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Canola (Brassica napus) is the major oilseed crop in Canada. Blackleg (Leptosphaeria maculans) causes yield losses annually, with a larger impact in the last two decades caused by breakdown of resistance in growers' fields. A 4‐year small‐plot field study from 2014 to 2017 in Manitoba investigated the impact of the B. napus–L. maculans interaction on the emergence of virulent isolates towards specific R genes. Doubled haploid B. napus 'Topas' introgression lines carrying single R genes were used, with a 2‐year canola–wheat rotation. Blackleg incidence was reduced by 40% in 2017 compared to 2014 for all R genes tested except Rlm4. In 2017, disease severity was reduced between 21% and 52% compared to 2014 for all R genes tested except Rlm2. There was a significant shift from AvrLm2 and AvrLm4 to virulent alleles avrLm2 and avrLm4, respectively, which led to the gain of virulence toward Rlm2 and Rlm4 Topas introgression lines within a year. Sequencing of the AvrLm2 gene of isolates from 2014 and 2015 revealed a shift of AvrLm2 to avrLm2 allele due to accumulation of point mutations. Masking of AvrLm3 phenotype by the presence of the AvrLm4‐7 allele was also confirmed by analysing phenotypes and genotypes of the isolates collected from 2014 to 2017. Based on these and previous findings, we demonstrate the need for an epidemiological model that takes into account complex molecular mechanisms allowing plant breeders to select appropriate R genes, including balanced cultivar rotation strategies to maintain blackleg resistance in the Canadian Prairies. [ABSTRACT FROM AUTHOR]

Research has been conducted on the ppk gene that encodes polyphosphate kinase. The null mutation in this bacterial pathogen gene of has been investigated.

The voltage-gated potassium channelKv1.3 is an established targetfor treatment of autoimmune diseases. Hence, there are intense effortsto develop immunosuppressant drugs from Kv1.3 blockers. ShK toxinfrom sea anemone is the most advanced peptide in this regard, butits lack of selectivity for Kv1.3 over Kv1.1 is an ongoing concern.The scorpion toxin HsTx1 is an equally potent blocker of Kv1.3, whichis also selective for Kv1.3. It is of interest to understand the molecularbasis of this selectivity as the lessons learned may suggest new avenuesfor enhancing the selectivity of other Kv1.3 blockers. Here we constructaccurate models of Kv1.x–HsTx1 complexes using docking andmolecular dynamics simulations. For each complex, the binding freeenergy of HsTx1 is determined from the potential of mean force calculations.Good agreement is found between the computed and experimental bindingfree energies, which increases confidence in the complex models. Comparisonof the binding modes of HsTx1 with Kv1.1 and Kv1.3 reveals that thelower affinity of HsTx1 for Kv1.1 is due to its inability to comeclose to the pore domain, which prevents the pore inserting lysinefrom making proper contacts with the tyrosine carbonyls in the selectivityfilter. [ABSTRACT FROM AUTHOR]

Toxins from marine animals offer novel drug leads for treatment of diseases involving ion channels. Computational methods could be very helpful in this endeavour in several ways, e.g., (i) constructing accurate models of the channel-toxin complexes using docking and molecular dynamics (MD) simulations; (ii) determining the binding free energies of toxins from umbrella sampling MD simulations; (iii) predicting the effect of mutations from free energy MD simulations. Using these methods, one can design new analogs of toxins with improved affinity and selectivity properties. Here we present a review of the computational methods and discuss their applications to marine toxins targeting potassium and sodium channels. Detailed examples from the potassium channel toxins--ShK from sea anemone and κ-conotoxin PVIIA--are provided to demonstrate capabilities of the computational methods to give accurate descriptions of the channel-toxin complexes and the energetics of their binding. An example is also given fromsodiumchannel toxins (µ-conotoxin GIIIA) to illustrate the differences between the toxin binding modes in potassium and sodium channels. [ABSTRACT FROM AUTHOR]

The voltage-gated potassium channel Kv1.3 is an attractivetargetfor treatment of autoimmune diseases. ShK toxin from sea anemone isone of the most potent blockers of Kv1.3, and therefore ShK and itsanalogues have been proposed as therapeutic leads for such diseases.Increasing the selectivity of the proposed leads for Kv1.3 over otherKv1 channels is a major issue in this endeavor. Here we study bindingof ShK toxin to Kv1 channels using free energy simulation methods.Homology models for Kv1.1 and Kv1.3 channels are constructed usingthe crystal structure of Kv1.2. The initial poses for the Kv1.x–ShKcomplexes are obtained using HADDOCK, which are then refined via moleculardynamics simulations. The binding mode in each complex is characterizedby identifying the strongly interacting residues, which compare wellwith available mutagenesis studies. For each complex, the potentialof mean force is calculated from umbrella sampling simulations, andthe corresponding absolute binding free energy is determined. Thecomputed binding free energies are in good agreement with the experimentaldata, which increases the confidence on the model complexes. The insightsgained on Kv1.x–ShK binding modes will be valuable in the developmentof new ShK analogues with better selectivity properties. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coil and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the β-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions. [ABSTRACT FROM AUTHOR]

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue while minimizing systemic drug exposure. beta-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity that allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. We evaluated the amino acid sequence of beta-lactamase from Enterobacter cloacae for the presence of human T-cell epitopes using a cell-based proliferation assay using samples from 65 community donors. We observed a low background response that is consistent with a lack of preexposure to this enzyme. beta-Lactamase was found to contain four CD4+ T-cell epitopes. For two of these epitopes, we identified single amino acid changes that result in significantly reduced proliferative responses while retaining stability and activity of the enzyme. The beta-lactamase variant containing both changes induces significantly less proliferation in human and mouse cell assays, and 5-fold lower levels of IgG1 in mice were observed after repeat administration of beta-lactamase variant with adjuvant. The beta-lactamase variant should be very suitable for the construction of ADEPT fusion proteins, as it combines high activity toward lactam prodrugs, high plasma stability, a monomeric architecture, and a relatively low risk of eliciting an immune response in patients.