Factors Affecting the Folding of Pseudomonas aeruginosa OprF Porin into the One-Domain Open Conformer

Pseudomonas aeruginosa OprF is a largely monomeric outer membrane protein that allows the slow, nonspecific transmembrane diffusion of solutes. This protein folds into two different conformers, with the majority conformer folding into a two-domain conformation that has no porin activity and the minority conformer into a one-domain conformation with high porin activity and presumably consisting of a large β barrel. We examined the factors that control the divergent folding pathways of OprF. OprF contains four Cys residues in the linker region connecting the N-terminal β-barrel domain and the C-terminal globular domain in the majority conformer. Prevention of disulfide bond formation either by expression of OprF in an Escherichia coli dsbA strain grown with dithiothreitol or by replacement of all Cys residues with serine (CS OprF) increased the specific pore-forming activity of OprF significantly. Replacement of Phe160 with Ile at the end of the β-barrel termination signal as well as replacement of Lys164 in the linker region with Gly, Cys, or Glu increased porin activity 2-fold. Improving a potential β-barrel termination signal in the periplasmic domain by replacement of Asp211 with asparagine also increased porin activity. The porin activity could be improved about 5-fold by the combination of these replacements. OprF mutants with higher porin activity were shown to contain more one-domain conformers by surface labeling of the A312C residue in intact cells, as this residue is located in the periplasmic domain in the two-domain conformers. Finally, when the OprF protein was expressed in an E. coli strain lacking the periplasmic chaperone Skp, the CS OprF protein exhibited increased pore-forming activity

Translating Clinical Findings into Knowledge in Drug Safety Evaluation - Drug Induced Liver Injury Prediction System (DILIps)

Drug-induced liver injury (DILI) is a significant concern in drug development due to the poor concordance between preclinical and clinical findings of liver toxicity. We hypothesized that the DILI types (hepatotoxic side effects) seen in the clinic can be translated into the development of predictive in silico models for use in the drug discovery phase. We identified 13 hepatotoxic side effects with high accuracy for classifying marketed drugs for their DILI potential. We then developed in silico predictive models for each of these 13 side effects, which were further combined to construct a DILI prediction system (DILIps). The DILIps yielded 60–70% prediction accuracy for three independent validation sets. To enhance the confidence for identification of drugs that cause severe DILI in humans, the “Rule of Three” was developed in DILIps by using a consensus strategy based on 13 models. This gave high positive predictive value (91%) when applied to an external dataset containing 206 drugs from three independent literature datasets. Using the DILIps, we screened all the drugs in DrugBank and investigated their DILI potential in terms of protein targets and therapeutic categories through network modeling. We demonstrated that two therapeutic categories, anti-infectives for systemic use and musculoskeletal system drugs, were enriched for DILI, which is consistent with current knowledge. We also identified protein targets and pathways that are related to drugs that cause DILI by using pathway analysis and co-occurrence text mining. While marketed drugs were the focus of this study, the DILIps has a potential as an evaluation tool to screen and prioritize new drug candidates or chemicals, such as environmental chemicals, to avoid those that might cause liver toxicity. We expect that the methodology can be also applied to other drug safety endpoints, such as renal or cardiovascular toxicity

Relevance of the Axis Spermidine/eIF5A for Plant Growth and Development

One key role of the essential polyamine spermidine in eukaryotes is to provide the 4-aminobutyl moiety group destined to the post-translational modification of a lysine in the highly conserved translation factor eIF5A. This modification is catalyzed by two sequential enzymatic steps leading to the activation of eIF5A by the conversion of one conserved lysine to the unusual amino acid hypusine. The active translation factor facilitates the sequence-specific translation of polyproline sequences that otherwise cause ribosome stalling. In spite of the well-characterized involvement of active eIF5A in the translation of proline repeat-rich proteins, its biological role has been recently elucidated only in mammals, and it is poorly described at the functional level in plants. Here we describe the alterations in plant growth and development caused by RNAi-mediated conditional genetic inactivation of the hypusination pathway in Arabidopsis thaliana by knocking-down the enzyme deoxyhypusine synthase. We have uncovered that spermidine-mediated activation of elF5A by hypusination is involved in several aspects of plant biology such as the control of flowering time, the aerial and root architecture, and root hair growth. In addition this pathway is required for adaptation to challenging growth conditions such as high salt and high glucose medium and to elevated concentrations of the plant hormone ABA. We have also performed a bioinformatic analysis of polyproline-rich containing proteins as putative eIF5A targets to uncover their organization in clusters of protein networks to find molecular culprits for the disclosed phenotypes. This study represents a first attempt to provide a holistic view of the biological relevance of the spermidine-dependent hypusination pathway for plant growth and development.We thank Susana Tarraga from the Proteomics Service and Javier Forment from the Bioinformatics Service, both at the Instituto de Biologia Molecular y Celular de Plantas, for their technical assistance and to Jaideep Mathur (University of Guelph) for help with the confocal microscope images. We want to acknowledge Jose Leon for providing ft-10 seeds, and to R. Mirmira for sending us aliquots of the anti-hypusine antibody prior to its commercialization. Special thanks to CSIRO, MPG (Max-Planck-Gesellschaft zur Forderung der Wissenschaften) and Dr. Ian Moore (University of Oxford) for providing the binary plasmid pOpOff2. We acknowledge funding from the Spanish MICINN/MINECO (BIO2009-11818 and BIO2011-23828). BB was a recipient of a VALi+d predoctoral (ACIF2010/085) contract of the Generalitat Valenciana.Belda Palazón, B.; Almendáriz, C.; Martí Sanchis, E.; Carbonell Gisbert, J.; Ferrando Monleón, AR. (2016). Relevance of the Axis Spermidine/eIF5A for Plant Growth and Development. Frontiers in Plant Science. 7(245):1-14. https://doi.org/10.3389/fpls.2016.00245S114724

