Exploring in the classroom the relationship between alcohol intake and behavioral disorders through an animal model.

Alcohol consumption has profound effects on behavior, such as impaired judgment, addiction or even death. It is estimated that alcohol contributes to around three million deaths worldwide, 13.5% of them in young people with ages between 20 and 39 years. Consequently, it is necessary to raise awareness among college and high school students of the risk related to alcohol drinking. The small nematode Caenorhabditis elegans is an animal widely used as a model organism to study nearly all aspects of Biochemistry. It is a powerful tool to test the potential bioactivity and molecular mechanisms of natural compounds and drugs in vivo. Therefore, it is an interesting topic to include in an undergraduate course of Biotechnology, Biochemistry or Biology students among other scientific vocations. C. elegans is also used as a neurobiological model to evaluate substances´ neurotoxicity and behavioral effects. The proposed experiment introduces students to the handling of this preclinical model and to the evaluation of behavioral alterations induced by chemicals in scientific research. The effects of different doses of ethanol on C. elegans behavior are studied using a versatile chemotaxis assay. This laboratory experiment is suitable for an undergraduate course. The practical session can be used in the global strategies of information and awareness of educational centres to mitigate the impact of alcohol abuse among students, both in formal courses or in Science fairs or exhibitions.

Are seven amino acid substitutions sufficient to explain the evolution of high l-DOPA 4,5-dioxygenase activity leading to betalain pigmentation? Revisiting the gain-of-function mutants of Bean et al. (2018).

This work revisits a publication by Bean et al. (2018) that reports seven amino acid substitutions are essential for the evolution of l-DOPA 4,5-dioxygenase (DODA) activity in Caryophyllales. In this study, we explore several concerns which led us to replicate the analyses of Bean et al. (2018). Our comparative analyses, with structural modelling, implicate numerous residues additional to those identified by Bean et al. (2018), with many of these additional residues occurring around the active site of BvDODAα1. We therefore replicated the analyses of Bean et al. (2018) to re-observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2-mut3 variant. Multiple in vivo assays, in both Saccharomyces cerevisiae and Nicotiana benthamiana, did not result in visible DODA activity in BvDODAα2-mut3, with betalain production always 10-fold below BvDODAα1. In vitro assays also revealed substantial differences in both catalytic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2-mut3 proteins, explaining their differing performance in vivo. In summary, we were unable to replicate the in vivo analyses of Bean et al. (2018), and our quantitative in vivo and in vitro analyses suggest a minimal effect of these seven residues in altering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Bean et al. (2018).

Consumption of commonly used artificial food dyes increases activity and oxidative stress in the animal model Caenorhabditis elegans.

In recent decades, the consumption of artificial colorants in foods and beverages has increased despite of concerns in the general population raised by studies that have shown possible injurious effects. In this study, tartrazine, sunset yellow, quinoline yellow, ponceau 4R, carmoisine and allura red were employed as pure compounds to explore their effects in vivo in the animal model Caenorhabditis elegans. The exposition of C. elegans to these artificial dyes produced damage related with aging such as oxidative stress and lipofuscin accumulation, as well as a heavy shortening of lifespan, alterations in movement patterns and alterations in the production of dopamine receptors. Besides, microarray analysis performed with worms treated with tartrazine and ponceau 4R showed how the consumption of synthetic colorants is able to alter the expression of genes involved in resistance to oxidative stress and neurodegeneration.

Characterization of betalain-loaded liposomes and its bioactive potential in vivo after ingestion.

Betalains are plant pigments characterized by showing a wide range of beneficial properties for health. Its bioactive potential has been studied for the first time after its encapsulation in liposomes and subsequent administration to the animal model Caenorhabditis elegans. Phenylalanine-betaxanthin and indoline carboxylic acid-betacyanin encapsulated at concentrations of 25 and 500 µM managed to reduce lipid accumulation and oxidative stress in the nematodes. Highly antioxidant betalains dopaxanthin and betanidin were also included in the survival analyses. The results showed that phenylalanine-betaxanthin was the most effective betalain by increasing the lifespan of C. elegans by 21.8%. In addition, the administration of encapsulated natural betanidin increased the nematodes' survival rate by up to 13.8%. The preservation of the bioactive properties of betalains manifested in this study means that the stabilization of the plant pigments through encapsulation in liposomes can be postulated as a new way for administration in pharmacological and food applications.

