Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve throughout the coronavirus disease-19 (COVID-19) pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2, BA.2.75 and EG.5.1. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75 and EG.5.1) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.

Grazing preferences of three species of amoebae on cyanobacteria and green algae.

Twenty species/isolates of cyanobacteria and green algae were isolated from cyanobacterial bloom samples in lakes associated with the upper Qu'Appelle River drainage system in southern Saskatchewan, Canada. Three amoebae species (Cochliopodium sp., Vannella sp. and Vermamoeba vermiformis) were also isolated from one of these samples, and were subjected to grazing assays to determine which species of cyanobacteria or algae could potentially serve as a food source. Amoeba grazing rates were quantified based on the diameter of the plaque after 12 days on agar plate assays, and by estimation of the amoeba population growth rate from the rate of increase of plaque area. The common cyanobacterial bloom-formers Dolichospermum sp. and Aphanizomenon flos-aquae supported high growth rates for all three amoebae, while green algae, with the exception of one green alga/amoeba combination, did not support growth of the tested amoebae. Many of the cyanobacterial and algal isolates that did not support amoebae growth were ingested, suggesting that ingestion did not determine grazing success. Overall, while the cyanobacteria Dolichospermum sp. and Aphanizomenon flos-aquae were suitable food sources for the amoebae, the other cyanobacteria were grazed in an unpredictable manner, with some species/strains grazed by some amoebae and some species not grazed at all.

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.

BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

Allosteric Communications between Domains Modulate the Activity of Matrix Metalloprotease-1.

An understanding of the structure-dynamics relationship is essential for understanding how a protein works. Prior research has shown that the activity of a protein correlates with intradomain dynamics occurring at picosecond to millisecond timescales. However, the correlation between interdomain dynamics and the function of a protein is poorly understood. Here, we show that communications between the catalytic and hemopexin domains of matrix metalloprotease-1 (MMP1) on type 1 collagen fibrils correlate with its activity. Using single-molecule Förster resonance energy transfer, we identified functionally relevant open conformations in which the two MMP1 domains are well separated, which were significantly absent for catalytically inactive point mutant (E219Q) of MMP1 and could be modulated by an inhibitor or an enhancer of activity. The observed relevance of open conformations resolves the debate about the roles of open and closed MMP1 structures in function. We fitted the histograms of single-molecule Förster resonance energy transfer values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We used a two-state Poisson process to describe the dynamics and calculate the kinetic rates from the fit parameters. All-atom and coarse-grained simulations reproduced some of the experimental features and revealed substrate-dependent MMP1 dynamics. Our results suggest that an interdomain separation facilitates opening up the catalytic pocket so that the collagen chains come closer to the MMP1 active site. Coordination of functional conformations at different parts of MMP1 occurs via allosteric communications that can take place via interactions mediated by collagen even if the linker between the domains is absent. Modeling dynamics as a Poisson process enables connecting the picosecond timescales of molecular dynamics simulations with the millisecond timescales of single-molecule measurements. Water-soluble MMP1 interacting with water-insoluble collagen fibrils poses challenges for biochemical studies that the single-molecule tracking can overcome for other insoluble substrates. Interdomain communications are likely important for multidomain proteins.

Modelling the Structure and Dynamics of Biological Pathways.

There is a need for formalised diagrams that both summarise current biological pathway knowledge and support modelling approaches that explain and predict their behaviour. Here, we present a new, freely available modelling framework that includes a biologist-friendly pathway modelling language (mEPN), a simple but sophisticated method to support model parameterisation using available biological information; a stochastic flow algorithm that simulates the dynamics of pathway activity; and a 3-D visualisation engine that aids understanding of the complexities of a system's dynamics. We present example pathway models that illustrate of the power of approach to depict a diverse range of systems.

Elemental Composition of Commercially Available Cannabis Rolling Papers.

With the recent legalization of cannabis in multiple jurisdictions and widespread use as a medical treatment, there has been an increased focus on product safety and the potential impacts of contaminants on human health. One factor that has received little attention is the possible exposure to potentially hazardous levels of toxic elements from rolling (smoking) papers. The elemental composition of rolling papers is largely unregulated, with a minority of jurisdictions regulating papers only when they are part of a final cannabis product. This study reports the concentrations of 26 elements in commercially available rolling papers and estimates potential maximum exposures relative to USP232 and ICH Q3D dosages in pharmaceutical compounds. Exposure estimates indicate that the concentrations of several elements in some products, particularly Cu, Cr, and V, may present a potential hazard to frequent users. Several elements, including Ag, Ca, Ba, Cu, Ti, Cr, Sb, and possibly others, are likely present in elevated quantities in some papers due to product design and manufacturing processes. Our results further suggest that Cu-based pigments are used by a number of manufacturers and that regular use of these products might result in exposures as high as 4.5-11 times the maximum exposure limits. Further research to quantify the contribution of rolling papers to elemental exposure under realistic smoking conditions is warranted.

