Cloning and expression of a gene from Isochrysis galbana modifying fatty acid profiles in Escherichia coli

An ester hydrolase gene from the microalga Isochrysis galbana was cloned and expressed in Escherichia coli BL21 Rosetta 2?. The full-length putative gene has 1,146 base pairs and codes for a 381-amino acid polypeptide. The predicted molecular mass of the deduced protein is approximately 42.31 kDa, with a theoretical pI of 9.37. Slight similarity and identity were observed between the microalga sequence and various α/β-fold hydrolases found in diverse phyla. The catalytic triad corresponds to residues Ser²⁵⁴, Asp³⁰⁹, and His³⁴¹, with the nucleophilic catalytic residue Ser²⁵⁴ located in the pentapeptide consensus motif G-X-S²⁵⁴-X-G. The activity of the enzyme was established by fatty acid profile analysis of the membrane lipids. The expression of the protein in E. coli shifted the fatty acid composition predominantly towards C16:1 and C18:1 fatty acids. This enzyme is called I. galbana thioesterase/carboxylesterase (or IgTeCe). This novel gene is shown to have a potential for use in metabolic engineering to enhance the lipid yields of microalgae.     

Addressing water needs of freshwater ecosystems in life cycle impact assessment of water consumption: state of the art and applicability of ecohydrological approaches to ecosystem quality characterization

Purpose

In recent history, human development overbalanced towards economic growth has often been accompanied by the degradation and reduction of freshwater resources at the expense of freshwater dependent ecosystems. For their subsistence and correct functioning, understanding environmental water requirements (EWR) represents an area of great interest for life cycle impact assessment (LCIA) and it has been only marginally explored. The aim of this paper is to investigate how this concept has evolved in ecological and hydrological literature and how it can be better integrated in LCIA, to identify potential options for improvement of LCIA indicators in the short, mid and long term.

Methods

To address the limitations of existing LCIA approaches in modelling EWR, four families of EWR methods have been reviewed, namely hydrological, hydraulic, habitat simulation and holistic methods. Based on existing scientific literature and their broad application, 24 methods have been selected and their suitability to be adopted in LCIA has been evaluated against nine criteria, with regard to data management issues, accuracy, scientific robustness, and potential for future development. A semi-quantitative performance score has been subsequently assigned for each criterion, showing the main strengths and weaknesses of selected methods.

Results and discussion

The underlying rationale of the chosen approaches is markedly different, likewise the input information needed and results applicability. Hydrological methods are well suited for the development of global models and they are the only ones currently considered in LCIA, although their applicability remains limited to water stress indicators. Habitat modelling is identified as an essential step for the development of mechanistic LCIA models and endpoint indicators. In this respect, hydraulic, habitat simulation and holistic methods are fit for the purpose. However, habitat simulation methods represent the best compromise between scientific robustness and applicability in LCIA. For this reason, a conceptual framework for the development of habitat-based characterization factors has been proposed. Among the evaluated habitat simulation methods, ESTIMHAB showed the best performance and was the method retained for the development of an LCIA model that will assess the consequences of water consumption on stream ecosystems.

Conclusions

This study identifies the advantages of specific modelling approaches for the assessment of water requirements for ecosystems. Selected methods could support the development of LCIA models at different levels. In the short-term for improving environmental relevance of water stress indicators, and in the mid/long-term to build up midpoint habitat indicators relating water needs of ecosystems with new endpoint metrics.

     

The Genomic Basis of Color Pattern Polymorphism in the Harlequin Ladybird

1. Download high-res image (251KB)
2. Download full-size image

     

Effects of auxins,cytokinins, carbohydrates and amino acids on somatic embryogenesis induction from shoot apices of pea

Studies were conducted to test the effects of various auxins, cytokinins, carbohydrates and amino acids on somatic embryogenesis from shoot apices of pea (Pisum sativum L.) cultured on a sole medium. Picloram (4.5 M) and 4-chlorophenoxyacetic acid (45 M) were the most effective auxins. Addition of cytokinins (benzyladenine, zeatin, kinetin) to auxin-containing medium reduced embryo production. Amino acids (glutamine, alanine, proline) did not improve somatic embryogenesis. Carbohydrate seemed to be a critical factor. Embryogenic efficiency and embryo development were promoted by high carbohydrate concentration. The best results were obtained with fructose (252–504 mM); the number of somatic embryos per cultured explant was 3- to 4-fold higher compared to the control (84 mM sucrose). From these results, an optimized induction medium is proposed.Abbreviations BA benzyladenine - 4-CPA 4-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige & Skoog - EE embryogenic explants - G globular somatic embryos     

