Toxicological assessment of the Achyrocline satureioides aqueous extract in the Caenorhabditis elegans alternative model.

Achyrocline satureioides, popularly called "marcela" in Brazil, is used in traditional medicine in South America. A. satureioides, inflorescences are used for many conditions, including to minimize the Sars-Cov-2 symptoms. Therefore, the aim of this study was to determine the toxicity profile of A. satureioides aqueous extract (ASAE), using the Caenorhabditis elegans (C. elegans) alternative model. Survival, reproduction, development, and transgenerational assays were performed. The effects of ASAE were investigated under conditions of thermal stress and presence of oxidant hydrogen peroxide (H2O2). In addition, C. elegans strains containing high antioxidant enzyme levels and elevated lineages of daf-16, skn-1 and daf-2 regulatory pathways were examined. The ASAE LC50 value was found to be 77.3 ± 4 mg/ml. The concentration of ASAE 10 mg/ml (frequently used in humans) did not exhibit a significant reduction in worm survival at either the L1 or L4 stage, after 24 or 72 hr treatment. ASAE did not markedly alter the body area. In N2 strain, ASAE (10 or 25 mg/ml) reversed the damage initiated by H2O2. In addition, ASAE protected the damage produced by H2O2 in strains containing significant levels of sod-3, gst-4 and ctl - 1,2,3, suggesting modulation in these antioxidant systems by this plant extract. ASAE exposure activated daf-16 and skn-1 stress response transcriptional pathways independently of daf-2, even under extreme stress. Data suggest that ASAE, at the concentrations tested in C. elegans, exhibits a reliable toxicity profile, which may contribute to consideration for safe use in humans.

Abalone peptide increases stress resilience and cost-free longevity via SKN-1-governed transcriptional metabolic reprogramming in C. elegans.

A major goal of healthy aging is to prevent declining resilience and increasing frailty, which are associated with many chronic diseases and deterioration of stress response. Here, we propose a loss-or-gain survival model, represented by the ratio of cumulative stress span to life span, to quantify stress resilience at organismal level. As a proof of concept, this is demonstrated by reduced survival resilience in Caenorhabditis elegans exposed to exogenous oxidative stress induced by paraquat or with endogenous proteotoxic stress caused by polyglutamine or amyloid-ß aggregation. Based on this, we reveal that a hidden peptide ("cryptide")-AbaPep#07 (SETYELRK)-derived from abalone hemocyanin not only enhances survival resilience against paraquat-induced oxidative stress but also rescues proteotoxicity-mediated behavioral deficits in C. elegans, indicating its capacity against stress and neurodegeneration. Interestingly, AbaPep#07 is also found to increase cost-free longevity and age-related physical fitness in nematodes. We then demonstrate that AbaPep#07 can promote nuclear localization of SKN-1/Nrf, but not DAF-16/FOXO, transcription factor. In contrast to its effects in wild-type nematodes, AbaPep#07 cannot increase oxidative stress survival and physical motility in loss-of-function skn-1 mutant, suggesting an SKN-1/Nrf-dependent fashion of these effects. Further investigation reveals that AbaPep#07 can induce transcriptional activation of immune defense, lipid metabolism, and metabolic detoxification pathways, including many SKN-1/Nrf target genes. Together, our findings demonstrate that AbaPep#07 is able to boost stress resilience and reduce behavioral frailty via SKN-1/Nrf-governed transcriptional reprogramming, and provide an insight into the health-promoting potential of antioxidant cryptides as geroprotectors in aging and associated conditions.

The nematode worm C. elegans chooses between bacterial foods as if maximizing economic utility.

