A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.     

In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein

Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.     

A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.     

Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol

Paclitaxel (Taxol) is a widely used anticancer isoprenoid produced by the secondary metabolism of yew (Taxus sp.) trees. However, only limited amounts of Taxol or related metabolites (taxoids) can be obtained from the currently available sources. In this work we have taken the first step toward genetically engineering the biosynthesis of taxoids in angiosperms. The first committed step in Taxol biosynthesis is the production of taxadiene from geranylgeranyl diphosphate (GGPP), catalyzed by the plastid-localized enzyme taxadiene synthase (TXS). A recombinant T. baccata TXS lacking the putative plastid targeting peptide and fused to a C-terminal histidine (His) tag was shown to be enzymatically active in Escherichia coli. Constitutive production of the full-length His-tagged enzyme in Arabidopsis thaliana plants led to the accumulation of taxadiene and concomitant growth retardation and decreased levels of photosynthetic pigment in transgenic plants. Although these phenotypes may derive from a toxic effect of taxadiene, the lower accumulation of endogenous plastid isoprenoid products such as carotenoids and chlorophylls in transgenic plants also suggests that the constitutive production of an active TXS enzyme might alter the balance of the GGPP pool. Induction of transgene expression using a glucocorticoid-mediated system consistently resulted in a more efficient recruitment of GGPP for the production of taxadiene, which reached levels 30-fold higher than those in plants constitutively expressing the transgene. This accomplishment illustrates the possibility of engineering the production of taxoids and other GGPP-derived isoprenoids in crop plants despite the constraints associated with limited knowledge with regard to regulation of GGPP availability.     

PARP-1 Regulates Metastatic Melanoma through Modulation of Vimentin-induced Malignant Transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.     

A standardized assessment of forest mammal communities reveals consistent functional composition and vulnerability across the tropics

The understanding of global diversity patterns has benefitted from a focus on functional traits and how they relate to variation in environmental conditions among assemblages. Distant communities in similar environments often share characteristics, and for tropical forest mammals, this functional trait convergence has been demonstrated at coarse scales (110–200 km resolution), but less is known about how these patterns manifest at fine scales, where local processes (e.g. habitat features and anthropogenic activities) and biotic interactions occur. Here, we used standardized camera trapping data and a novel analytical method that accounts for imperfect detection to assess how the functional composition of terrestrial mammal communities for two traits – trophic guild and body mass – varies across 16 protected areas in tropical forests and three continents, in relation to the extent of protected habitat and anthropogenic pressures. We found that despite their taxonomic differences, communities generally have a consistent trophic guild composition, and respond similarly to these factors. Insectivores were found to be sensitive to the size of protected habitat and surrounding human population density. Body mass distribution varied little among communities both in terms of central tendency and spread, and interestingly, community average body mass declined with proximity to human settlements. Results indicate predicted trait convergence among assemblages at the coarse scale reflects consistent functional composition among communities at the local scale, suggesting that broadly similar habitats and selective pressures shaped communities with similar trophic strategies and responses to drivers of change. These similarities provide a foundation for assessing assemblages under anthropogenic threats and sharing conservation measures.