Biosynthesis of a novel polymeric chitosan-betaxanthin and characterization of the first sugar-derived betalains and their effects in the in vivo model Caenorhabditis elegans.

Betaxanthins are nitrogenous plant pigments belonging to the family of betalains and they are known for their health-promoting effects and fluorescent properties. A novel biotechnological approach in the synthesis of these compounds has allowed the synthesis of high amounts of known betalains and of novel, tailor-made betalains through the condensation of the structural unit - betalamic acid - with amine groups of different compounds. Here we describe the synthesis and characterization of chitosan-betaxanthin, the first fluorescent polymeric betaxanthin which forms nanoparticles and that might combine the fluorescent properties of betalains and the properties of chitosan, a sugar polymer widely used with medical purposes. In addition, glucosamine, the structural unit of chitosan, and its stereoisomer galactosamine were shown to condense in solution with betalamic acid. This produced novel molecules with spectral and in vivo antioxidant and anti-aging properties similar to those of biological betaxanthins, which are the first sugar-derived betaxanthins described.

Lifespan extension in Caenorhabditis elegans by oxyresveratrol supplementation in hyper-branched cyclodextrin-based nanosponges.

In this work, the increase of the Caenorhabditis elegans (C. elegans) lifespan extension using hyper-branched cyclodextrin-based nanosponges (CD-NS) complexing oxyresveratrol (OXY), and the possible inhibition of C. elegans phosphodiesterase type 4 (PDE4) were evaluated. The titration displacement of fluorescein was used to calculate the apparent complexation constant (KF) between CD-NS and OXY. Moreover, PDE4 was expressed in E. coli, purified and refolded in presence of cyclodextrins (CDs) to study its possible inhibition as pharmacological target of OXY. The apparent activity was characterized and the inhibitory effect of OXY on PDE4 displayed a competitive in vitro inhibition corroborated in silico. A maximum increase of the in vivo life expectancy of about 9.6% of using OXY/CD-NS complexes in comparison with the control was obtained, in contrast to the 6.5% obtained with free OXY. No effect on lifespan or toxicity with CD-NS alone was found. These results as a whole represent new opportunities to use OXY and CD-NS in lifespan products.

Antitumoral Drug Potential of Tryptophan-Betaxanthin and Related Plant Betalains in the Caenorhabditis elegans Tumoral Model.

Betalains are plants pigments identified as potent antioxidant molecules, naturally present in foods like beetroot and prickly pears. Although activities described for betalain-containing formulations include cancer prevention and treatment, the use of extracts instead of purified pigments has avoided the investigation of the real chemopreventive and chemotherapeutic potential of these phytochemicals. Three betalain-rich extracts and six individual pure betalains were used in this work to characterize the activity and to explore possible molecular mechanisms. The animal model Caenorhabditis elegans (tumoral strain JK1466) was used to evaluate the effect of betalains as chemotherapeutics drugs. An objective evaluation method of tumor growth in C. elegans has been developed to assess the possible antitumoral activity of the different treatments. This protocol allowed a fast and reliable screening of possible antitumoral drugs. Among the betalains tested, tryptophan-betaxanthin reduced tumor size by 56.4% and prolonged the animal's lifespan by 9.3%, indicating high effectiveness and low toxicity. Structure-activity relationships are considered. Assays with mutant strains of C. elegans showed that the mechanism underlying these effects was the modulation of the DAF-16 transcription factor and the insulin signaling pathway. Our results indicate that tryptophan-betaxanthin and related betalains are strong candidates as antitumoral molecules in cancer treatment.

Betalain health-promoting effects after ingestion in Caenorhabditis elegans are mediated by DAF-16/FOXO and SKN-1/Nrf2 transcription factors.

Betalain-rich extracts have been used for many years by their nutraceutical potential. However, the study of their bioactivities has always been hampered by their difficult obtention. To explain their mode of action, seventeen pure betalains were tested in vivo using the animal model C. elegans. Four betalains, named indicaxanthin, indoline carboxylic acid-betacyanin, phenylalanine-betaxanthin, and dopaxanthin, behaved as extraordinary in vivo antioxidants and anti-aging compounds, by increasing the lifespan of C. elegans up to 16.82%, 16.65%, 16.53%, and 12.93%, respectively. The first microarrays performed with betalains and biological confirmation with different mutant strains showed that this life extension is due to a reduction of oxidative stress and the activation of the transcription factors DAF-16/FOXO and SKN-1/Nrf2. They are involved in longevity and oxidative stress resistance pathways and lead to overexpression of HSPs genes, involved in resistance to cancer and Alzheimer's, opening novel research lines in the search for effective plant-based treatments.