A spatially resolved single-cell lung atlas integrated with clinical and blood signatures distinguishes COVID-19 disease trajectories.

COVID-19 is characterized by a broad range of symptoms and disease trajectories. Understanding the correlation between clinical biomarkers and lung pathology during acute COVID-19 is necessary to understand its diverse pathogenesis and inform more effective treatments. Here, we present an integrated analysis of longitudinal clinical parameters, peripheral blood markers, and lung pathology in 142 Brazilian patients hospitalized with COVID-19. We identified core clinical and peripheral blood signatures differentiating disease progression between patients who recovered from severe disease compared with those who succumbed to the disease. Signatures were heterogeneous among fatal cases yet clustered into two patient groups: "early death" (<15 days until death) and "late death" (>15 days). Progression to early death was characterized systemically and in lung histopathological samples by rapid endothelial and myeloid activation and the presence of thrombi associated with SARS-CoV-2+ macrophages. In contrast, progression to late death was associated with fibrosis, apoptosis, and SARS-CoV-2+ epithelial cells in postmortem lung tissue. In late death cases, cytotoxicity, interferon, and T helper 17 (TH17) signatures were only detectable in the peripheral blood after 2 weeks of hospitalization. Progression to recovery was associated with higher lymphocyte counts, TH2 responses, and anti-inflammatory-mediated responses. By integrating antemortem longitudinal blood signatures and spatial single-cell lung signatures from postmortem lung samples, we defined clinical parameters that could be used to help predict COVID-19 outcomes.

Mass Spectrometry-Based Untargeted Metabolomics Reveals the Importance of Glycosylated Flavones in Patterned Lentil Seed Coats.

Lentil seed coats are rich in antioxidant polyphenols that are important for plant defense and have potential as valorized byproducts. Although biochemical differences among lentil seed coat colors have been previously studied, differences among seed coat patterns remain largely unexplored. This study used mass spectrometry-based untargeted metabolomics to investigate polyphenol differences among lentil seed coat patterns to search for biochemical pathways potentially responsible for seed coat pattern differences. Comparing patterned with non-patterned green lentil seed coats, 28 significantly upregulated metabolites were found in patterned seed coats; 19 of them were identified as flavones. Flavones were virtually absent in non-patterned seed coats, thereby strongly suggesting a blockage in their flavone biosynthetic pathway. Although the black pattern is not readily discernible on black seed coats, many of the same flavones found in green marbled seed coats were also found in black seed coats, indicating that black seed coats likely have a marbled pattern.

Monitoring of COVID-19 in wastewater across the Eastern Upper Peninsula of Michigan.

Wastewater-based epidemiology is being used as a tool to monitor the spread of COVID-19 and provide an early warning for the presence or increase of clinical cases in a community. The majority of wastewater-based epidemiology for COVID-19 tracking has been utilized in sewersheds that service populations in the tens-to-hundreds of thousands. Few studies have been conducted to assess the usefulness of wastewater in predicting COVID-19 clinical cases specifically in rural areas. This study collected samples from 16 locations across the Eastern Upper Peninsula of Michigan from June to December 2021. Sampling locations included 12 rural municipalities, a Tribal housing community and casino, a public university, three municipalities that also contained a prison, and a small island with heavy tourist traffic. Samples were analyzed for SARS-CoV-2 N1, N2, and variant gene copies using reverse transcriptase droplet digital polymerase chain reaction (RT-ddPCR). Wastewater N1 and N2 gene copies and clinical case counts were correlated to determine if wastewater results were predictive of clinical cases. Significant correlation between N1 and N2 gene copies and clinical cases was found for all sites (⍴= 0.89 to 0.48). N1 and N2 wastewater results were predictive of clinical case trends within 0-7 days. The Delta variant was detected in the Pickford and St. Ignace samples more than 12-days prior to the first reported Delta clinical cases in their respective counties. Locations with low correlation could be attributed to their high rates of tourism. This is further supported by the high correlation seen in the public university, which is a closed population. Long-term wastewater monitoring over a large, rural geographic area is useful for informing the public of potential outbreaks in the community regardless of asymptomatic cases and access to clinical testing.

Focusing the GWAS Lens on days to flower using latent variable phenotypes derived from global multienvironment trials.