In value-based decision making, options are selected according to subjective values assigned by the individual to available goods and actions. Despite the importance of this faculty of the mind, the neural mechanisms of value assignments, and how choices are directed by them, remain obscure. To investigate this problem, we used a classic measure of utility maximization, the Generalized Axiom of Revealed Preference, to quantify internal consistency of food preferences in Caenorhabditis elegans, a nematode worm with a nervous system of only 302 neurons. Using a novel combination of microfluidics and electrophysiology, we found that C. elegans food choices fulfill the necessary and sufficient conditions for utility maximization, indicating that nematodes behave as if they maintain, and attempt to maximize, an underlying representation of subjective value. Food choices are well-fit by a utility function widely used to model human consumers. Moreover, as in many other animals, subjective values in C. elegans are learned, a process we find requires intact dopamine signaling. Differential responses of identified chemosensory neurons to foods with distinct growth potentials are amplified by prior consumption of these foods, suggesting that these neurons may be part of a value-assignment system. The demonstration of utility maximization in an organism with a very small nervous system sets a new lower bound on the computational requirements for utility maximization and offers the prospect of an essentially complete explanation of value-based decision making at single neuron resolution in this organism.

Intestine-specific removal of DAF-2 nearly doubles lifespan in Caenorhabditis elegans with little fitness cost.

Twenty-nine years following the breakthrough discovery that a single-gene mutation of daf-2 doubles Caenorhabditis elegans lifespan, it remains unclear where this insulin/IGF-1 receptor gene is expressed and where it acts to regulate ageing. Using knock-in fluorescent reporters, we determined that daf-2 and its downstream transcription factor daf-16 are expressed ubiquitously. Using tissue-specific targeted protein degradation, we determined that intracellular DAF-2-to-DAF-16 signaling in the intestine plays a major role in lifespan regulation, while that in the hypodermis, neurons, and germline plays a minor role. Notably, intestine-specific loss of DAF-2 activates DAF-16 in and outside the intestine, causes almost no adverse effects on development and reproduction, and extends lifespan by 94% in a way that partly requires non-intestinal DAF-16. Consistent with intestine supplying nutrients to the entire body, evidence from this and other studies suggests that altered metabolism, particularly down-regulation of protein and RNA synthesis, mediates longevity by reduction of insulin/IGF-1 signaling.

Assessment of Neuronal Cell Death in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a powerful experimental platform for cell biology studies. The molecular mechanisms that mediate cell death and neurodegeneration have been characterized extensively in the nematode. In addition, the availability of a wide arsenal of genetic and molecular tools and methodologies renders C. elegans an organism of choice for modeling human neurodegenerative diseases. Indeed, neuronal necrosis can readily be observed and examined in vivo, in the worm. In this chapter, we describe the two main approaches that are routinely used for monitoring and quantifying neuronal cell death in C. elegans. The first is based on direct visualization of dying cells via Nomarski differential interference contrast (DiC) microscopy, and the second on the assessment of neuronal survival by fluorescence microscopy.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Rapid assessment of the temporal function and phenotypic reversibility of neurodevelopmental disorder risk genes in Caenorhabditis elegans.

Recent studies have indicated that some phenotypes caused by decreased function of select neurodevelopmental disorder (NDD) risk genes can be reversed by restoring gene function in adulthood. However, few of the hundreds of risk genes have been assessed for adult phenotypic reversibility. We developed a strategy to rapidly assess the temporal requirements and phenotypic reversibility of NDD risk gene orthologs using a conditional protein degradation system and machine-vision phenotypic profiling in Caenorhabditis elegans. We measured how degrading and re-expressing orthologs of EBF3, BRN3A and DYNC1H1 at multiple periods throughout development affect 30 morphological, locomotor, sensory and learning phenotypes. We found that phenotypic reversibility was possible for each gene studied. However, the temporal requirements of gene function and degree of rescue varied by gene and phenotype. This work highlights the critical need to assess multiple windows of degradation and re-expression and a large number of phenotypes to understand the many roles a gene can have across the lifespan. This work also demonstrates the benefits of using a high-throughput model system to prioritize NDD risk genes for re-expression studies in other organisms.

Modeling protein dynamics in Caenorhabditis elegans embryos reveals that the PLK-1 gradient relies on weakly coupled reaction-diffusion mechanisms.

SignificanceIntracellular gradients have essential roles in cell and developmental biology, but their formation is not fully understood. We have developed a computational approach facilitating interpretation of protein dynamics and gradient formation. We have combined this computational approach with experiments to understand how Polo-Like Kinase 1 (PLK-1) forms a cytoplasmic gradient in Caenorhabditis elegans embryos. Although the PLK-1 gradient depends on the Muscle EXcess-5/6 (MEX-5/6) proteins, we reveal differences in PLK-1 and MEX-5 gradient formation that can be explained by a model with two components, PLK-1 bound to MEX-5 and unbound PLK-1. Our combined approach suggests that a weak coupling between PLK-1 and MEX-5 reaction-diffusion mechanisms dictates the dynamic exchange of PLK-1 with the cytoplasm, explaining PLK-1 high diffusivity and smooth gradient.

