Response of wheat restricted‐tillering and vigorous growth traits to variables of climate change

The response of wheat to the variables of climate change includes elevated CO₂, high temperature, and drought which vary according to the levels of each variable and genotype. Independently, elevated CO₂, high temperature, and terminal drought affect wheat biomass and grain yield, but the interactive effects of these three variables are not well known. The aim of this study was to determine the effects of elevated CO₂ when combined with high temperature and terminal drought on the high‐yielding traits of restricted‐tillering and vigorous growth. It was hypothesized that elevated CO₂ alone, rather than combined with high temperature, ameliorates the effects of terminal drought on wheat biomass and grain yield. It was also hypothesized that wheat genotypes with more sink capacity (e.g. high‐tillering capacity and leaf area) have more grain yield under combined elevated CO₂, high temperature, and terminal drought. Two pairs of sister lines with contrasting tillering and vigorous growth were grown in poly‐tunnels in a four‐factor completely randomized split‐plot design with elevated CO₂ (700 µL L^?1), high day time temperature (3 °C above ambient), and drought (induced from anthesis) in all combinations to test whether elevated CO₂ ameliorates the effects of high temperature and terminal drought on biomass accumulation and grain yield. For biomass and grain yield, only main effects for climate change variables were significant. Elevated CO₂ significantly increased grain yield by 24–35% in all four lines and terminal drought significantly reduced grain yield by 16–17% in all four lines, while high temperature (3 °C above the ambient) had no significant effect. A trade‐off between yield components limited grain yield in lines with greater sink capacity (free‐tillering lines). This response suggests that any positive response to predicted changes in climate will not overcome the limitations imposed by the trade‐off in yield components.     

Mixed-mode loading of the cement-bone interface: a finite element study

While including the cement-bone interface of complete cemented hip reconstructions is crucial to correctly capture their response, its modelling is often overly simplified. In this study, the mechanical mixed-mode response of the cement-bone interface is investigated, taking into account the effects of the well-defined microstructure that characterises the interface. Computed tomography-based plain strain finite element analyses models of the cement-bone interface are built and loaded in multiple directions. Periodic boundaries are considered and the failure of the cement and bone fractions by cracking of the bulk components are included. The results compare favourably with experimental observations. Surprisingly, the analyses reveal that under shear loading no failure occurs and considerable normal compression is generated to prevent interface dilation. Reaction forces, crack patterns and stress fields provide more insight into the mixed-mode failure process. Moreover, the cement-bone interface analyses provide details which can serve as a basis for the development of a cohesive law.     

Strategies for bacterial expression of protein-peptide complexes: application to solubilization of papillomavirus E6

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif.     

Genetic predictors of response to photodynamictherapy

In Western countries, therapeutic management of patients affected by choroidal neovascularization (CNV) secondary to different typologies of macular degeneration represents a major health care problem. Age-related macular degeneration is the disease most frequently associated with CNV development. Schematically, CNVs can be distinguished into classic and occult subtypes, which are characterized by variable natural history and different responsiveness to some therapeutic procedures. At present, the dramatic vision loss due to CNV can be mainly treated by two interventional strategies, which are utilizable in either single or combined modalities: photodynamic therapy with verteporfin (PDT-V), and intravitreal administration of drugs acting against vascular endothelial growth factor. The combined use of PDT-V and anti-angiogenic drugs represents one of the most promising strategies against neovascular macular degeneration, but it unavoidably results in an expensive increase in health resource utilization. However, the positive data from several studies serve as a basis for reconsidering the role of PDT-V, which has undergone a renaissance prompted by the need for a more rational therapeutic approach toward CNV. New pharmacogenetic knowledge of PDT-V points to exploratory prospects to optimize the clinical application of this intriguing photothrombotic procedure. In fact, a Medline search provides data regarding the role of several single nucleotide polymorphisms (SNPs) as genetic predictors of CNV responsiveness to PDT-V. Specifically, correlations between SNPs and different levels of PDT-V efficacy have been detected by examining the gene variants influencing (i) thrombo-coagulative pathways, i.e. methylenetetrahydrofolate reductase (MTHFR) 677C>T (rs1801133), factor V (F5) 1691G>A (rs6025), prothrombin (F2) 20210G>A (rs1799963), and factor XIII-A (F13A1) 185G>T (rs5985); (ii) complement activation and/or inflammatory processes, i.e. complement factor H (CFH) 1277T>C (rs1061170), high-temperature requirement factor A1 (HTRA1) promoter -512G>A (rs11200638), and two variants of the C-reactive protein (CRP) gene (rs2808635 and rs876538); and (iii) production and bioavailability of vascular endothelial growth factor (VEGFA -2578C>A [rs699947] and rs2146323). This article critically evaluates both the clinical plausibility and the opportunity to utilize the most important SNP-response interactions of PDT-V for an effective upgrade of the current anti-CNV therapeutic scenario. In addition, the pharmacogenetics of a very severe post-PDT-V adverse event, i.e. a decrease in acute vision, is briefly discussed. A comprehensive appraisal of the findings reviewed in this article should be carefully considered to design future trials aimed at verifying (after proper genotypic stratification of the enrolled patients) whether these innovative pharmacogenetic approaches will be able to improve the multifaceted interventional management of neovascular macular degeneration.     

High hydrophobic amino acid exposure is responsible of the neurotoxic effects induced by E200K or D202N disease-related mutations of the human prion protein

Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a β-rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in α-helix, hPrP90-231 E200K is spontaneously refolded in a β-structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides.     

Elucidation of the Molecular Mechanisms of Action of the Natural Antimicrobial Peptide Subtilosin Against the Bacterial Vaginosis-associated Pathogen Gardnerella vaginalis

Subtilosin A is a 35-amino acid long cyclical peptide produced by Bacillus amyloliquefaciens that has potent antimicrobial activity against a variety of human pathogens, including the bacterial vaginosis-related Gardnerella vaginalis. The specific mode of action of subtilosin against G. vaginalis was elucidated by studying its effects on the proton motive force's (PMF) components: transmembrane electric potential (ΔΨ), transmembrane pH gradient (ΔpH), and intracellular ATP levels. The addition of subtilosin to G. vaginalis cells caused an immediate and total depletion of the ΔpH, but had no effect on the ΔΨ. Subtilosin also triggered an instant but partial efflux of intracellular ATP that was twofold higher than that of the positive control bacteriocin, nisin. Taken together, these data suggest that subtilosin inhibits G. vaginalis growth by creating transient pores in the cells' cytoplasmic membrane, leading to an efflux of intracellular ions and ATP and eventually cell death.