Adaptation constraints within crop species have resulted in limited genetic diversity in some breeding programs and areas where new crops have been introduced, for example, for lentil (Lens culinaris Medik.) in North America. An improved understanding of the underlying genetics involved in phenology-related traits is valuable knowledge to aid breeders in overcoming limitations associated with unadapted germplasm and expanding their genetic diversity by introducing new, exotic material. We used a large, 18 site-year, multienvironment dataset phenotyped for phenology-related traits across nine locations and over 3 yr along with accompanying latent variable phenotypes derived from a photothermal model and principal component analysis (PCA) of days from sowing to flower (DTF) data for a lentil diversity panel (324 accessions), which has also been genotyped with an exome capture array. Genome-wide association studies (GWAS) on DTF across multiple environments helped confirm associations with known flowering-time genes and identify new quantitative trait loci (QTL), which may contain previously unknown flowering time genes. Additionally, the use of latent variable phenotypes, which can incorporate environmental data such as temperature and photoperiod as both GWAS traits and as covariates, strengthened associations, revealed additional hidden associations, and alluded to potential roles of the associated QTL. Our approach can be replicated with other crop species, and the results from our GWAS serve as a resource for further exploration into the complex nature of phenology-related traits across the major growing environments for cultivated lentil.

SARS-CoV-2 variant evasion of monoclonal antibodies based on in vitro studies.

Monoclonal antibodies (mAbs) offer a treatment option for individuals with severe COVID-19 and are especially important in high-risk individuals where vaccination is not an option. Given the importance of understanding the evolution of resistance to mAbs by SARS-CoV-2, we reviewed the available in vitro neutralization data for mAbs against live variants and viral constructs containing spike mutations of interest. Unfortunately, evasion of mAb-induced protection is being reported with new SARS-CoV-2 variants. The magnitude of neutralization reduction varied greatly among mAb-variant pairs. For example, sotrovimab retained its neutralization capacity against Omicron BA.1 but showed reduced efficacy against BA.2, BA.4 and BA.5, and BA.2.12.1. At present, only bebtelovimab has been reported to retain its efficacy against all SARS-CoV-2 variants considered here. Resistance to mAb neutralization was dominated by the action of epitope single amino acid substitutions in the spike protein. Although not all observed epitope mutations result in increased mAb evasion, amino acid substitutions at non-epitope positions and combinations of mutations also contribute to evasion of neutralization. This Review highlights the implications for the rational design of viral genomic surveillance and factors to consider for the development of novel mAb therapies.

Identification of allosteric fingerprints of alpha-synuclein aggregates in matrix metalloprotease-1 and substrate-specific virtual screening with single molecule insights.

Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previous reports showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. This paper reports quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is unavailable, binding poses were predicted by molecular docking of MMP1 with aSyn using ClusPro. MMP1 dynamics were simulated using predicted binding poses and compared with the experimental interdomain dynamics to identify an appropriate pose. The selected aSyn-MMP1 binding pose near aSyn residue K45 was simulated and analyzed to define conformational changes at the catalytic site. Allosteric residues in aSyn-bound MMP1 exhibiting strong correlations with the catalytic motif residues were compared with allosteric residues in free MMP1, and aSyn-specific residues were identified. The allosteric residues in aSyn-bound MMP1 are K281, T283, G292, G327, L328, E329, R337, F343, G345, N346, Y348, G353, Q354, D363, Y365, S366, S367, F368, P371, R372, V374, K375, A379, F391, A394, R399, M414, F419, V426, and C466. Shannon entropy was defined to quantify MMP1 dynamics. Virtual screening was performed against a site on selected aSyn-MMP1 binding poses, which showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for potential substrate-specific control of MMP1 activity in the future. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

A Framework for Investigating Rules of Life by Establishing Zones of Influence.

The incredible complexity of biological processes across temporal and spatial scales hampers defining common underlying mechanisms driving the patterns of life. However, recent advances in sequencing, big data analysis, machine learning, and molecular dynamics simulation have renewed the hope and urgency of finding potential hidden rules of life. There currently exists no framework to develop such synoptic investigations. Some efforts aim to identify unifying rules of life across hierarchical levels of time, space, and biological organization, but not all phenomena occur across all the levels of these hierarchies. Instead of identifying the same parameters and rules across levels, we posit that each level of a temporal and spatial scale and each level of biological organization has unique parameters and rules that may or may not predict outcomes in neighboring levels. We define this neighborhood, or the set of levels, across which a rule functions as the zone of influence. Here, we introduce the zone of influence framework and explain using three examples: (a) randomness in biology, where we use a Poisson process to describe processes from protein dynamics to DNA mutations to gene expressions, (b) island biogeography, and (c) animal coloration. The zone of influence framework may enable researchers to identify which levels are worth investigating for a particular phenomenon and reframe the narrative of searching for a unifying rule of life to the investigation of how, when, and where various rules of life operate.

