Miskowski, Jennifer et al. (2015) International Worm Meeting "Investigating the mechanism of action for the anthelmintic activity of CL-5."

Sagstetter, Mary, Monte, Aaron, Miskowski, Jennifer, Brown, Kiersten

[International Worm Meeting, 2015]

Established and emerging drug resistance in infective organisms is a growing problem that threatens humans, livestock, and crops and causes morbidity, mortality, and economic woes. The need to identify pharmaceuticals with novel modes of action is imperative. To this end, an interdisciplinary team of scientists at the University of Wisconsin-La Crosse pursued the rich reservoir of metabolites produced in plants and fungi as a potential source of medicinal agents. A compound with strong antimicrobial activity was first isolated from Comptonia peregrina (sweet fern) and used as a template for the synthesis of hundreds of derivatives. This novel library of compounds has wide-ranging activities against different organisms, including the nematode Caenorhabditis elegans.Although non-parasitic, C. elegans has long been a model system for parasitic worms, called helminths. Anthelmintic drugs affect C. elegans and helminths similarly, and the mechanisms of action for commercially available anthelmintics have been elucidated in C. elegans. Previously, we reported the identification of multiple compounds from our library that affect worm motility and/or survival based on two microscale, liquid-based assays. Work to date has primarily focused on the compound CL-5, which showed the strongest activity. CL-5 is effective against mutant strains that are resistant to the major anthelmintics on the market, including ivermectin, benomyl, and levamisole. These data suggest that CL-5 acts via a different molecular mechanism. Recent experimentation has been driven by the fact that CL-5, along with other compounds in the library, is structurally related to resveratrol. Resveratrol alone does not affect the worms in our assay, nor does it attenuate or augment the effects of CL-5. Resveratrol is reported to lower oxidative stress in C. elegans, thereby increasing longevity (1). Preliminary experiments show that treatment with CL-5 leads to increased expression of sod-3::GFP, an indirect indicator of oxidative stress. Current experiments aim to detect reactive oxygen species directly and these results will be reported.1. Chen, W. et al. Influence of resveratrol on oxidative stress resistance and life span in Caenorhabditis elegans. J Pharm Pharmacol. 65(5): 682-88 (2013).

Gross, Megan et al. (2013) International Worm Meeting "Update: Screening Potential Anthelmintic Compounds for Novel Activity."

Monte, Aaron, Smith, Michael, Gross, Megan, Miskowski, Jennifer

[International Worm Meeting, 2013]

Parasitic worms, called helminths, infect plants, animals, and humans worldwide leading to a decreased food supply, economic hardship, and significant levels of morbidity and mortality. Anthelmintics that combat these infections are represented by only five major classes of compounds. Misuse of these pharmaceuticals has contributed to widespread anthelmintic resistance in worms that infect livestock and emerging drug resistance in human-infecting helminths. The identification of new means to target helminths is imperative. The non-parasitic nematode Caenorhabditis elegans has long been a model system for helminths. Previously, our group screened a series of novel, synthetic compounds for anthelmintic activity in C. elegans. These compounds are derivatives of a natural product stilbene that have generated great interest due to their broad anti-microbial effects. Two microassays were used: the motility assay that screened for paralysis and the developmental assay that screened for developmental delays, developmental arrests, a decrease in fecundity, or death. Six compounds demonstrated significant anthelmintic activity and were prioritized for further study. This research project aims to determine if these six stilbenoid derivatives act via a novel mechanism. To this end, the compounds are being tested against mutant C. elegans that are resistant to existing anthelmintics using the two assays described above, along with an NGM plate-based assay. Work to date has primarily focused on the compound CL-5, which showed the strongest activity. Ivermectin is one of the most widely used anthelmintics today, and ivermectin resistance has been documented in helminth strains. Interestingly, CL-5 affects ivermectin-resistant C. elegans in a dose-dependent manner, similar to wildtype worms. Whereas ivermectin acts on ligand-gated chloride channels, the anthelmintic benomyl disturbs the microtubule cytoskeleton. Benomyl-resistant C. elegans are also sensitive to CL-5 and exhibit developmental defects similar to CL-5-treated N2 worms. We are currently testing mutant strains that are resistant to levamisole and emodepside. In addition, we are pursuing some other interesting effects of CL-5 treatment.

Torfeh E et al. (2019) Sci Rep "Monte-Carlo dosimetry and real-time imaging of targeted irradiation consequences in 2-cell stage Caenorhabditis elegans embryo."

Muggiolu G, Simon M, Deves G, Bourret S, Incerti S, Torfeh E, Vianna F, Seznec H, Barberet P

[Sci Rep, 2019]

Charged-particle microbeams (CPMs) provide a unique opportunity to investigate the effects of ionizing radiation on living biological specimens with a precise control of the delivered dose, i.e. the number of particles per cell. We describe a methodology to manipulate and micro-irradiate early stage C. elegans embryos at a specific phase of the cell division and with a controlled dose using a CPM. To validate this approach, we observe the radiation-induced damage, such as reduced cell mobility, incomplete cell division and the appearance of chromatin bridges during embryo development, in different strains expressing GFP-tagged proteins in situ after irradiation. In addition, as the dosimetry of such experiments cannot be extrapolated from random irradiations of cell populations, realistic three-dimensional models of 2 cell-stage embryo were imported into the Geant4 Monte-Carlo simulation toolkit. Using this method, we investigate the energy deposit in various chromatin condensation states during the cell division phases. The experimental approach coupled to Monte-Carlo simulations provides a way to selectively irradiate a single cell in a rapidly dividing multicellular model with a reproducible dose. This method opens the way to dose-effect investigations following targeted irradiation.