Habitat Composition and Connectivity Predicts Bat Presence and Activity at Foraging Sites in a Large UK Conurbation

Background: Urbanization is characterized by high levels of sealed land-cover, and small, geometrically complex, fragmented land-use patches. The extent and density of urbanized land-use is increasing, with implications for habitat quality, connectivity and city ecology. Little is known about densification thresholds for urban ecosystem function, and the response of mammals, nocturnal and cryptic taxa are poorly studied in this respect. Bats (Chiroptera) are sensitive to changing urban form at a species, guild and community level, so are ideal model organisms for analyses of this nature. Methodology/Principal Findings: We surveyed bats around urban ponds in the West Midlands conurbation, United Kingdom (UK). Sites were stratified between five urban land classes, representing a gradient of built land-cover at the 1 km 2 scale. Models for bat presence and activity were developed using land-cover and land-use data from multiple radii around each pond. Structural connectivity of tree networks was used as an indicator of the functional connectivity between habitats. All species were sensitive to measures of urban density. Some were also sensitive to landscape composition and structural connectivity at different spatial scales. These results represent new findings for an urban area. The activity of Pipistrellus pipistrellus (Schreber 1774) exhibited a non-linear relationship with the area of built land-cover, being much reduced beyond the threshold of,60 % built surface. The presence of tree networks appears to mitigate the negative effects of urbanization for this species

Biochemical quantitation of the eIF5A hypusination in Arabidopsis thaliana uncovers ABA-dependent regulation

The eukaryotic translation elongation factor eIF5A is the only protein known to contain the unusual amino acid hypusine which is essential for its biological activity. This post-translational modification is achieved by the sequential action of the enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The crucial molecular function of eIF5A during translation has been recently elucidated in yeast and it is expected to be fully conserved in every eukaryotic cell, however the functional description of this pathway in plants is still sparse. The genetic approaches with transgenic plants for either eIF5A overexpression or antisense have revealed some activities related to the control of cell death processes but the molecular details remain to be characterized. One important aspect of fully understanding this pathway is the biochemical description of the hypusine modification system. Here we have used recombinant eIF5A proteins either modified by hypusination or non-modified to establish a bi-dimensional electrophoresis (2D-E) profile for the three eIF5A protein isoforms and their hypusinated or unmodified proteoforms present in Arabidopsis thaliana. The combined use of the recombinant 2D-E profile together with 2D-E/western blot analysis from whole plant extracts has provided a quantitative approach to measure the hypusination status of eIF5A. We have used this information to demonstrate that treatment with the hormone abscisic acid produces an alteration of the hypusine modification system in Arabidopsis thaliana. 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Metabolic inactivation of estrogens in breast tissue by UDP-glucuronosyltransferase enzymes: an overview

The breast tissue is the site of major metabolic conversions of estradiol (E(2)) mediated by specific cytochromes P450 hydroxylations and methylation by catechol-O-methytransferase. In addition to E(2 )itself, recent findings highlight the significance of 4-hydroxylated estrogen metabolites as chemical mediators and their link to breast cancer development and progression, whereas, in opposition, 2-methoxylated estrogens appear to be protective. Recent data also indicate that breast tissue possesses enzymatic machinery to inactivate and eliminate E(2 )and its oxidized and methoxylated metabolites through conjugation catalyzed by UDP-glucuronosyltransferases (UGTs), which involves the covalent addition of glucuronic acid. In opposition to other metabolic pathways of estrogen, the UGT-mediated process leads to the formation of glucuronides that are devoid of biologic activity and are readily excreted from the tissue into the circulation. This review addresses the most recent findings on the identification of UGT enzymes that are responsible for the glucuronidation of E(2 )and its metabolites, and evidence regarding their potential role in breast cancer

Global lake responses to climate change

Climate change is one of the most severe threats to global lake ecosystems. Lake surface conditions, such as ice cover, surface temperature, evaporation and water level, respond dramatically to this threat, as observed in recent decades. In this Review, we discuss physical lake variables and their responses to climate change. Decreases in winter ice cover and increases in lake surface temperature modify lake mixing regimes and accelerate lake evaporation. Where not balanced by increased mean precipitation or inflow, higher evaporation rates will favour a decrease in lake level and surface water extent. Together with increases in extreme-precipitation events, these lake responses will impact lake ecosystems, changing water quantity and quality, food provisioning, recreational opportunities and transportation. Future research opportunities, including enhanced observation of lake variables from space (particularly for small water bodies), improved in situ lake monitoring and the development of advanced modelling techniques to predict lake processes, will improve our global understanding of lake responses to a changing climate