DARTpaths, an in silico platform to investigate molecular mechanisms of compounds.

SUMMARY: Xpaths is a collection of algorithms that allow for the prediction of compound-induced molecular mechanisms of action by integrating phenotypic endpoints of different species; and proposes follow-up tests for model organisms to validate these pathway predictions. The Xpaths algorithms are applied to predict developmental and reproductive toxicity (DART) and implemented into an in silico platform, called DARTpaths. AVAILABILITY AND IMPLEMENTATION: All code is available on GitHub https://github.com/Xpaths/dartpaths-app under Apache license 2.0, detailed overview with demo is available at https://www.vivaltes.com/dartpaths/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cell shape and Wnt signaling redundantly control the division axis of C. elegans epithelial stem cells.

Tissue-specific stem cells combine proliferative and asymmetric divisions to balance self-renewal with differentiation. Tight regulation of the orientation and plane of cell division is crucial in this process. Here, we study the reproducible pattern of anterior-posterior-oriented stem cell-like divisions in the Caenorhabditis elegans seam epithelium. In a genetic screen, we identified an alg-1 Argonaute mutant with additional and abnormally oriented seam cell divisions. ALG-1 is the main subunit of the microRNA-induced silencing complex (miRISC) and was previously shown to regulate the timing of postembryonic development. Time-lapse fluorescence microscopy of developing larvae revealed that reduced alg-1 function successively interferes with Wnt signaling, cell adhesion, cell shape and the orientation and timing of seam cell division. We found that Wnt inactivation, through mig-14 Wntless mutation, disrupts tissue polarity but not anterior-posterior division. However, combined Wnt inhibition and cell shape alteration resulted in disordered orientation of seam cell division, similar to the alg-1 mutant. Our findings reveal additional alg-1-regulated processes, uncover a previously unknown function of Wnt ligands in seam tissue polarity, and show that Wnt signaling and geometric cues redundantly control the seam cell division axis.

3'sialyllactose and 6'sialyllactose enhance performance in endurance-type exercise through metabolic adaptation.

Human milk oligosaccharides (HMOs) belong to a group of multifunctional glycans that are abundantly present in human breast milk. While health effects of neutral oligosaccharides have been investigated extensively, a lot remains unknown regarding health effects of acidic oligosaccharides, such as the two sialyllactoses (SLs), 3'sialyllactose (3'SL), and 6'sialyllactose (6'SL). We utilized Caenorhabditis elegans (C. elegans) to investigate the effects of SLs on exercise performance. Using swimming as an endurance-type exercise, we found that SLs decrease exhaustion, signifying an increase in endurance that is strongest for 6'SL. Through an unbiased metabolomics approach, we identified changes in energy metabolism that correlated with endurance performance. Further investigation suggested that these metabolic changes were related to adaptations of muscle mitochondria that facilitated a shift from beta oxidation to glycogenolysis during exercise. We found that the effect of SLs on endurance performance required AMPK- (aak-1/aak-2) and adenosine receptor (ador-1) signaling. We propose a model where SLs alter the metabolic status in the gut, causing a signal from the intestine to the nervous system toward muscle cells, where metabolic adaptation increases exercise performance. Together, our results underline the potential of SLs in exercise-associated health and contribute to our understanding of the molecular processes involved in nutritionally-induced health benefits.

Coordination of cell proliferation and differentiation: finding a GEM in the root?

How cells acquire specific fates in conjunction with cell division is a major developmental question. In a recent issue of Nature, Caro and colleagues describe the Arabidopsis protein GEM, which interacts both with DNA-replication and transcriptional regulators (Caro et al., 2007). The results are surprisingly reminiscent of the dual functions reported for Geminin in animals.

C. elegans as a test system to study relevant compounds that contribute to the specific health-related effects of different cannabis varieties.

