The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles on toxin oligomerization and pore formation

The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a β-sheet. Regarding to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the β-sheet allowing insertion of β-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects in Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, N-terminal region is able to induce aggregation of Cyt1Aa toxin in solution. Finally, Cterminal domain composed mainly of β-strands, is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation while the C-terminal domain in the interaction of the toxin with the lipid membrane.Research was funded in part through grants from the National Institutes of Health, 1R01 AI066014, DGAPA/UNAM IN218608 and IN210208-N, CONACyT U48631-Q 478. IRdE received a José Castillejo postdoctoral grant, and a mobility grant for teaching and research staff of UPNA, Spain

Dominant Negative Mutants of Bacillus thuringiensis Cry1Ab Toxin Function as Anti-Toxins: Demonstration of the Role of Oligomerization in Toxicity

BACKGROUND:Bacillus thuringiensis Cry toxins, that are used worldwide in insect control, kill insects by a mechanism that depends on their ability to form oligomeric pores that insert into the insect-midgut cells. These toxins are being used worldwide in transgenic plants or spray to control insect pests in agriculture. However, a major concern has been the possible effects of these insecticidal proteins on non-target organisms mainly in ecosystems adjacent to agricultural fields. METHODOLOGY/PRINCIPAL FINDINGS:We isolated and characterized 11 non-toxic mutants of Cry1Ab toxin affected in different steps of the mechanism of action namely binding to receptors, oligomerization and pore-formation. These mutant toxins were analyzed for their capacity to block wild type toxin activity, presenting a dominant negative phenotype. The dominant negative phenotype was analyzed at two levels, in vivo by toxicity bioassays against susceptible Manduca sexta larvae and in vitro by pore formation activity in black lipid bilayers. We demonstrate that some mutations located in helix alpha-4 completely block the wild type toxin activity at sub-stoichiometric level confirming a dominant negative phenotype, thereby functioning as potent antitoxins. CONCLUSIONS/SIGNIFICANCE:This is the first reported case of a Cry toxin dominant inhibitor. These data demonstrate that oligomerization is a fundamental step in Cry toxin action and represent a potential mechanism to protect special ecosystems from the possible effect of Cry toxins on non-target organisms

The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins

The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Sequencing and de novo transcriptome assembly of Anthopleura dowii Verrill (1869), from Mexico

Next-generation technologies for determination of genomics and transcriptomics composition have a wide range of applications. Moreover, the development of tools for big data set analysis has allowed the identification of molecules and networks involved in metabolism, evolution or behavior. By natural habitats aquatic organisms have implemented molecular strategies for survival, including the production and secretion of toxic compounds for their predators; therefore these organisms are possible sources of proteins or peptides with potential biotechnological application. In the last decade anthozoans, mainly octocorals but also sea anemones, have been proben to be a source of natural products. Members of the genus Anthopleura are one of the best known and most studied sea anemones because they are common constituents of rocky intertidal communities and show interesting ecological and biological phenomena (e.g. intraespecific competition, symbiosis, etc.); however, many aspects of these taxa remain in need to be analyzed. This work describes the transcriptome sequencing of Anthopleura dowii Verrill, 1869 (Cnidaria: Anthozoa: Actiniaria); this is the first report of this kind for these species. The data set used to construct the transcriptome has been deposited on NCBI's database. Illumina sequence reads are available under BioProject accession number PRJNA329297 and Sequence Read Archive under accession number SRP078992

Graphene-Based Biosensors for Molecular Chronic Inflammatory Disease Biomarker Detection

Chronic inflammatory diseases, such as cancer, diabetes mellitus, stroke, ischemic heart diseases, neurodegenerative conditions, and COVID-19 have had a high number of deaths worldwide in recent years. The accurate detection of the biomarkers for chronic inflammatory diseases can significantly improve diagnosis, as well as therapy and clinical care in patients. Graphene derivative materials (GDMs), such as pristine graphene (G), graphene oxide (GO), and reduced graphene oxide (rGO), have shown tremendous benefits for biosensing and in the development of novel biosensor devices. GDMs exhibit excellent chemical, electrical and mechanical properties, good biocompatibility, and the facility of surface modification for biomolecular recognition, opening new opportunities for simple, accurate, and sensitive detection of biomarkers. This review shows the recent advances, properties, and potentialities of GDMs for developing robust biosensors. We show the main electrochemical and optical-sensing methods based on GDMs, as well as their design and manufacture in order to integrate them into robust, wearable, remote, and smart biosensors devices. We also describe the current application of such methods and technologies for the biosensing of chronic disease biomarkers. We also describe the current application of such methods and technologies for the biosensing of chronic disease biomarkers with improved sensitivity, reaching limits of detection from the nano to atto range concentration

Shade coffee plantations favor specialization, decrease robustness and increase foraging in hummingbird-plant networks

Agroforestry systems, such as shade coffee plantations, favor the maintenance of woody vegetation, which promotes the presence of pollinators such as hummingbirds. Many shade coffee plantations in Mesoamerica have been abandoned due to the fall in coffee prices and undergone succession processes that increase vegetation complexity. Alternatively, they have been replaced by cattle pastures, which negatively affect hummingbird-plant interactions. Here, we describe the structure of hummingbird-plant interaction networks in three types of land use―late-successional forests, coffee plantations, and cattle pastures―that were derived from a cloud forest in southern Mexico. For one year (2019–2020), we collected bimonthly quantitative data on hummingbird-plant interactions for each type of land use. We found that the network of each land use had a heterogeneous structure, and most species had few linkages. The late forests and coffee plantations had more species, pairs of interactions, and modularity than the cattle pastures. The cattle pasture network had the greatest robustness due to the presence of generalist hummingbirds, which are important for network cohesion in a great disturbance scenario. Furthermore, hummingbird visits were influenced by plant traits, such as foliage height diversity. The study findings suggest that the conversion of natural habitats have implications for the conservation of plant-pollinator interactions and that generalist pollinator species are key to disturbance resiliency