The Functional Assessment of LRRK2 in Caenorhabditis elegans Mechanosensory Neurons.

The nematode Caenorhabditis elegans (C. elegans) is a powerful model organism to systematically analyze the functions of genes of interest in vivo. Especially, C. elegans nervous system is suitable for morphological and functional analyses of neuronal genes due to its optical transparency of the body and the well-established anatomy including neural connections. The C. elegans ortholog of Parkinson's disease-associated gene LRRK2, named lrk-1, has been shown to play a role in the regulation of axonal morphology in a subset of neurons. Here I describe the detailed methodologies for the assessment of LRK-1/LRRK2 function as well as the analysis of genetic interaction involving lrk-1/LRRK2 by performing live imaging of C. elegans mechanosenrory neurons.

CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6.

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules.

Metabolic Assessment of Lipid Abundance and Distribution.

Lipids are an essential macromolecule used for diverse functions including use for structural components of membranes, energy storage, and signaling molecules. The regulation of cellular lipid stores is critical for maintaining organismal metabolic homeostasis. Lipid homeostasis can decline with age, which can lead to poor health outcomes and accelerate the progression of disease states. C. elegans represents an excellent model to study age-related decline in lipid homeostasis due to its short lifespan and remarkably well-conserved metabolic pathways. Due to their ease of use and similarities, there have been numerous developments in methodologies to study intracellular lipid abundance and tissue distribution in C. elegans.

Assessment of nanopolystyrene toxicity under fungal infection condition in Caenorhabditis elegans.

Due to the potential of release and accumulation in the environment, nanoplastics have attracted an increasing attention. In this study, we investigated the effect of exposure to nanopolystyrene (30 nm) in nematode Caenorhabditis elegans after the fungal infection. After Candida albicans infection, exposure to nanopolystyrene (10 and 100 µg/L) for 24-h could cause the more severe toxicity on lifespan and locomotion behavior compared with fungal infection alone. The more severe activation of oxidative stress and suppression of SOD-3:GFP expression and mitochondrial unfolded protein response (mt UPR) were associated with this observed toxicity enhancement induced by nanopolystyrene exposure. Moreover, the more severe C. albicans colony formation and suppression of innate immune response as indicated by the alteration in expression of anti-microbial genes (abf-2, cnc-4, cnc-7, and fipr-22/23) further contributed to the formation of this toxicity enhancement induced by nanopolystyrene exposure. Our results demonstrated that short-term exposure to nanopolystyrene in the range of µg/L potentially enhances the adverse effects of fungal infection on organisms.

Cafestol increases fat oxidation and energy expenditure in Caenorhabditis elegans via DAF-12-dependent pathway.

Cafestol, a coffee diterpene, is a known agonist of farnesoid X receptors (FXR), which are involved in cholesterol homeostasis. FXR plays critical roles in other lipid metabolic pathways, including fat oxidation; however, little is known about cafestol's effects on fatty acid metabolism. Thus, the goal was to investigate cafestol's effects on fatty acid metabolism using Caenorhabditis elegans. Cafestol at 60 µM reduced fat accumulation and increased locomotor activity (an indicator of energy expenditure) by 20% and 38%, respectively, compared to the control. Cafestol's effects were dependent on daf-12 (a functional homolog of the human FXR) with upregulation of ech-1.1 (a homolog of enoyl-CoA hydratase involved in fatty acid ß-oxidation) and tub-1 (an ortholog of the human TUBBY involved in the neurological regulation of energy expenditure) without any effects on lipogenesis, lipolysis or lipid uptake and transport. Therefore, cafestol increased fat oxidation and energy expenditure via DAF-12-dependent pathway in C. elegans.

Assessment and Maintenance of Unigametic Germline Inheritance for C. elegans.

