Peptoids advance multidisciplinary research and undergraduate education in parallel: Sequence effects on conformation and lipid interactions

Peptoids are versatile peptidomimetic molecules with wide-ranging applications from drug discovery to materials science. An understanding of peptoid sequence features that contribute to both their three-dimensional structures and their interactions with lipids will expand functions of peptoids in varied fields. Furthermore, these topics capture the enthusiasm of undergraduate students who prepare and study diverse peptoids in laboratory coursework and/or in faculty led research. Here, we present the synthesis and study of 21 peptoids with varied functionality, including 19 tripeptoids and 2 longer oligomers. We observed differences in fluorescence spectral features for 10 of the tripeptoids that correlated with peptoid flexibility and relative positioning of chromophores. Interactions of representative peptoids with sonicated glycerophospholipid vesicles were also evaluated using fluorescence spectroscopy. We observed evidence of conformational changes effected by lipids for select peptoids. We also summarize our experiences engaging students in peptoid-based projects to advance both research and undergraduate educational objectives in parallel.     

Predictors of Phytophthora diversity and community composition in natural areas across diverse Australian ecoregions

Comprehensive understanding of the patterns and drivers of microbial diversity at a landscape scale is in its infancy, despite the recent ease by which soil communities can be characterized using massively parallel amplicon sequencing. Here we report on a comprehensive analysis of the drivers of diversity distribution and composition of the ecologically and economically important Phytophthora genus from 414 soil samples collected across Australia. We assessed 22 environmental and seven categorical variables as potential predictors of Phytophthora species richness, α and β diversity, including both phylogenetically and non‐phylogenically explicit methods. In addition, we classified each species as putatively native or introduced and examined the distribution with respect to putative origin. The two most widespread species, P. multivora and P. cinnamomi, are introduced, though five of the ten most widely distributed species are putatively native. Introduced taxa comprised over 54% of Australia's Phytophthora diversity and these species are known pathogens of annual and perennial crop habitats as well as urban landscapes and forestry. Patterns of composition were most strongly predicted by bioregion (R² = 0.29) and ecoregion (R² = 0.26) identity; mean precipitation of warmest quarter, mean temperature of the wettest quarter and latitude were also highly significant and described approximately 21, 14 and 13% of variation in NMDS composition, respectively. We also found statistically significant evidence for phylogenetic over‐dispersion with respect to key climate variables.This study provides a strong baseline for understanding biogeographical patterns in this important genus as well the impact of key plant pathogens and invasive Phytophthora species in natural ecosystems.     

Artificial selection shifts flowering phenology and other correlated traits in an autotetraploid herb

There is mounting evidence that plants are responding to anthropogenic climate change with shifts in flowering phenologies. We conducted a three-generation artificial selection experiment on flowering time in Campanulastrum americanum, an autotetraploid herb, to determine the potential for adaptive evolution of this trait as well as possible costs associated with enhanced or delayed flowering. Divergent selection for earlier and later flowering resulted in a 25-day difference in flowering time. Experiment-wide heritability was 0.31 and 0.23 for the initiation of flowering in early and late lines, respectively. Selection for earlier flowering resulted in significant correlated responses in other traits including smaller size, fewer branches, smaller floral displays, longer fruit maturation times, fewer seeds per fruit and slower seed germination. Results suggest that although flowering time shows the potential to adapt to a changing climate, phenological shifts may be associated with reduced plant fitness possibly hindering evolutionary change.     

Larvicidal potential of the polyol sweeteners erythritol and xylitol in two filth fly species

The house fly, Musca domestica (L.) (Diptera: Muscidae), and the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), are two filth flies responsible for significant economic losses in animal production. Although some chemical control products target adults of both species, differences in mouthpart morphology and behavior necessitates distinct modalities for each. For these reasons, larvicides are an attractive means of chemical control. We assessed the potential of the polyol sweeteners erythritol and xylitol as larvicides to the house fly and stable fly. LC₅₀ values of erythritol against 2^nd instar larvae were 34.94 mg/g media (house fly) and 22.10 mg/g media (stable fly). For xylitol, LC₅₀ values were 74.91 mg/g media (house fly) and 41.58 mg/g media (stable fly). When given a choice, neither species showed a preference for ovipositing in media treated with either sweetener at various concentrations or in media without sweetener. Significantly lower development from egg to adult was observed when the 2^nd instar LC₅₀ equivalent of each sweetener was present in the media compared to controls. Erythritol and xylitol both have larvicidal qualities, however their effective concentrations would necessitate creative product formulation and deployment methods to control all stages of developing flies.     