Berriz GF et al. (2003) Bioinformatics "Characterizing gene sets with FuncAssociate."

King OD, Berriz GF, Roth FP, Sander C, Bryant B

[Bioinformatics, 2003]

SUMMARY: FuncAssociate is a web-based tool to help researchers use Gene Ontology attributes to characterize large sets of genes derived from experiment. Distinguishing features of FuncAssociate include the ability to handle ranked input lists, and a Monte Carlo simulation approach that is more appropriate to determine significance than other methods, such as Bonferroni or idk p-value correction. FuncAssociate currently supports 10 organisms (Vibrio cholerae, Shewanella oneidensis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Caenorhaebditis elegans, Drosophila melanogaster, Mus musculus, Rattus norvegicus and Homo sapiens). AVAILABILITY: FuncAssociate is freely accessible at http://llama.med.harvard.edu/Software.html. Source code (in Perl and C) is freely available to academic users ''as is''.

Hong F et al. (2006) Biometrics "Functional hierarchical models for identifying genes with different time-course expression profiles."

Hong F, Li H

[Biometrics, 2006]

Time-course studies of gene expression are essential in biomedical research to understand biological phenomena that evolve in a temporal fashion. We introduce a functional hierarchical model for detecting temporally differentially expressed (TDE) genes between two experimental conditions for cross-sectional designs, where the gene expression profiles are treated as functional data and modeled by basis function expansions. A Monte Carlo EM algorithm was developed for estimating both the gene-specific parameters and the hyperparameters in the second level of modeling. We use a direct posterior probability approach to bound the rate of false discovery at a pre-specified level and evaluate the methods by simulations and application to microarray time-course gene expression data on Caenorhabditis elegans developmental processes. Simulation results suggested that the procedure performs better than the two-way ANOVA in identifying TDE genes, resulting in both higher sensitivity and specificity. Genes identified from the C. elegans developmental data set show clear patterns of changes between the two experimental conditions.

Barbieri, Sofia et al. (2021) International Worm Meeting "The dynamic partnered dance between PLK-1 and MEX-5: interpreting gradient formation with computational modelling."

Griffin, Erik E., Gotta, Monica, Nurni Ravi, Aparna, Barbieri, Sofia

[International Worm Meeting, 2021]

In C. elegans embryos, PLK-1 is pivotal for cell division and it orchestrates polarity establishment together with its binding partner, MEX-5. To achieve this, their localization/activity must be precisely regulated. MEX-5 enrichment at the anterior cytoplasm results from a change in its diffusivity following uneven phosphorylation along the embryo axis. We know PLK-1 relocalization to the anterior depends on MEX-5. However, the biological and physical mechanisms behind the dynamics of this protein are still poorly described. To address this, PLK-1 and MEX-5 gradient formation was measured in two CRISPR strains and significant discrepancies were revealed between the two proteins in terms of: 1) gradient steepness, as PLK-1 forms a less steep gradient compared to MEX-5; 2) dynamics, with PLK-1 gradient establishment delayed and slower; 3) diffusivity, as PLK-1 diffusion coefficient does not correspond to MEX-5's one from anterior to posterior. To shed light on PLK-1 dynamics, and how it is intertwined to MEX-5, we developed a novel Monte Carlo simulation framework able to recreate the protein motions in the C. elegans one-cell embryo. Thanks to our computational approach, we were able to postulate on the biological mechanisms behind MEX-5 and PLK-1 dynamics during the whole cell division, from early embryos to the steady-state before cytokinesis. The simulations succeed in reproducing PLK-1 gradient formation, in agreement with experimental measurements, if: 1) PLK-1 binds to phosphorylated MEX-5; 2) the binding is triggered after a defined time delay; 3) PLK-1 dynamically interacts with MEX-5, leading to a continuous replenishment of a pool of unbound PLK-1. The Monte Carlo framework we propose can eventually be applied to other polarity-related factors or mutants in which polarization is perturbed, to understand if it can be traced back to a failure in PLK-1 localization. Finally, conditions where gradient formation is altered, like after stress, can be simulated.

Dolinski C et al. (2008) Mem Inst Oswaldo Cruz "Molecular and morphological characterization of heterorhabditid entomopathogenic nematodes from the tropical rainforest in Brazil."