The recent work of Besseling and Bringmann (2016) identified a molecular intervention for C. elegans in which premature segregation of maternal and paternal chromosomes in the fertilized oocyte can produce viable animals exhibiting a non-Mendelian inheritance pattern. Overexpression in embryos of a single protein regulating chromosome segregation (GPR-1) provides a germline derived clonally from a single parental gamete. We present a collection of strains and cytological assays to consistently generate and track non-Mendelian inheritance. These tools allow reproducible and high-frequency (>80%) production of non-Mendelian inheritance, the facile and simultaneous homozygosis for all nuclear chromosomes in a single generation, the precise exchange of nuclear and mitochondrial genomes between strains, and the assessments of non-canonical mitosis events. We show the utility of these strains by demonstrating a rapid assessment of cell lineage requirements (AB versus P1) for a set of genes (lin-2, lin-3, lin-12, and lin-31) with roles in C. elegans vulval development.

Label-free molecular mapping and assessment of glycogen in C. elegans.

Caenorhabditis elegans is an animal model frequently used in research on the effects of metabolism on organismal aging. This comes with a requirement for methods to investigate metabolite content, turnover, and distribution. The aim of our study was to assess the use of a label-free approach to determine both content and distribution of glycogen, the storage form of glucose, in C. elegans. To this end, we grew C. elegans worms under three different dietary conditions for 24-48 h, representing starvation, regular diet and a high glucose diet, followed by analysis of glycogen content. Glycogen analysis was performed on fixed individual whole worms using Raman micro-spectroscopy (RMS). Results were confirmed by comparison with two conventional assays, i.e. iodine staining of worms and enzymatic determination of glycogen. RMS was further used to assess overall lipid and protein content and distribution in the same samples used for glycogen analysis. Expectedly, both glycogen and lipid content were highest in worms grown on a high glucose diet, lower in regularly fed, and lowest in starved nematodes. In summary, RMS is a method suitable for analysis of glycogen content in C. elegans that has the advantage over established methods that (i) individual worms (rather than hundreds per sample) can be analyzed, (ii) glycogen distribution can be assessed at subcellular resolution and (iii) the distribution patterns of other macromolecules can be assessed from the same worms. Thus, RMS has the potential to be used as a sensitive, accurate, cost-effective and high throughput method to evaluate glycogen stores in C. elegans.

Myrf ER-Bound Transcription Factors Drive C. elegans Synaptic Plasticity via Cleavage-Dependent Nuclear Translocation.

Synaptic refinement is a critical step in nervous system maturation, requiring a carefully timed reorganization and refinement of neuronal connections. We have identified myrf-1 and myrf-2, two C. elegans homologs of Myrf family transcription factors, as key regulators of synaptic rewiring. MYRF-1 and its paralog MYRF-2 are functionally redundant specifically in synaptic rewiring. They co-exist in the same protein complex and act cooperatively to regulate synaptic rewiring. We find that the MYRF proteins localize to the ER membrane and that they are cleaved into active N-terminal fragments, which then translocate into the nucleus to drive synaptic rewiring. Overexpression of active forms of MYRF is sufficient to accelerate synaptic rewiring. MYRF-1 and MYRF-2 are the first genes identified to be indispensable for promoting synaptic rewiring in C. elegans. These findings reveal a molecular mechanism underlying synaptic rewiring and developmental circuit plasticity.

Inference of Functionally-Relevant N-acetyltransferase Residues Based on Statistical Correlations.

Over evolutionary time, members of a superfamily of homologous proteins sharing a common structural core diverge into subgroups filling various functional niches. At the sequence level, such divergence appears as correlations that arise from residue patterns distinct to each subgroup. Such a superfamily may be viewed as a population of sequences corresponding to a complex, high-dimensional probability distribution. Here we model this distribution as hierarchical interrelated hidden Markov models (hiHMMs), which describe these sequence correlations implicitly. By characterizing such correlations one may hope to obtain information regarding functionally-relevant properties that have thus far evaded detection. To do so, we infer a hiHMM distribution from sequence data using Bayes' theorem and Markov chain Monte Carlo (MCMC) sampling, which is widely recognized as the most effective approach for characterizing a complex, high dimensional distribution. Other routines then map correlated residue patterns to available structures with a view to hypothesis generation. When applied to N-acetyltransferases, this reveals sequence and structural features indicative of functionally important, yet generally unknown biochemical properties. Even for sets of proteins for which nothing is known beyond unannotated sequences and structures, this can lead to helpful insights. We describe, for example, a putative coenzyme-A-induced-fit substrate binding mechanism mediated by arginine residue switching between salt bridge and π-π stacking interactions. A suite of programs implementing this approach is available (psed.igs.umaryland.edu).