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.     

Discrete Element Framework for Modelling Extracellular Matrix,Deformable Cells and Subcellular Components

This paper presents a framework for modelling biological tissues based on discrete particles. Cell components (e.g. cell membranes, cell cytoskeleton, cell nucleus) and extracellular matrix (e.g. collagen) are represented using collections of particles. Simple particle to particle interaction laws are used to simulate and control complex physical interaction types (e.g. cell-cell adhesion via cadherins, integrin basement membrane attachment, cytoskeletal mechanical properties). Particles may be given the capacity to change their properties and behaviours in response to changes in the cellular microenvironment (e.g., in response to cell-cell signalling or mechanical loadings). Each particle is in effect an ‘agent’, meaning that the agent can sense local environmental information and respond according to pre-determined or stochastic events. The behaviour of the proposed framework is exemplified through several biological problems of ongoing interest. These examples illustrate how the modelling framework allows enormous flexibility for representing the mechanical behaviour of different tissues, and we argue this is a more intuitive approach than perhaps offered by traditional continuum methods. Because of this flexibility, we believe the discrete modelling framework provides an avenue for biologists and bioengineers to explore the behaviour of tissue systems in a computational laboratory.     

Genome-wide Association Study Identifies Shared Risk Loci Common to Two Malignancies in Golden Retrievers

Dogs, with their breed-determined limited genetic background, are great models of human disease including cancer. Canine B-cell lymphoma and hemangiosarcoma are both malignancies of the hematologic system that are clinically and histologically similar to human B-cell non-Hodgkin lymphoma and angiosarcoma, respectively. Golden retrievers in the US show significantly elevated lifetime risk for both B-cell lymphoma (6%) and hemangiosarcoma (20%). We conducted genome-wide association studies for hemangiosarcoma and B-cell lymphoma, identifying two shared predisposing loci. The two associated loci are located on chromosome 5, and together contribute ~20% of the risk of developing these cancers. Genome-wide p-values for the top SNP of each locus are 4.6×10^-7 and 2.7×10^-6, respectively. Whole genome resequencing of nine cases and controls followed by genotyping and detailed analysis identified three shared and one B-cell lymphoma specific risk haplotypes within the two loci, but no coding changes were associated with the risk haplotypes. Gene expression analysis of B-cell lymphoma tumors revealed that carrying the risk haplotypes at the first locus is associated with down-regulation of several nearby genes including the proximal gene TRPC6, a transient receptor Ca²⁺-channel involved in T-cell activation, among other functions. The shared risk haplotype in the second locus overlaps the vesicle transport and release gene STX8. Carrying the shared risk haplotype is associated with gene expression changes of 100 genes enriched for pathways involved in immune cell activation. Thus, the predisposing germ-line mutations in B-cell lymphoma and hemangiosarcoma appear to be regulatory, and affect pathways involved in T-cell mediated immune response in the tumor. This suggests that the interaction between the immune system and malignant cells plays a common role in the tumorigenesis of these relatively different cancers.     

Doxycycline reduces the migration of tuberous sclerosis complex‐2 null cells ‐ effects on RhoA‐GTPase and focal adhesion kinase

Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2‐deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2‐positive and TSC2‐negative mouse embryonic fibroblasts (MEF), 323‐TSC2‐positive and 323‐TSC2‐null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time‐lapse microscopy and manual cell counts respectively. RhoA‐GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2‐negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2‐null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2–59 μM. Doxycycline decreased RhoA‐GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2‐null cell migration. Thus doxycycline has potential as an anti‐migratory agent in the treatment of diseases with TSC2 dysfunction.