Hard to kill: Inactivation of Plasmodiophora brassicae resting spores using chemical disinfectants

Biosafety practices, such as bioexclusion via sanitization, can prevent the spread of infectious soilborne threats such as the clubroot pathogen, Plasmodiophora brassicae. Twenty three chemical disinfectants were evaluated for efficacy against P. brassicae resting spores. Evans blue staining was used to directly measure the viability of P. brassicae resting spores after 20 min exposures to ten concentrations of each of the 23 chemical disinfectants. Only nine disinfectants were capable of greater than 95% inactivation, and only five were capable of inactivating >99% of resting spores. Bleach (sodium hypochlorite) and Spray Nine® were the most effective disinfectants for inactivation of clubroot resting spores. AES 2500, SaniDate® and ethanol also inactivated >99% of resting spores, but only at very high concentrations. A time course experiment showed that 10 to 12 min contact time was sufficient for ≥ 95% resting spore inactivation with Spray Nine® and sodium hypochlorite, but ≥ 30 min contact was required for other disinfectants evaluated. These results will assist in guiding management recommendations for sanitization aimed at bioexclusion and biocontainment of P. brassicae.

Capitalizing on transcriptome profiling to optimize and identify targets for promoting early murine folliculogenesis in vitro

In vitro ovarian follicle culture is an active area of research towards providing fertility options for survivors of childhood cancer. Late-stage murine follicles (multilayer secondary and onwards) can be cultured successfully to maturity to obtain a meiotically competent oocyte for fertilization, but primordial and primary follicles usually die in culture because many key components of early follicle development are still unknown and difficult to mimic in vitro. To engineer a biomimetic three-dimensional culture system with high efficacy and reproducibility for the clinic, detailed mechanisms of early folliculogenesis must be uncovered. Previous studies have shown that primary murine follicles co-cultured in groups, in contrast to single follicles cultured in isolation, can reach preovulatory size and produce competent oocytes, but the factors accounting for the synergy of follicle co-culture are still unknown. To probe the underlying mechanisms of successful follicle co-culture, we conducted a time-course experiment for murine follicles encapsulated in 0.3% alginate hydrogels and compared between two conditions: groups of 5 (5X) versus groups of 10 (10X). For every 2 days during the course of 12 days, follicles were dissociated and somatic cells were isolated for microarray-based gene expression analysis (n = 380 follicles for 5X and n = 430 follicles for 10X). Gene activities in follicles co-cultured in larger groups (10X) had a distinct transcriptomic profile of key genes and pathways such as prolactin signaling and angiogenesis-related genes when compared to cells from follicles co-cultured in the smaller cohort (5X). To benchmark the results for follicles grown in culture, we compared our microarray data to data from murine follicles freshly isolated from the ovary at comparable stages of development previously published by Bernabé et al. Comparison of these datasets identified similarities and differences between folliculogenesis in the native microenvironment and the engineered in vitro system. A more detailed understanding of follicle growth in vitro will not only allow for better culture methods but also advance the field towards providing improved fertility options for survivors of childhood cancer.

142 Transcriptome Analysis Captures Radiation Exposure in a Dose-sensitive Manner and Predicts Short-term Survival in a Mouse Model

