Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNF_Y. CNF_Y consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNF_Y, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool.     

Genital system development of Williamia radiata (Gastropoda, Siphonariidae)

The adult anatomy and development of the genital system of the limpet Williamia radiata (Siphonariidae, Gastropoda) was investigated by means of 3D computer reconstruction and visualization of serial resin and paraplast light microscopical sections. As is characteristic for siphonariid species, the adult genital system consists of a single duct, the spermoviduct leading from the nidamental glandular system to the anteriorly located genital atrium with opening. The copulatory complex as well as the bursa copulatrix also open into the common genital atrium. The genital system develops from three separate anlagen. The posterior one appears first at a body length of 0.7 mm and gives rise to the ovotestis and part of the hermaphrodite duct. The nidamental glandular complex, the fertilization pouch-spermatheca complex, part of the hermaphrodite duct, the posterior part of the spermoviduct, and the bursa copulatrix develop later from the pallial anlage. Finally, the anterior anlage is formed on the right side of the head and gives rise to the genital atrium, the copulatory complex, and the anterior spermoviduct. This formation of the genital system from three, locally separated anlagen, differs strikingly from that of most other species of the Euthyneura. In both the Nudibranchia (Opisthobranchia) and the Stylommatophora (Pulmonata) development proceeds from a single site. We regarded this as a secondary condition as a result of derived features like heterochronies in development in these taxa. Comparison of development with that of other species of the Pulmonata allows conclusions on homology. The homology of the bursa copulatrix within the pulmonates is confirmed. The two separate chambers inside the spermoviduct of W. radiata correspond to oviduct and vas deferens of the freshwater Basommatophora.     

Trait-based approaches reveal fungal adaptations to nutrient-limiting conditions

The dependency of microbial activity on nutrient availability in soil is only partly understood, but highly relevant for nutrient cycling dynamics. In order to achieve more insight on microbial adaptations to nutrient limiting conditions, precise physiological knowledge is needed. Therefore, we developed an experimental system assessing traits of 16 saprobic fungal isolates in nitrogen (N) limited conditions. We tested the hypotheses that (1) fungal traits are negatively affected by N deficiency to a similar extent and (2) fungal isolates respond in a phylogenetically conserved fashion. Indeed, mycelial density, spore production and fungal activity (respiration and enzymatic activity) responded similarly to limiting conditions by an overall linear decrease. By contrast, mycelial extension and hyphal elongation peaked at lowest N supply (C:N 200), causing maximal biomass production at intermediate N contents. Optimal N supply rates differed among isolates, but only the extent of growth reduction was phylogenetically conserved. In conclusion, growth responses appeared as a switch from explorative growth in low nutrient conditions to exploitative growth in nutrient-rich patches, as also supported by responses to phosphorus and carbon limitations. This detailed trait-based pattern will not only improve fungal growth models, but also may facilitate interpretations of microbial responses observed in field studies.     

Misuse,perceived risk,and safety issues of household insecticides: Qualitative findings from focus groups in Arequipa,Peru

BackgroundThe current body of research on insecticide use in Peru deals primarily with application of insecticides offered through Ministry of Health-led campaigns against vector-borne disease. However, there is a gap in the literature regarding the individual use, choice and perceptions of insecticides which may influence uptake of public health-based vector control initiatives and contribute to the thousands of deaths annually from acute pesticide poisoning in Peru.MethodsResidents (n = 49) of the Alto Selva Alegre and CC districts of peri-urban Arequipa participated in seven focus group discussions (FGD). Using a FGD guide, two facilitators led the discussion and conducted a role-playing activity. this activity, participants insecticides (represented by printed photos of insecticides available locally) and pretended to “sell” the insecticides to other participants, including describing their qualities as though they were advertising the insecticide. The exercise was designed to elicit perceptions of currently available insecticides. The focus groups also included questions about participants’ preferences, use and experiences related to insecticides outside the context of this activity. Focus group content was transcribed, and qualitative data were analyzed with Atlas.ti and coded using an inductive process to generate major themes related to use and choice of insecticides, and perceived risks associated with insecticide use.ResultsThe perceived risks associated with insecticides included both short- and long-term health impacts, and safety for children emerged as a priority. However, in some cases insecticides were reportedly applied in high-risk ways including application of insecticides directly to children and bedding. Some participants attempted to reduce the risk of insecticide use with informal, potentially ineffective personal protective equipment and by timing application when household members were away. Valued insecticide characteristics, such as strength and effectiveness, were often associated with negative characteristics such as odor and health impacts. “Agropecuarios” (agricultural supply stores) were considered a trusted source of information about insecticides and their health risks.ConclusionsIt is crucial to characterize misuse and perceptions of health impacts and risks of insecticides at the local level, as well as to find common themes and patterns across populations to inform national and regional programs to prevent acute insecticide poisoning and increase community participation in insecticide-based vector control campaigns. We detected risky practices and beliefs about personal protective equipment, risk indicators, and safety levels that could inform such preventive campaigns, as well as trusted information sources such as agricultural stores for partnerships in disseminating information.     

Characterization of maximal enzyme catalytic rates in central metabolism of Arabidopsis thaliana

Availability of plant‐specific enzyme kinetic data is scarce, limiting the predictive power of metabolic models and precluding identification of genetic factors of enzyme properties. Enzyme kinetic data are measured in vitro, often under non‐physiological conditions, and conclusions elicited from modeling warrant caution. Here we estimate maximal in vivo catalytic rates for 168 plant enzymes, including photosystems I and II, cytochrome‐b6f complex, ATP‐citrate synthase, sucrose‐phosphate synthase as well as enzymes from amino acid synthesis with previously undocumented enzyme kinetic data in BRENDA. The estimations are obtained by integrating condition‐specific quantitative proteomics data, maximal rates of selected enzymes, growth measurements from Arabidopsis thaliana rosette with and fluxes through canonical pathways in a constraint‐based model of leaf metabolism. In comparison to findings in Escherichia coli, we demonstrate weaker concordance between the plant‐specific in vitro and in vivo enzyme catalytic rates due to a low degree of enzyme saturation. This is supported by the finding that concentrations of nicotinamide adenine dinucleotide (phosphate), adenosine triphosphate and uridine triphosphate, calculated based on our maximal in vivo catalytic rates, and available quantitative metabolomics data are below reported values and, therefore, indicate undersaturation of respective enzymes. Our findings show that genome‐wide profiling of enzyme kinetic properties is feasible in plants, paving the way for understanding resource allocation.     

Fostering accessible online education using Galaxy as an e-learning platform

The COVID-19 pandemic is shifting teaching to an online setting all over the world. The Galaxy framework facilitates the online learning process and makes it accessible by providing a library of high-quality community-curated training materials, enabling easy access to data and tools, and facilitates sharing achievements and progress between students and instructors. By combining Galaxy with robust communication channels, effective instruction can be designed inclusively, regardless of the students’ environments.