BACKGROUND: The medicinal effects of cannabis varieties on the market cannot be explained solely by the presence of the major cannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Evidence for putative entourage effects caused by other compounds present in cannabis is hard to obtain due to the subjective nature of patient experience data. Caenorhabditis elegans (C. elegans) is an objective test system to identify cannabis compounds involved in claimed health and entourage effects. METHODS: From a medicinal cannabis breeding program by MariPharm BV, the Netherlands a set of 12 varieties were selected both THC rich varieties as well as CBD rich varieties. A consecutive extraction process was applied resulting in a non-polar (cannabinoid-rich) and polar (cannabinoid-poor) extract of each variety. The test model C. elegans was exposed to these extracts in a broad set of bioassays for appetite control, body oscillation, motility, and nervous system function. RESULTS: Exposing C. elegans to extracts with a high concentration of cannabinoids (> 1 µg/mL) reduces the life span of C. elegans dramatically. Exposing the nematodes to the low-cannabinoid (< 0.005 µg/mL) polar extracts, however, resulted in significant effects with respect to appetite control, body oscillation, motility, and nervous system-related functions in a dose-dependent and variety-dependent manner. DISCUSSION: C. elegans is a small, transparent organism with a complete nervous system, behavior and is due to its genetic robustness and short life cycle highly suitable to unravel entourage effects of Cannabis compounds. Although C. elegans lacks an obvious CB1 and CB2 receptor it has orthologs of Serotonin and Vanilloid receptor which are also involved in (endo)cannabinoid signaling. CONCLUSION: By using C. elegans, we were able to objectively distinguish different effects of different varieties despite the cannabinoid content. C. elegans seems a useful test system for studying entourage effects, for targeted medicinal cannabis breeding programs and product development.

The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.

Local accumulation of the plant growth regulator auxin mediates pattern formation in Arabidopsis roots and influences outgrowth and development of lateral root- and shoot-derived primordia. However, it has remained unclear how auxin can simultaneously regulate patterning and organ outgrowth and how its distribution is stabilized in a primordium-specific manner. Here we show that five PIN genes collectively control auxin distribution to regulate cell division and cell expansion in the primary root. Furthermore, the joint action of these genes has an important role in pattern formation by focusing the auxin maximum and restricting the expression domain of PLETHORA (PLT) genes, major determinants for root stem cell specification. In turn, PLT genes are required for PIN gene transcription to stabilize the auxin maximum at the distal root tip. Our data reveal an interaction network of auxin transport facilitators and root fate determinants that control patterning and growth of the root primordium.

Serum Metabolomic Analysis of Coronary Heart Disease Patients with Stable Angina Pectoris Subtyped by Traditional Chinese Medicine Diagnostics Reveals Biomarkers Relevant to Personalized Treatments.

To improve the treatment of patients with coronary heart disease (CHD), personalized treatments based on potential biomarkers could make a difference. To investigate if such potential biomarkers could be found for CHD inhomogeneous, we combined traditional Chinese medicine based diagnosis with untargeted and targeted metabolomics analyses. Shi and Xu patient subtype groups of CHD with angina pectoris were identified. Different metabolites including lipids, fatty acids and amino acids were further analyzed with targeted metabolomics and mapped to disease-related pathways. The long-chain unsaturated lipids ceramides metabolism, bile acid metabolism were differentially affected in the Xu subtype groups. While, Shi-subtype patients seemed to show inflammation, anomalous levels of bioactive phospholipids and antioxidant molecules. Furthermore, variations in the endothelial damage response and energy metabolism found based on ELISA analysis are the key divergence points between different CHD subtypes. The results showed Xu subtype patients might benefit from long-chain unsaturated lipids ceramides as therapeutic targets. Shi subtype patients might benefit more from levels of polyunsaturated fatty acid consumption and treatments that help in restoring energy balance. Metabolic differences can be essential for treatment protocols. Thus, patient group specific differences can serve as important information to refine current treatment approaches in a personalized manner.

Towards a reporting guideline for developmental and reproductive toxicology testing in C. elegans and other nematodes.