Introduction A mass exposure to irradiation would be a challenge to health care systems. A simple tool or test that indicates the intensity of the absorbed radiation or the chances of survivability would be invaluable in this scenario. To identify biomarkers that potentially could provide novel biodosimetry tools, we have conducted a radiation dose-response and time-course experiment in mice that includes an assessment of the transcriptome of the skin. Methods Groups of mice (n=5) received whole-body X-ray exposures (0, 1, 3, 6, or 20Gy) and skin biopsies were obtained from each animal at times post-irradiation (h2, Days 4, 7, 21, 28). Biopsies were collected from the 20Gy cohort for only days 0, 4, and 7. Total RNA was isolated and microarrays were performed and analyzed using custom R scripts to obtain lists of probe sets differentially expressed. Changes in gene expression at Benjamini-Hochberg FDR adjusted P < 0.05 and FC >2 were deemed significant. Analyses were performed comparing the different doses of X-ray exposure over all time points. Results Mice in the 20Gy group were euthanized by d7 and the dose was considered lethal. Animals in 1, 3, and 6Gy groups completed the full experiment to d28. Sammon plot analysis of transcriptomes showed clear separation of samples based on the irradiation levels and time after exposure. The clearest separation was between samples of lethal and sublethal doses. Samples from animals exposed to sublethal doses separated more based on timepoints rather than the IR dose suggesting a level of similarity in the progression of the response to sublethal doses. Downregulation was the dominant modulation in the significantly differentially transcribed genes (SDTGs) in the 20Gy group. Temporal changes in ratios of upregulated/downregulated SDTGs (P < 0.05 and FC > 2) revealed further the difference between of transcriptome responses after exposure to lethal and sublethal doses and indicated a delayed peaks response with increasing IR doses within the sublethal range. About 59% of the SDTGs in 20gy were common to all timepoints while no more than 11% were common at the same duration in the other groups. Ratios of the number of SDTGs at h2 to those common to all TPs decreased in a dose-dependent manner with potential radiation dosimetric applications. Conclusions These results demonstrate a solid ability in detecting IR exposure, differentiating lethal and sublethal exposures, and differentiating among the exposure to sublethal doses.

Degradation of aflatoxin B1 by atoxigenic Aspergillus flavus biocontrol agents

Aflatoxins are potent Aspergillus mycotoxins that contaminate food and feed, thereby impacting health and trade. Biopesticides with atoxigenic A. flavus as active ingredients are used to reduce aflatoxin contamination in crops. The mechanism of aflatoxin biocontrol is primarily attributed to competitive exclusion but sometimes aflatoxin is reduced by greater amounts than can be explained by displacement of aflatoxin-producing fungi on the crop. Objectives of this study were to 1) evaluate the ability of atoxigenic A. flavus genotypes to degrade aflatoxin B1 (AFB1) and 2) characterize impacts of temperature, time, and nutrient availability on AFB1 degradation by atoxigenic A. flavus. Aflatoxin-contaminated maize was inoculated with atoxigenic isolates in three separate experiments that included different atoxigenic genotypes, temperature, and time as variables. Atoxigenic genotypes varied in aflatoxin degradation, but all degraded AFB1 > 44% after seven days at 30°C. The optimum temperature for AFB1 degradation was 25-30°C which is similar to the optimum range for AFB1 production. In a time-course experiment, atoxigenics degraded 40% of AFB1 within three days, and 80% of aflatoxin was degraded by day 21. Atoxigenic isolates were able to degrade and utilize AFB1 as a sole carbon source in a chemically defined medium, but quantities of AFB1 degraded declined as glucose concentrations increased. Degradation may be an additional mechanism through which atoxigenic A. flavus biocontrol products reduce aflatoxin contamination pre- and/or post-harvest. Thus, selection of optimal atoxigenic active ingredients can include assessment of both competitive ability in agricultural fields and their ability to degrade aflatoxins.

Molecular Identification and Dual Functions of Two Different CXC Chemokines in Nile Tilapia (Oreochromis niloticus) against Streptococcus agalactiae and Flavobacterium columnare

Two CXC chemokines in Nile tilapia (On-CXC1 and On-CXC2) were identified at both the genomic and proteomic levels. A southern blot analysis and comparison searching in Ensembl confirmed the typical structure of the CXC chemokine genes and provided evidence for unusual mechanisms used to generate the two different CXC chemokine transcripts that have not been reported in other vertebrate species so far. The expression levels of On-CXC1 and On-CXC2 were analyzed by quantitative real-time PCR. These two mRNAs were detected in various tissues of normal Nile tilapia, especially in the spleen, heart, and head kidney, indicating a homeostatic function in immunosurveillance. A time-course experiment clearly demonstrated that these two transcripts were effectively enhanced in the head kidney, spleen and trunk kidney of Nile tilapia 6, 12 and 24 h after injection with Streptococcus agalactiae but were down-regulated in all tested tissues at 48 h, reflecting the fact that they have short half-lives during the crucial response to pathogens that is characteristic of CXC chemokine genes in other vertebrates. Functional analyses obviously exhibited that these two CXC chemokines at concentrations of 1–10 μg strongly inactivated S. agalactiae and Flavobacterium columnare and effectively induced phagocytosis of leukocytes in vitro.

