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.     

Allometric and Group Differences in the Xenarthran Femur

The aim of this study is to analyze shape variation in the xenarthran femur to gain insights into their behavior and locomotion. Specimens of both Cingulata (armadillos and glyptodonts) and Pilosa (anteaters and sloths) were studied and within each group body mass varies by several orders of magnitude. The main focus of the analysis was allometric variation in femoral shape in the three groups studied, armadillos, glyptodonts, and pilosans. Three dimensional coordinates were recorded for 40 homologous landmarks on each of 51 xenarthran femurs. The data were analyzed by geometric morphometric methods, and form space analysis was used to identify the allometric variation in each of the three groups. Across all groups, larger specimens tended to have larger articular surfaces, more robust femora generally, and the shape of the femoral condyles was more suited to extended postures. In addition, in larger specimens the medial condyle was much larger than the lateral condyle and the third trochanter was located more distally. The larger armadillo femora had a greater trochanter located considerably proximal to the femoral head and this is thought to improve femoral extension, but in glyptodonts and pilosans the larger specimens had a greater trochanter that was far lateral to the femoral head and this is interpreted as enhancing femoral rotation.     

Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.     

Cooperative Anti-Invasive Effect of Cdc42/Rac1 Activation and ROCK Inhibition in SW620 Colorectal Cancer Cells with Elevated Blebbing Activity

Rho GTPases are key regulators of tumour cell invasion and therefore constitute attractive targets for the design of anticancer agents. Several strategies have been developed to modulate their increased activities during cancer progression. Interestingly, none of these approaches took into account the existence of the well-known antagonistic relationship between RhoA and Rac1. In this study, we first compared the invasiveness of a collection of colorectal cancer cell lines with their RhoA, Rac1 and Cdc42 activities. A marked decrease of active Cdc42 and Rac1 correlated with the high invasive potential of the cell lines established from metastatic sites of colorectal adenocarcinoma (LoVo, SKCo1, SW620 and CoLo205). Conversely, no correlation between RhoA activity and invasiveness was detected, whereas the activity of its kinase effector ROCK was higher in cancer cell lines with a more invasive phenotype. In addition, invasiveness in these colon cancer cell lines was correlated with a typical round and blebbing morphology. We then tested whether treatment with PDGF to restore Cdc42 and Rac1 activities and/or with Y27632, a chemical inhibitor of ROCK, could decrease the invasiveness of SW620 cells. The association of both treatments substantially decreased the invasive potential of SW620 cells and this effect was accompanied by loss of membrane blebbing, restoration of a more elongated cell morphology and re-establishment of E-cadherin-dependent adherens junctions. This study paves the road to the development of therapeutic strategies in which different Rho GTPase modulators are combined to modulate the cross-talk between Rho GTPases and their specific input in metastatic progression.     

Trypanosoma cruzi IV Causing Outbreaks of Acute Chagas Disease and Infections by Different Haplotypes in the Western Brazilian Amazonia

Background

Chagas disease is an emergent tropical disease in the Brazilian Amazon Region, with an increasing number of cases in recent decades. In this region, the sylvatic cycle of Trypanosoma cruzi transmission, which constitutes a reservoir of parasites that might be associated with specific molecular, epidemiological and clinical traits, has been little explored. The objective of this work is to genetically characterize stocks of T. cruzi from human cases, triatomines and reservoir mammals in the State of Amazonas, in the Western Brazilian Amazon.

Methodology/Principal Findings

We analyzed 96 T. cruzi samples from four municipalities in distant locations of the State of Amazonas. Molecular characterization of isolated parasites from cultures in LIT medium or directly from vectors or whole human blood was performed by PCR of the non-transcribed spacer of the mini-exon and of the 24 S alfa ribosomal RNA gene, RFLP and sequencing of the mitochondrial cytochrome c oxidase subunit II (COII) gene, and by sequencing of the glucose-phosphate isomerase gene. The T. cruzi parasites from two outbreaks of acute disease were all typed as TcIV. One of the outbreaks was triggered by several haplotypes of the same DTU. TcIV also occurred in isolated cases and in Rhodnius robustus. Incongruence between mitochondrial and nuclear phylogenies is likely to be indicative of historical genetic exchange events resulting in mitochondrial introgression between TcIII and TcIV DTUs from Western Brazilian Amazon. TcI predominated among triatomines and was the unique DTU infecting marsupials.