Tracking SARS-CoV-2 mutations and variants through the COG-UK-Mutation Explorer.

COG-UK Mutation Explorer (COG-UK-ME, https://sars2.cvr.gla.ac.uk/cog-uk/-last accessed date 16 March 2022) is a web resource that displays knowledge and analyses on SARS-CoV-2 virus genome mutations and variants circulating in the UK, with a focus on the observed amino acid replacements that have an antigenic role in the context of the human humoral and cellular immune response. This analysis is based on more than 2 million genome sequences (as of March 2022) for UK SARS-CoV-2 data held in the CLIMB-COVID centralised data environment. COG-UK-ME curates these data and displays analyses that are cross-referenced to experimental data collated from the primary literature. The aim is to track mutations of immunological importance that are accumulating in current variants of concern and variants of interest that could alter the neutralising activity of monoclonal antibodies (mAbs), convalescent sera, and vaccines. Changes in epitopes recognised by T cells, including those where reduced T cell binding has been demonstrated, are reported. Mutations that have been shown to confer SARS-CoV-2 resistance to antiviral drugs are also included. Using visualisation tools, COG-UK-ME also allows users to identify the emergence of variants carrying mutations that could decrease the neutralising activity of both mAbs present in therapeutic cocktails, e.g. Ronapreve. COG-UK-ME tracks changes in the frequency of combinations of mutations and brings together the curated literature on the impact of those mutations on various functional aspects of the virus and therapeutics. Given the unpredictable nature of SARS-CoV-2 as exemplified by yet another variant of concern, Omicron, continued surveillance of SARS-CoV-2 remains imperative to monitor virus evolution linked to the efficacy of therapeutics.

Bee Visitation and Fruit Quality in Berries Under Protected Cropping Vary Along the Length of Polytunnels.

Wild and managed bees provide effective crop pollination services worldwide. Protected cropping conditions are thought to alter the ambient environmental conditions in which pollinators forage for flowers, yet few studies have compared conditions at the edges and center of growing tunnels. We measured environmental variables (temperature, relative humidity, wind speed, white light, and UV light) and surveyed activity of the managed honey bee, Apis mellifera L.; wild stingless bee, Tetragonula carbonaria Smith; and wild sweat bee, Homalictus urbanus Smith, along the length of 32 multiple open-ended polyethylene growing tunnels. These were spaced across 12 blocks at two commercial berry farms, in Coffs Harbour, New South Wales and Walkamin, North Queensland, Australia. Berry yield, fresh weight, and other quality metrics were recorded at discrete increments along the length of the tunnels. We found a higher abundance and greater number of flower visits by stingless bees and honey bees at the end of tunnels, and less frequent visits to flowers toward the middle of tunnels. The center of tunnels experienced higher temperatures and reduced wind speed. In raspberry, fruit shape was improved with greater pollinator abundance and was susceptible to higher temperatures. In blueberry, per plant yield and mean berry weight were positively associated with pollinator abundance and were lower at the center of tunnels than at the edge. Fruit quality (crumbliness) in raspberries was improved with a greater number of visits by sweat bees, who were not as susceptible to climatic conditions within tunnels. Understanding bee foraging behavior and changes to yield under protected cropping conditions is critical to inform the appropriate design of polytunnels, aid pollinator management within them, and increase economic gains in commercial berry crops.

Activity-dependent interdomain dynamics of matrix metalloprotease-1 on fibrin.

The roles of protein conformational dynamics and allostery in function are well-known. However, the roles that interdomain dynamics have in function are not entirely understood. We used matrix metalloprotease-1 (MMP1) as a model system to study the relationship between interdomain dynamics and activity because MMP1 has diverse substrates. Here we focus on fibrin, the primary component of a blood clot. Water-soluble fibrinogen, following cleavage by thrombin, self-polymerize to form water-insoluble fibrin. We studied the interdomain dynamics of MMP1 on fibrin without crosslinks using single-molecule Forster Resonance Energy Transfer (smFRET). We observed that the distance between the catalytic and hemopexin domains of MMP1 increases or decreases as the MMP1 activity increases or decreases, respectively. We modulated the activity using (1) an active site mutant (E219Q) of MMP1, (2) MMP9, another member of the MMP family that increases the activity of MMP1, and (3) tetracycline, an inhibitor of MMP1. We fitted the histograms of smFRET values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We modeled the dynamics as a two-state Poisson process and calculated the kinetic rates from the histograms and autocorrelations. Activity-dependent interdomain dynamics may enable allosteric control of the MMP1 function.