Analysis of model organism viability through an interspecies pathway comparison pipeline using the dynamic impact approach

BackgroundComputational biologists investigate gene expression time-series data using estimation, clustering, alignment, and enrichment methods to make biological sense of the data and provide compelling visualization. While there is an abundance of microarray and RNA-seq data available, interpreting the data while capturing the dynamism of a time-course experiment remains a difficult challenge. Advancements in RNA-seq technologies have allowed us to collect extensive profiles of diverse developmental processes but also requires additional methods for analysis and data integration to capture the increased dynamism. An approach that can both capture the dynamism and direction of change in a time-course experiment in a holistic manner and simultaneously identify which biological pathways are significantly altered is necessary for the interpretation of systems biology data. In addition, there is a need for a method to evaluate the viability of model organisms across different treatments and conditions. By comparing effects of a specific treatment (e.g., a drug) on the target pathway between multiple species and determining pathways with a similar response to biological cues between organisms, we can determine the best animal model for that treatment for future studies.MethodsHere, we present Dynamic Impact Approach with Normalization (DIA-norm), a dynamic pathway analysis tool for the analysis of time-course data without unsupervised dimensionality reduction. We analyzed five datasets of mesenchymal stem cells retrieved from the Gene Expression Omnibus data repository (3 human, 1 mouse cell line, 1 pig) which were differentiated in vitro towards adipogenesis. In the first step, DIA-norm calculated an impact and flux score for each biological term using p-value and fold change. In the second step, these scores were normalized and interpolated using cubic spline. Cross-correlation was then performed between all the data sets with r≥0.6 as a benchmark for high correlation as r = 0.7 is the limit of experimental reproducibility.ResultsDIA-norm predicted that the pig was a better model for humans than a mouse for the study of adipogenesis. The pig model had a higher number of correlating pathways with humans (64.5 to 30.5) and higher average correlation (r = 0.51 vs r = 0.46) as compared to mouse model vs human. While not a definitive conclusion, the results are in accordance with prior phylogenetic and disease studies in which pigs are a good model for studying humans, specifically regarding obesity. In addition, DIA-norm identified a larger number of biologically important pathways (approximately 2x number of pathways) versus a comparable enrichment analysis tool, DAVID. DIA-norm also identified some possible pathways of interests for adipogenesis, namely, nitrogen metabolism (r = 0.86), where there is little to no existing literature.ConclusionDIA-norm captured 80+% of biological important pathways and achieved high pathway correlation between species for the vast majority of important adipogenesis pathways. DIA-norm can be used for both time-series pathway analysis and the determination of a model organism. Our findings indicate that DIA-norm can be used to study the effect of any treatment, including drugs, on specific pathways between multiple species to determine the best animal model for that treatment for future studies. The reliability of DIA-norm to provide biological insights compared to enrichment approach tools has been demonstrated in the selected transcriptomic studies by identifying a higher number of total and biologically relevant pathways. DIA-norm’s final advantage was its easily interpretable graphical outputs that aid in visualizing dynamic changes in expression.

Evidence for pre-climacteric activation of AOX transcription during cold-induced conditioning to ripen in European pear (Pyrus communis L.)