Conclusion/Significance

DTU TcIV, rarely associated with human Chagas disease in other areas of the Amazon basin, is the major strain responsible for the human infections in the Western Brazilian Amazon, occurring in outbreaks as single or mixed infections by different haplotypes.     

Removal of surfactant dodecylbenzenesulfonate by consecutive use of ozonation and biodegradation

Successful surfactant removal from wastewater is often limited by the high concentration of the surfactant. The use of advanced oxidation processes can be the key to aid biological treatment of water containing high amounts of surfactants. The present study analyzes the biodegradation of the anionic surfactant sodium dodecylbenzenesulfonate (SDBS) and the effects of its combination with ozonation. SDBS pre‐ozonation favors the metabolism by microorganisms. Experimental results indicate that the application of a concentration of up to 60 μM of ozone for 60 min, prior to contact with microorganisms, increases the percentage of SDBS removed by biodegradation alone. These results demonstrate that the removal of SDBS and of the total organic carbon is increased by the consecutive use of ozonation and biodegradation.     

Lack of association of the G22A polymorphism of the ADA gene in patients with ankylosing spondylitis

Genes located outside the HLA region (6p21) have been considered as candidates for susceptibility to ankylosing spondylitis. We tested the hypothesis that the G22A polymorphism of the adenosine deaminase gene (ADA; 20q13.11) is associated with ankylosing spondylitis in 166 Brazilian subjects genotyped for the HLA*27 gene (47 patients and 119 controls matched for gender, age and geographic origin). The HLA-B*27 gene and the G22A ADA polymorphism were identified by PCR with sequence-specific oligonucleotide probes and PCR-RFLP, respectively. There were no significant differences in frequencies of ADA genotypes [odds ratio (OR) = 1.200, 95% confidence interval (CI) = 0.3102-4.643, P > 0.8] and ADA*01 and ADA*02 alleles (OR = 1.192, 95%CI = 0.3155-4.505, P > 0.8) in patients versus controls. We conclude that the G22A polymorphism is not associated with ankylosing spondylitis.     

Myosin II contributes to fusion pore expansion during exocytosis

During exocytosis, the fusion pore expands to allow release of neurotransmitters and hormones to the extracellular space. To understand the process of synaptic transmission, it is of outstanding importance to know the properties of the fusion pore and how these properties affect the release process. Many proteins have been implicated in vesicle fusion; however, there is little evidence for proteins involved in fusion pore expansion. Myosin II has been shown to participate in the transport of vesicles and, surprisingly, in the final phases of exocytosis, affecting the kinetics of catecholamine release in adrenal chromaffin cells as measured by amperometry. Here, we have studied single vesicle exocytosis in chromaffin cells overexpressing an unphosphorylatable form (T18AS19A RLC-GFP) of myosin II that produces an inactive protein by patch amperometry. This method allows direct determination of fusion pore expansion by measuring its conductance, whereas the release of catecholamines is recorded simultaneously by amperometry. Here we demonstrated that the fusion pore is of critical importance to control the release of catecholamines during single vesicle secretion in chromaffin cells. We proved that myosin II acts as a molecular motor on the fusion pore expansion by hindering its dilation when it lacks the phosphorylation sites.     

Monitoring of black mangrove restoration with nursery-reared seedlings on an arid coastal lagoon

Black mangrove (Avicennia germinans) seedlings (n=555) were grown from field-collected propagules for 3 months in a new type of terrestrial nursery. They were grown in clusters of five plants, and then they were transplanted to a clear-cut zone in a lagoon fringed by a mangrove forest at Laguna de Balandra, Baja California Sur, Mexico. Survival and plant development of transplants were monitored at 6-monthly intervals for 2 years. After 1 month, the survival of seedlings was 96%, later stabilizing at approximately 77%. After 24 months, 74% of the plants were still alive. The best cluster, showing maximum growth under mangrove swamp conditions in this arid zone, was a two-plant cluster. The lagoon has a low natural regeneration rate of 48 plants per 350 m² per 6 years of monitoring. This study shows the feasibility of restoring destroyed arid-coast lagoons with black mangroves.