the Quilombola. We conclude that future interventions in the region need to build on the new, functional links between sustainable livelihoods and biodiversity, where less restrictive state policies leave room for new opportunities in self-organization and innovation.     

Identification of Plasmodium berghei Oocyst Rupture Protein 2 (ORP2) domains involved in sporozoite egress from the oocyst

Sporozoites are the infective form of malaria parasites which are transmitted from the mosquito salivary glands to a new host in a mosquito blood meal. The sporozoites develop inside the sporogonic oocyst and it is crucial for the continuation of the life cycle that the oocyst ruptures to release sporozoites. We recently described two Plasmodium Oocyst Rupture Proteins (ORP1 and ORP2), localized at the oocyst capsule, that are each essential for rupture of the oocysts. Both ORPs contain a histone fold domain implicated in the mechanism of oocyst rupture, possibly through the formation of a heterodimer between the two histone fold domains. To gain an understanding of the function of the different regions of the ORP2 protein, we generated deletion mutants. We monitored oocyst formation and rupture as well as sporozoites in the salivary gland. Our results show that different regions of ORP2 play independent roles in sporozoite egress. Deleting the N-terminal histone fold domain of ORP2 blocked sporozoite egress from the oocyst. Progressive deletions from the C-terminal resulted in no or significantly impaired sporozoite egress.     

Unwinding of the Substrate Transmembrane Helix in Intramembrane Proteolysis

Intramembrane-cleaving proteases (I-CLiPs) activate pools of single-pass helical membrane protein signaling precursors that are key in the physiology of prokaryotic and eukaryotic cells. Proteases typically cleave peptide bonds within extended or flexible regions of their substrates, and thus the mechanism underlying the ability of I-CLiPs to hydrolyze the presumably α-helical transmembrane domain (TMD) of these membrane proteins is unclear. Using deep-ultraviolet resonance Raman spectroscopy in combination with isotopic labeling, we show that although predominantly in canonical α-helical conformation, the TMD of the established I-CLiP substrate Gurken displays 3₁₀-helical geometry. As measured by microscale thermophoresis, this substrate binds with high affinity to the I-CLiPs GlpG rhomboid and MCMJR1 presenilin homolog in detergent micelles. Binding results in deep-ultraviolet resonance Raman spectra, indicating conformational changes consistent with unwinding of the 3₁₀-helical region of the substrate’s TMD. This 3₁₀-helical conformation is key for intramembrane proteolysis, as the substitution of a single proline residue in the TMD of Gurken by alanine suppresses 3₁₀-helical content in favor of α-helical geometry and abolishes cleavage without affecting binding to the I-CLiP. Complemented by molecular dynamics simulations of the TMD of Gurken, our vibrational spectroscopy data provide biophysical evidence in support of a model in which the transmembrane region of cleavable I-CLiP substrates displays local deviations in canonical α-helical conformation characterized by chain flexibility, and binding to the enzyme results in conformational changes that facilitate local unwinding of the transmembrane helix for cleavage.     

Vitis vinifera berry metabolic composition during maturation: Implications of defoliation

Leaves are an important contributor toward berry sugar and nitrogen (N) accumulation, and leaf area, therefore, affects fruit composition during grapevine (Vitis vinifera) berry ripening. The aim of this study was to investigate the impact of leaf presence on key berry quality attributes in conjunction with the accumulation of primary berry metabolites. Shortly after the start of véraison (berry ripening), potted grapevines were defoliated (total defoliation and 25% of the control), and the accumulation of berry soluble solids, N and anthocyanins were compared to that of a full leaf area control. An untargeted approach was undertaken to measure the content in primary metabolites by gas chromatography/mass spectrometry. Partial and full defoliation resulted in reduced berry sugar and anthocyanin accumulation, while total berry N content was unaffected. The juice yeast assimilable N (YAN), however, increased upon partial and full defoliation. Remobilized carbohydrate reserves allowed accumulation of the major berry sugars during the absence of leaf photoassimilation. Berry anthocyanin biosynthesis was strongly inhibited by defoliation, which could relate to the carbon (C) source limitation and/or increased bunch exposure. Arginine accumulation, likely resulting from reserve translocation, contributed to increased YAN upon defoliation. Furthermore, assessing the implications on various products of the shikimate pathway suggests the C flux through this pathway to be largely affected by leaf source limitation during fruit maturation. This study provides a novel investigation of impacts of leaf C and N source presence during berry maturation, on the development of key berry quality parameters as underlined by alterations in primary metabolism.