Implementation of reliable methodologies allowing Reduction, Refinement, and Replacement (3Rs) of animal testing is a process that takes several decades and is still not complete. Reliable methods are essential for regulatory hazard assessment of chemicals where differences in test protocol can influence the test outcomes and thus affect the confidence in the predictive value of the organisms used as an alternative for mammals. Although test guidelines are common for mammalian studies, they are scarce for non-vertebrate organisms that would allow for the 3Rs of animal testing. Here, we present a set of 30 reporting criteria as the basis for such a guideline for Developmental and Reproductive Toxicology (DART) testing in the nematode Caenorhabditis elegans. Small organisms like C. elegans are upcoming in new approach methodologies for hazard assessment; thus, reliable and robust test protocols are urgently needed. A literature assessment of the fulfilment of the reporting criteria demonstrates that although studies describe methodological details, essential information such as compound purity and lot/batch number or type of container is often not reported. The formulated set of reporting criteria for C. elegans testing can be used by (i) researchers to describe essential experimental details (ii) data scientists that aggregate information to assess data quality and include data in aggregated databases (iii) regulators to assess study data for inclusion in regulatory hazard assessment of chemicals.

Application of Caenorhabditis elegans (nematode) and Danio rerio embryo (zebrafish) as model systems to screen for developmental and reproductive toxicity of Piperazine compounds.

To enable selection of novel chemicals for new processes, there is a recognized need for alternative toxicity screening assays to assess potential risks to man and the environment. For human health hazard assessment these screening assays need to be translational to humans, have high throughput capability, and from an animal welfare perspective be harmonized with the principles of the 3Rs (Reduction, Refinement, Replacement). In the area of toxicology a number of cell culture systems are available but while these have some predictive value, they are not ideally suited for the prediction of developmental and reproductive toxicology (DART). This is because they often lack biotransformation capacity, multicellular or multi- organ complexity, for example, the hypothalamus pituitary gonad (HPG) axis and the complete life cycle of whole organisms. To try to overcome some of these limitations in this study, we have used Caenorhabditis elegans (nematode) and Danio rerio embryos (zebrafish) as alternative assays for DART hazard assessment of some candidate chemicals being considered for a new commercial application. Nematodes exposed to Piperazine and one of the analogs tested showed a slight delay in development compared to untreated animals but only at high concentrations and with Piperazine as the most sensitive compound. Total brood size of the nematodes was also reduced primarily by Piperazine and one of the analogs. In zebrafish Piperazine and analogs showed developmental delays. Malformations and mortality in individual fish were also scored. Significant malformations were most sensitively identified with Piperazine, significant mortality was only observed in Piperazine and only at the higest dose. Thus, Piperazine seemed the most toxic compound for both nematodes and zebrafish. The results of the nematode and zebrafish studies were in alignment with data obtained from conventional mammalian toxicity studies indicating that these have potential as developmental toxicity screening systems. The results of these studies also provided reassurance that none of the Piperazines tested are likely to have any significant developmental and/or reproductive toxicity issues to humans when used in their commercial applications.

The RETINOBLASTOMA-RELATED gene regulates stem cell maintenance in Arabidopsis roots.

The maintenance of stem cells in defined locations is crucial for all multicellular organisms. Although intrinsic factors and signals for stem cell fate have been identified in several species, it has remained unclear how these connect to the ability to reenter the cell cycle that is one of the defining properties of stem cells. We show that local reduction of expression of the RETINOBLASTOMA-RELATED (RBR) gene in Arabidopsis roots increases the amount of stem cells without affecting cell cycle duration in mitotically active cells. Conversely, induced RBR overexpression dissipates stem cells prior to arresting other mitotic cells. Overexpression of D cyclins, KIP-related proteins, and E2F factors also affects root stem cell pool size, and genetic interactions suggest that these factors function in a canonical RBR pathway to regulate somatic stem cells. Expression analysis and genetic interactions position RBR-mediated regulation of the stem cell state downstream of the patterning gene SCARECROW.

SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem.

Stem cells self-renew and produce daughter cells that differentiate. How stem cells are specified and maintained is a central question in developmental biology. Plant stem cells occupy a small region or niche in larger zones of mitotic activity called meristems. Here we provide molecular evidence that in the Arabidopsis root meristem, the stem cell population depends on a central group of cells, the quiescent center (QC), which positions the stem cell niche. We show that the putative transcription factor SCARECROW (SCR), first identified by its role in radial patterning, is required cell-autonomously for distal specification of the QC, which in turn regulates stem cell fate of immediately surrounding cells.