Machado IR, Dolinski C, Winter CE, Kamitani FL

[Mem Inst Oswaldo Cruz, 2008]

Despite massive losses of primary forest, the Amazonian rainforest remains an extremely rich source of biodiversity. In recent years, entomopathogenic nematodes (EPNs) have been isolated from soil in various parts of the world and used successfully as biological control agents against numerous insect pests. Therefore, a sampling in the rainforest of Monte Negro, Rondonia, Brazil was conducted with the aim of discovering new strains and/or species of EPNs for future development as biological control agents. From 156 soil samples taken at nine collecting sites, 19 isolates were obtained, all of them belonging to the genus Heterorhabditis. Four strains were subjected to detailed morphological and molecular evaluation. Based on morphometrics and internal transcribed spacer (ITS) sequence data, the strains LPP1, LPP2 and LPP4 were identified as Heterorhabditis indica, whereas LPP7 was considered Heterorhabditis baujardi. Comparative analysis of the ITS1 sequence of H. indica and H. baujardi isolates showed a polymorphic site for the restriction enzyme Tth 111 that could be used to distinguish the two species. Consequently, strains LPP1, LPP2, LPP3, LPP4, and LPP9 were identified as H. indica, whereas LPP5, LPP7, LPP8 and LPP10 were identified as H. baujardi.

Rivas E et al. (2000) Bioinformatics "Secondary structure alone is generally not statistically significant for the detection of noncoding RNAs."

Rivas E, Eddy SR

[Bioinformatics, 2000]

MOTIVATION: Several results in the literature suggest that biologically interesting RNAs have secondary structures that are more stable than expected by chance. Based on these observations, we developed a scanning algorithm for detecting noncoding RNA genes in genome sequences, using a fully probabilistic version of the Zuker minimum-energy folding algorithm. RESULTS: Preliminary results were encouraging, but certain anomalies led us to do a carefully controlled investigation of this class of methods. Ultimately, our results argue that for the probabilistic model there is indeed a statistical effect, but it comes mostly from local base-composition bias and not from RNA secondary structure. For the thermodynamic implementation (which evaluates statistical significance by doing Monte Carlo shuffling in fixed-length sequence windows, thus eliminating the base-composition effect) the signals for noncoding RNAs are still usually indistinguishable from noise, especially when certain statistical artifacts resulting from local base-composition inhomogeneity are taken into account. We conclude that although a distinct, stable secondary structure is undoubtedly important in most noncoding RNAs, the stability of most noncoding RNA secondary structures is not sufficiently different from the predicted stability of a random sequence to be useful as a general genefinding approach.

Yu W et al. Dose Response "Study on Caenorhabditis Elegans as a Combined Model of Microdosimetry and Biology."

Gao J, Sun L, Long H, Chen N, Yu W, Tu Y, Wang Y

[Dose Response]

Microdosimetry is a tool for the investigation of microscopic energy deposition of ionizing radiation. This work used <i>Caenorhabditis</i> elegans as a model to estimate the microdosimetric deposition level at the ⁶⁰Co gamma radiation. Monte Carlo software PHITS was employed to establish irradiated nematodes model. The dose deposition of the entire body and gonad irradiated to 100 Gy was calculated. The injury levels of radiation were evaluated by the detection of biological indicators. The result of microdosimetric experiment suggested that the dose of whole body of nematodes was estimated to be 99.9 +/- 57.8 Gy, ranging from 19.6 to 332.2 Gy. The dose of gonad was predicted to be 129.4 +/- 558.8 Gy (9.5-6597 Gy). The result of biological experiment suggested that there were little changes in the length of nematodes after irradiation. However, times of head thrash per minute and the spawning yield in 3 consecutive days decreased 27.1% and 94.7%, respectively. Nematodes in the irradiated group displayed heterogeneity. Through contour analysis, trends of behavior kinematics and reproductive capacity of irradiated nematodes proved to be consistent with the dose distribution levels estimated by microdosimetric model. Finally, <i>C</i>. elegans presented a suitable combined model of microdosimetry and biology for studying radiation.

Malone B et al. (2017) Nucleic Acids Res "Bayesian prediction of RNA translation from ribosome profiling."

GroBhans H, Atanassov I, Dieterich C, Li X, Malone B, Aeschimann F

[Nucleic Acids Res, 2017]

Ribosome profiling via high-throughput sequencing (ribo-seq) is a promising new technique for characterizing the occupancy of ribosomes on messenger RNA (mRNA) at base-pair resolution. The ribosome is responsible for translating mRNA into proteins, so information about its occupancy offers a detailed view of ribosome density and position which could be used to discover new translated open reading frames (ORFs), among other things. In this work, we propose Rp-Bp, an unsupervised Bayesian approach to predict translated ORFs from ribosome profiles. We use state-of-the-art Markov chain Monte Carlo techniques to estimate posterior distributions of the likelihood of translation of each ORF. Hence, an important feature of Rp-Bp is its ability to incorporate and propagate uncertainty in the prediction process. A second novel contribution is automatic Bayesian selection of read lengths and ribosome P-site offsets (BPPS). We empirically demonstrate that our read length selection technique modestly improves sensitivity by identifying more canonical and non-canonical ORFs. Proteomics- and quantitative translation initiation sequencing-based validation verifies the high quality of all of the predictions. Experimental comparison shows that Rp-Bp results in more peptide identifications and proteomics-validated ORF predictions compared to another recent tool for translation prediction.