Genomic selection for lentil breeding: Empirical evidence.

Genomic selection (GS) is a marker-based selection initially suggested for livestock breeding and is being encouraged for crop breeding. Several statistical models are used to implement GS; however, none have been tested for use in lentil (Lens culinaris Medik.) breeding. This study was conducted to compare the accuracy of different GS models and prediction scenarios based on empirical data and to make recommendations for designing genomic selection strategies for lentil breeding. We evaluated nine single-trait (ST) models, two multiple-trait (MT) models, and a model that incorporates genotype × environment interaction (GEI) using populations from a lentil diversity panel and two recombinant inbred lines (RILs). The lines in all populations were phenotyped for five phenological traits and genotyped using a custom exome capture assay. Within-population, across-population, and across-environment genomic predictions were made. Prediction accuracy varied among the evaluated models, populations, prediction scenarios, and traits. Single-trait models showed similar accuracy in the absence of large effect quantitative trait loci (QTL) but BayesB outperformed all models when there were QTL with relatively large effects. Models that accounted for GEI and MT-GS models increased prediction accuracy for a low heritability trait by up to 66 and 14%, respectively. Moderate to high accuracies were obtained for within-population (range of .36-.85) and across-environment (range of .19-.89) predictions but across-population prediction accuracy was very low. Results suggest that GS can be implemented in lentil breeding to make predictions within populations and across environments, but across-population prediction should not be considered when the population size is small.

HNF4A and GATA6 Loss Reveals Therapeutically Actionable Subtypes in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3ß) a key regulator of glycolysis. Pharmacological inhibition of GSK3ß results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3ß inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC.

A graphical and computational modeling platform for biological pathways.

A major endeavor of systems biology is the construction of graphical and computational models of biological pathways as a means to better understand their structure and function. Here, we present a protocol for a biologist-friendly graphical modeling scheme that facilitates the construction of detailed network diagrams, summarizing the components of a biological pathway (such as proteins and biochemicals) and illustrating how they interact. These diagrams can then be used to simulate activity flow through a pathway, thereby modeling its dynamic behavior. The protocol is divided into four sections: (i) assembly of network diagrams using the modified Edinburgh Pathway Notation (mEPN) scheme and yEd network editing software with pathway information obtained from published literature and databases of molecular interaction data; (ii) parameterization of the pathway model within yEd through the placement of 'tokens' on the basis of the known or imputed amount or activity of a component; (iii) model testing through visualization and quantitative analysis of the movement of tokens through the pathway, using the network analysis tool Graphia Professional and (iv) optimization of model parameterization and experimentation. This is the first modeling approach that combines a sophisticated notation scheme for depicting biological events at the molecular level with a Petri net-based flow simulation algorithm and a powerful visualization engine with which to observe the dynamics of the system being modeled. Unlike many mathematical approaches to modeling pathways, it does not require the construction of a series of equations or rate constants for model parameterization. Depending on a model's complexity and the availability of information, its construction can take days to months, and, with refinement, possibly years. However, once assembled and parameterized, a simulation run, even on a large model, typically takes only seconds. Models constructed using this approach provide a means of knowledge management, information exchange and, through the computation simulation of their dynamic activity, generation and testing of hypotheses, as well as prediction of a system's behavior when perturbed.

Plant essential oils and potassium metabisulfite as repellents for Drosophila suzukii (Diptera: Drosophilidae).

Spotted wing drosophila, Drosophila suzukii, is a globally invasive pest of soft-skinned fruit. Females oviposit into ripening fruit and larvae cause direct destruction of tissues. As many plant essential oils are permitted food additives, they may provide a safe means of protecting fruit from D. suzukii infestation in both conventional and organic production systems. Twelve oils and potassium metabisulfite (KMS) were screened in the laboratory as repellents for D. suzukii flies. Most essential oils deterred D. suzukii flies from cotton wicks containing attractive raspberry juice. Peppermint oil was particularly effective, preventing almost all flies from contacting treated wicks and remaining 100% repellent for 6 d post-application. Thyme oil was unique because it caused high male mortality and reduced the number of responding flies compared to other oils. KMS was not found to be repellent to D. suzukii, but may have fumigant properties, particularly at high concentrations. Peppermint oil appears to be the best candidate for field testing to determine the effectiveness and feasibility of using essential oils as part of a push-pull management strategy against D. suzukii. This is the first time that essential oils have been evaluated and proven effective in preventing fruit-infesting flies from contacting attractive stimuli.