Light Emission in Betalains: From Fluorescent Flowers to Biotechnological Applications.

The discovery of visible fluorescence in the plant pigments betalains revealed the existence of fluorescent patterns in flowers of plants of the order Caryophyllales, where betalains substitute anthocyanins. The serendipitous initial discovery led to a systemized characterization of the role of different substructures on the photophysical phenomenon. Strong fluorescence is general to all members of the family of betaxanthins linked to the structural property that the betalamic acid moiety is connected to an amine group. This property has led to bioinspired tailor-made probes and to the development of novel biotechnological applications in screening techniques or microscopy labeling. Here, we comprehensively review the photophysics, photochemistry, and photobiology of betalain fluorescence and describe all current applications.

First Betalain-Producing Bacteria Break the Exclusive Presence of the Pigments in the Plant Kingdom.

The biosynthesis of antioxidant pigments, namely, betalains, was believed to be restricted to Caryophyllales plants. This paper changes this paradigm, and enzyme mining from bacterial hosts promoted the discovery of bacterial cultures producing betalains. The spectrum of possible sources of betalain pigments in nature is broadened by our description of the first betalain-forming bacterium, Gluconacetobacter diazotrophicus The enzyme-specific step is the extradiol cleavage of the precursor amino acid l-dihydroxyphenylalanine (l-DOPA) to form the structural unit betalamic acid. Molecular and functional work conducted led to the characterization of a novel dioxygenase, a polypeptide of 17.8 kDa with a Km of 1.36 mM, with higher activity and affinity than those of its plant counterparts. Its superior activity allowed the first experimental characterization of the early steps in the biosynthesis of betalains by fully characterizing the presence and time evolution of 2,3- and 4,5-seco-DOPA intermediates. Furthermore, spontaneous chemical reactions are characterized and incorporated into a comprehensive enzymatic-chemical mechanism that yields the final pigments.IMPORTANCE Several studies have demonstrated the health-promoting effects of betalains due to their high antioxidant capacity and their positive effect on the dose-dependent inhibition of cancer cells and their proliferation. To date, betalains were restricted to plants of the order Caryophyllales and some species of fungi, but the present study reveals the first betalain-producing bacterium, as well as the first steps in the formation of pigments. This finding demonstrates that betalain biosynthesis can be expanded to prokaryotes.

Extension of life-span using a RNAi model and in vivo antioxidant effect of Opuntia fruit extracts and pure betalains in Caenorhabditis elegans.

Betalains are nitrogenous plant pigments known for their high antioxidant capacity. For the first time, this antioxidant nature has been studied in an in vivo system using the model organism Caenorhabditis elegans. The oxidative stress caused in the fluorescent strain TJ375 (hsp-16.2::GFP) was reversed by the presence of both natural and semi-synthetic betalains, with an ED50 value around 25 µM for betacyanins and up to 10 µM for betaxanthins, with indicaxanthin, the major pigments in prickly pear fruits, as the most effective betalain. The effect of model betalains on the lifespan of the wild-type N2 strain was carefully studied using the automatic platform "Lifespan Machine". In a search for different approaches to suppress progeny, pop-1 RNAi was used to avoid FUdR use. The presence of betalains in the medium, both as pure compounds and as enriched Opuntia extracts significantly increased the lifespan of C. elegans.

Development of Betalain Producing Callus Lines from Colored Quinoa Varieties (Chenopodium quinoa Willd).

Betalains are water-soluble plant pigments of hydrophilic nature with promising bioactive potential. Among the scarce edible sources of betalains is the grain crop quinoa (Chenopodium quinoa Willd), with violet, red, and yellow grains being colored by these pigments. In this work, callus cultures have been developed from differently colored plant varieties. Stable callus lines exhibited color and pigment production when maintained on Murashige and Skoog medium supplemented with the plant growth regulators 6-benzylaminopurine (8.88 µM) and 2,4-dichlorophenoxyacetic acid (6.79 µM) with a reduction of the nitrogen source to 5.91 mM. Pigment analysis by HPLC-DAD and ESI-MS/MS fully describes the content of individual pigments in the cell lines and allows the first report on the pigments present in quinoa seedlings. Phyllocactin and vulgaxanthin I are described as novel pigments in the species and show the potential of C. quinoa culture lines in the production of compounds of nutritional value.