Variations in protein expression related to human eosinophil heterogeneity

In hypereosinophilic patients, eosinophil heterogeneity has been assessed mainly according to morphologic and biologic criteria. In order to investigate the molecular basis of such heterogeneity, biochemical analysis was performed on various eosinophil subpopulations fractionated on metrizamide gradients. Whole cell extracts from purified eosinophils disrupted with a nonionic (NP-40) detergent were successively analyzed by SDS-PAGE and two-dimensional electrophoresis (isoelectric focusing or nonequilibrium pH gradient electrophoresis in the first dimension). Hypodense eosinophils that sediment in the lightest density gradients (18 to 22% metrizamide solution) differed from other purified eosinophils (intermediate and normodense eosinophils respectively collected in 22 to 23% and 23 to 25% metrizamide solutions). Comparative analysis of protein patterns on both monodimensional and bidimensional electrophoresis showed that a basic protein of Mr 51 kDa, present on normodense or intermediate eosinophils, was poorly detected in the case of hypodense eosinophils. In contrast, two other proteins with apparent Mr of about 23 kDa and 41 kDa were exclusively or predominantly identified in these latter cell fractions. Immunochemical analysis with polyclonal antibodies against eosinophil basic proteins and enzymatic assays revealed that the 51-kDa polypeptide could be related to an eosinophil peroxidase-like molecule. In addition, the two proteins detected only in hypodense eosinophils might be related to proteins newly synthesized by in vivo activated eosinophils. Our results suggest that variations in protein expression might represent a good marker of in vivo activation.     

Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL.

Erwinia chrysanthemi secretes, by the type II secretory pathway, a large number of enzymes, including cellulases and pectinases. This process requires the products of the out genes, which are widely conserved in Gram-negative bacteria. The Out proteins are thought to form a membrane-associated multiprotein complex. Here, we investigated interaction between OutE, the putative ATP binding component, and OutL, an inner membrane protein. We showed, by limited proteolysis, genetic suppression and the yeast two-hybrid system, that OutE and OutL interact directly. Analysis of truncated forms of OutE demonstrated that the N terminus of OutE (residues 1-97) is important for the OutE/OutL interaction. Moreover, results from the yeast two-hybrid system suggested that OutE and OutL are each able to form homomultimers. The region required for homomultimerisation of OutE is located in its C terminus. Limited proteolysis assay indicated that OutE induces a conformational change in OutL, in both its cytoplasmic and periplasmic domains. Moreover, the secretion process requires a conformational change in OutE which depends on both the interaction with OutL and on the presence of an intact Walker A motif in OutE. Our results support the view that interaction occurring on the cytoplasmic side influences the events occurring in the outer membrane. We discuss a model in which OutE uses ATP to control the assembly of the type II secretion machinery.     

Spermine-NBD as fluorescent probe for studies of the polyamine transport system in Leishmania donovani

This study describes the synthesis of fluorescent probes as potential substrates for the polyamine transport system (PTS) of Leishmania donovani. A competitive radioassay was used to determine the most efficient probe. We observed that the conjugate spermine-nitrobenzofurazan (Spm-NBD) was able to compete with [³H]-spermidine in L. donovani at a potent IC₅₀ of 60 µM.     

Intranasal vaccine from whole Leishmania donovani antigens provides protection and induces specific immune response against visceral leishmaniasis

Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.     

Monitoring Insect Transposable Elements in Large Double-Stranded DNA Viruses Reveals Host-to-Virus and Virus-to-Virus Transposition

The mechanisms by which transposable elements (TEs) can be horizontally transferred between animals are unknown, but viruses are possible candidate vectors. Here, we surveyed the presence of host-derived TEs in viral genomes in 35 deep sequencing data sets produced from 11 host–virus systems, encompassing nine arthropod host species (five lepidopterans, two dipterans, and two crustaceans) and six different double-stranded (ds) DNA viruses (four baculoviruses and two iridoviruses). We found evidence of viral-borne TEs in 14 data sets, with frequencies of viral genomes carrying a TE ranging from 0.01% to 26.33% for baculoviruses and from 0.45% to 7.36% for iridoviruses. The analysis of viral populations separated by a single replication cycle revealed that viral-borne TEs originating from an initial host species can be retrieved after viral replication in another host species, sometimes at higher frequencies. Furthermore, we detected a strong increase in the number of integrations in a viral population for a TE absent from the hosts’ genomes, indicating that this TE has undergone intense transposition within the viral population. Finally, we provide evidence that many TEs found integrated in viral genomes (15/41) have been horizontally transferred in insects. Altogether, our results indicate that multiple large dsDNA viruses have the capacity to shuttle TEs in insects and they underline the potential of viruses to act as vectors of horizontal transfer of TEs. Furthermore, the finding that TEs can transpose between viral genomes of a viral species sets viruses as possible new niches in which TEs can persist and evolve.