An integrative C. elegans protein-protein interaction network with reliability assessment based on a probabilistic graphical model.

In Caenorhabditis elegans, a large number of protein-protein interactions (PPIs) are identified by different experiments. However, a comprehensive weighted PPI network, which is essential for signaling pathway inference, is not yet available in this model organism. Therefore, we firstly construct an integrative PPI network in C. elegans with 12,951 interactions involving 5039 proteins from seven molecular interaction databases. Then, a reliability score based on a probabilistic graphical model (RSPGM) is proposed to assess PPIs. It assumes that the random number of interactions between two proteins comes from the Bernoulli distribution to avoid multi-links. The main parameter of the RSPGM score contains a few latent variables which can be considered as several common properties between two proteins. Validations on high-confidence yeast datasets show that RSPGM provides more accurate evaluation than other approaches, and the PPIs in the reconstructed PPI network have higher biological relevance than that in the original network in terms of gene ontology, gene expression, essentiality and the prediction of known protein complexes. Furthermore, this weighted integrative PPI network in C. elegans is employed on inferring interaction path of the canonical Wnt/ß-catenin pathway as well. Most genes on the inferred interaction path have been validated to be Wnt pathway components. Therefore, RSPGM is essential and effective for evaluating PPIs and inferring interaction path. Finally, the PPI network with RSPGM scores can be queried and visualized on a user interactive website, which is freely available at .

Adsorbable organic bromine compounds (AOBr) in aquatic samples: a nematode-based toxicogenomic assessment of the exposure hazard.

Elevated levels of adsorbable organic bromine compounds (AOBr) have been detected in German lakes, and cyanobacteria like Microcystis, which are known for the synthesis of microcystins, are one of the main producers of natural organobromines. However, very little is known about how environmental realistic concentrations of organobromines impact invertebrates. Here, the nematode Caenorhabditis elegans was exposed to AOBr-containing surface water samples and to a Microcystis aeruginosa-enriched batch culture (MC-BA) and compared to single organobromines and microcystin-LR exposures. Stimulatory effects were observed in certain life trait variables, which were particularly pronounced in nematodes exposed to MC-BA. A whole genome DNA-microarray revealed that MC-BA led to the differential expression of more than 2000 genes, many of which are known to be involved in metabolic, neurologic, and morphologic processes. Moreover, the upregulation of cyp- and the downregulation of abu-genes suggested the presence of chronic stress. However, the nematodes were not marked by negative phenotypic responses. The observed difference in MC-BA and microcystin-LR (which impacted lifespan, growth, and reproduction) exposed nematodes was hypothesized to be likely due to other compounds within the batch culture. Most likely, the exposure to low concentrations of organobromines appears to buffer the effects of toxic substances, like microcystin-LR.

Galvez-Markov network transferability indices: review of classic theory and new model for perturbations in metabolic reactions.

Topological Indices (TIs) are numerical parameters useful to carry out Quantitative Structure-Property Relationships (QSPR) analysis and predict the effect of perturbations in many types of Complex Networks. This work, focuses on a very powerful class of TIs called Galvez charge transfer indices. First, we review the classic concept and some applications of these indices. Next, we review the Galvez-Markov TIs of order k (GMk), a recent generalization to these TIs introduced by us. We also reviewed some previous examples of calculation of GMk values for different classes of networks, including metabolic networks. Here, we also demonstrated that Galvez- Markov TIs are useful to predict perturbations and the transferability of biochemical patterns forms metabolic networks of species to others. We report a linear QSPR-Perturbation theory model that predicts more than 300,000 perturbations in metabolic networks with 85 - 99% of good classification in training and validation series.