Mapping determinants of cytokine signaling via protein engineering

Cytokines comprise a large family of secreted ligands that are critical for the regulation of immune homeostasis. Cytokines initiate signaling via dimerization or oligomerization of the cognate receptor subunits, triggering the activation of the Janus Kinases (JAKs)/ signal transducer and activator of transcription (STATs) pathway and the induction of specific gene expression programs and bioactivities. Deregulation of cytokines or their downstream signaling pathways are at the root of many human disorders including autoimmunity and cancer. Identifying and understanding the mechanistic principles that govern cytokine signaling will, therefore, be highly important in order to harness the therapeutic potential of cytokines. In this review, we will analyze how biophysical (ligand-receptor binding geometry and affinity) and cellular (receptor trafficking and intracellular abundance of signaling molecules) parameters shape the cytokine signalosome and cytokine functional pleiotropy; from the initial cytokine binding to its receptor to the degradation of the cytokine receptor complex in the proteasome and/or lysosome. We will also discuss how combining advanced protein engineering with detailed signaling and functional studies has opened promising avenues to tackle complex questions in the cytokine signaling field

Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host.

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.post-print10768 K

Design and construction best practices for dugout earth dams in northeast British Columbia

In the Peace River Region in northeast British Columbia, dugout reservoirs, which classify as dams under the Water Sustainability Act, are used to store fresh water for oil and gas activities. These dams are constructed using native silt and clay soils with low strength properties and susceptibility to erosion and freeze-thaw. Site visits were conducted in August 2018 and May 2019 at seven dams. Soil samples were collected for laboratory testing to determine material properties for the soils used to construct the dams. Aerial images were captured with drones for topographic analysis using photogrammetry to determine as-built conditions. Numerical modelling was conducted to analyze seepage and stability under steady-state conditions. Probabilistic and sensitivity analyses were used to assess the impact of strength properties and dam geometry. Observed as-built conditions were compared with designs and existing best practices for construction. Where recommended best practices are not followed, the methods used were evaluated. The objective is to make recommendations for best practices for design and construction of dugout earth dams storing fresh water for oil and gas activities in northeast BC. Seven key areas of dam performance were analyzed at each of the seven dams: embankment stability, hydrotechnical considerations, internal seepage, surface erosion protection, construction, appurtenant structures, and maintenance. Embankment slopes were typically designed to follow recommendations but were often constructed steeper than designed. Erosion protection blankets are not as effective as riprap for protection against wave action. The value of cohesion varies for similar soils, and embankment stability is very sensitive to this parameter. Blanket drains are effective for managing seepage and preventing piping. Existing guidelines for freeboard and spillways are typically followed and are found to be appropriate. Lift thickness and compaction equipment varied, and dams should be overbuilt to ensure that adequate freeboard is maintained after consolidation settlement.Applied Science, Faculty ofEngineering, School of (Okanagan)Graduat

Enhanced resilience of older <i>C</i>. <i>glabrata</i> cells.

<p>(A) Older cells contributed to increased virulence in <i>Galleria</i> (n = 20 worms compared by Log-Rank test). (B) CFU counts continuously increased after <i>Galleria mellonella</i> infection with old BG2 cells (10 generations). Young BG2 cells (0–3 generations) were cleared before establishing infection within the first few hours post infection (n = 25 worms per time point). (C) Resistance to neutrophil-mediated killing was observed in older cells (experiments were run in duplicates, with six replicates, and compared by Student’s t-test). (D and E) NET induction was higher by old (14 and 28 generations) compared to young <i>C</i>. <i>glabrata</i> cells, but still significantly lower compared to that seen with hyphal <i>C</i>. <i>albicans</i>. % NETs indicates neutrophil nuclei >1,000 μm² over total neutrophils, and upper panel in D shows sytox staining of nuclei (experiments were run in duplicates, with six replicates, and compared by Student’s t-test). (F) Neutrophil Elastase nuclear localization was higher in old (14 and 28 generations) compared to young <i>C</i>. <i>glabrata</i> cells and unstimulated neutrophils (10 neutrophils were analyzed per condition and compared by one-way ANOVA) (G) H₂O₂ disc diffusion assay shows smaller zone of inhibition for both 14 and 28 generation old cells (experiments were run in triplicates and compared by Student’s t-test). (H) Epithelial cell adhesion assays demonstrate decreased adhesion of older cells (experiments were run in triplicates and compared by Student’s t-test). <i>*P</i> < 0.05, **<i>P <</i> 0.01, ***<i>P <</i> 0.001, ****<i>P <</i> 0.0001.</p

Phenotypic characterization of RLS in <i>C</i>. <i>glabrata</i>.

<p>(A) A box-and-whiskers plot demonstrates significant variability in RLSs of <i>C</i>. <i>glabrata</i> strains, shown as box plots with percentiles (n = 20 cells per experiment and 2–6 experiments compared by Log-Rank test). (B) Calcofluor stain reliably identifies generational age of <i>C</i>. <i>glabrata</i> (n = 100 cells). (C) Distinct doubling times during different lifespan phases, and (D) various cell morphologies were captured at time of death. (E) shows thicker cell wall in older BG2 cells by TEM (n = 100 cells compared by Student’s t-test). (F) <i>C</i>. <i>glabrata</i> demonstrated variable virulence in <i>G</i>. <i>mellonella</i> (n = 20 worms compared by Log-Rank test).</p

Gene ontology enrichment analysis for genes upregulated in old <i>C</i>. <i>glabrata</i> cells.

<p>Gene ontology enrichment analysis for genes upregulated in old <i>C</i>. <i>glabrata</i> cells.</p

Enhanced resilience of older <i>C</i>. <i>glabrata</i> cells stems from cell wall remodeling.

<p>(A) <i>C</i>. <i>glabrata</i> cells aged under sub-therapeutic fluconazole were more resistant to growth inhibition than younger cells when subjected to various concentrations of fluconazole for 4 h (experiments were run in triplicates and compared by Student’s t-test). (B) Older <i>C</i>. <i>glabrata</i> cells contain a slightly higher quantity of β1,3-glucans, but comparable amounts of β1,6-glucans and chitin (experiments were run in triplicates and compared by Student’s t-test). (C) Lipid analysis by GC-MS indicated that younger <i>C</i>. <i>glabrata</i> cells contain a higher amount of ergosterol compared to 14-generation-old cells (experiment was run in triplicates). <i>*P</i> < 0.05, **<i>P <</i> 0.01, ***<i>P <</i> 0.001.</p