European pears (Pyrus communis L.) require a range of cold-temperature exposure to induce ethylene biosynthesis and fruit ripening. Physiological and hormonal responses to cold temperature storage in pear have been well characterized, but the molecular underpinnings of these phenomena remain unclear. An established low-temperature conditioning model was used to induce ripening of ‘D’Anjou’ and ‘Bartlett’ pear cultivars and quantify the expression of key genes representing ripening-related metabolic pathways in comparison to non-conditioned fruit. Physiological indicators of pear ripening were recorded, and fruit peel tissue sampled in parallel, during the cold-conditioning and ripening time-course experiment to correlate gene expression to ontogeny. Two complementary approaches, Nonparametric Multi-Dimensional Scaling and efficiency-corrected 2-(ΔΔCt), were used to identify genes exhibiting the most variability in expression. Interestingly, the enhanced alternative oxidase (AOX) transcript abundance at the pre-climacteric stage in ‘Bartlett’ and ‘D’Anjou’ at the peak of the conditioning treatments suggests that AOX may play a key and a novel role in the achievement of ripening competency. There were indications that cold-sensing and signaling elements from ABA and auxin pathways modulate the S1-S2 ethylene transition in European pears, and that the S1-S2 ethylene biosynthesis transition is more pronounced in ‘Bartlett’ as compared to ‘D’Anjou’ pear. This information has implications in preventing post-harvest losses of this important crop.

Exposure to Atmospheric Ultrafine Particles Induces Severe Lung Inflammatory Response and Tissue Remodeling in Mice

Exposure to particulate matter (PM) is leading to various respiratory health outcomes. Compared to coarse and fine particles, less is known about the effects of chronic exposure to ultrafine particles, despite their higher number and reactivity. In the present study, we performed a time-course experiment in mice to better analyze the lung impact of atmospheric ultrafine particles, with regard to the effects induced by fine particles collected on the same site. Trace element and PAH analysis demonstrated the almost similar chemical composition of both particle fractions. Mice were exposed intranasally to FF or UFP according to acute (10, 50 or 100 µg of PM) and repeated (10 µg of PM 3 times a week during 1 or 3 months) exposure protocols. More particle-laden macrophages and even greater chronic inflammation were observed in the UFP-exposed mice lungs. Histological analyses revealed that about 50% of lung tissues were damaged in mice exposed to UFP for three months versus only 35% in FF-exposed mice. These injuries were characterized by alveolar wall thickening, macrophage infiltrations, and cystic lesions. Taken together, these results strongly motivate the update of current regulations regarding ambient PM concentrations to include UFP and limit their emission.

Optimal foraging and physiological responses to the risk of predation: how fecal cortisol concentrations from trapped Allenby’s gerbil (Gerbillus andersoni allenbyi) relate to foraging under the risk of predation

We studied the influence of manipulating predation risk on Allenby’s gerbil ( Gerbillus andersoni allenbyi) held in a large, outdoor enclosure. We measured giving up densities (GUDs), apprehension, time allocation to foraging, harvest strategy (grab and go (GAG) vs. eat at tray (EAT)), and fecal cortisol concentration. First we established the time necessary for cortisol and corticosterone concentrations to change significantly from baseline after a stressful experience. To do this we collected feces from gerbils 2, 4, 6, or 8 hours after being handled (treatment) or not (control). After 8 h, fecal cortisol, but not corticosterone, concentration was significantly higher in treatment animals. We used the results from the hormone time course experiment to design the predation experiment. We used a dog, trained to harass gerbils, to increase predation risk for the gerbils. We predicted that fecal cortisol concentrations would increase directly in the face of predation risk, or indirectly, due to reduced foraging time because of perceived predation risk that, in turn, leads to increased hunger levels. As predicted, in the presence of a predator, GUDs were higher, time allocation lower, and GAG foraging was used more in treatment animals than in controls, but we found no change in apprehension. There was no difference in cortisol concentration between predator present and no-predator treatments. However, individuals that tended to have higher average fecal cortisol concentrations also tended, on average, to spend more time foraging. This indicates a relationship between stress hormones and optimal foraging. This relationship is potentially causal. While nightly changes in behavior may not be related to stress hormones, over course time scales, stress hormones may be driving gerbils to forage more.