The diversification and lineage-specific expansion of nitric oxide signaling in Placozoa: insights in the evolution of gaseous transmission.

Nitric oxide (NO) is a ubiquitous gaseous messenger, but we know little about its early evolution. Here, we analyzed NO synthases (NOS) in four different species of placozoans-one of the early-branching animal lineages. In contrast to other invertebrates studied, Trichoplax and Hoilungia have three distinct NOS genes, including PDZ domain-containing NOS. Using ultra-sensitive capillary electrophoresis assays, we quantified nitrites (products of NO oxidation) and L-citrulline (co-product of NO synthesis from L-arginine), which were affected by NOS inhibitors confirming the presence of functional enzymes in Trichoplax. Using fluorescent single-molecule in situ hybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions of Trichoplax. These data suggest both the compartmentalized release of NO and a greater diversity of cell types in placozoans than anticipated. NO receptor machinery includes both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, although Trichoplax and Hoilungia exemplify the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling in Placozoa is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa and the preservation of conservative NOS architecture from prokaryotic ancestors.

Microchemical identification of enantiomers in early-branching animals: Lineage-specific diversification in the usage of D-glutamate and D-aspartate.

D-amino acids are unique and essential signaling molecules in neural, hormonal, and immune systems. However, the presence of D-amino acids and their recruitment in early animals is mostly unknown due to limited information about prebilaterian metazoans. Here, we performed the comparative survey of L-/D-aspartate and L-/D-glutamate in representatives of four phyla of early-branching Metazoa: cnidarians (Aglantha); placozoans (Trichoplax), sponges (Sycon) and ctenophores (Pleurobrachia, Mnemiopsis, Bolinopsis, and Beroe), which are descendants of ancestral animal lineages distinct from Bilateria. Specifically, we used high-performance capillary electrophoresis for microchemical assays and quantification of the enantiomers. L-glutamate and L-aspartate were abundant analytes in all species studied. However, we showed that the placozoans, cnidarians, and sponges had high micromolar concentrations of D-aspartate, whereas D-glutamate was not detectable in our assays. In contrast, we found that in ctenophores, D-glutamate was the dominant enantiomer with no or trace amounts of D-aspartate. This situation illuminates prominent lineage-specific diversifications in the recruitment of D-amino acids and suggests distinct signaling functions of these molecules early in the animal evolution. We also hypothesize that a deep ancestry of such recruitment events might provide some constraints underlying the evolution of neural and other signaling systems in Metazoa.

Glycine as a signaling molecule and chemoattractant in Trichoplax (Placozoa): insights into the early evolution of neurotransmitters.

The origin and early evolution of neurotransmitter signaling in animals are unclear due to limited comparative information, primarily about prebilaterian animals. Here, we performed the comparative survey of signal molecules in placozoans - the simplest known free-living animals without canonical synapses, but with complex behaviors. First, using capillary electrophoresis with laser-induced fluorescence detection, we performed microchemical analyses of transmitter candidates in Trichoplax adhaerens - the classical reference species in comparative biology. We showed that the endogenous level of glycine (about 3 mM) was significantly higher than for other candidates such as L-glutamate, L-aspartate, or gamma-aminobutyric acid. Neither serotonin nor dopamine were detected. The absolute glycine concentrations in Trichoplax were even higher than we measured in ctenophores (Beroe) and cnidarians (Aequorea). We found that at millimolar concentrations of glycine (similar to the endogenous level), induced muscle-like contractions in free behaving animals. But after long incubation (24 h), 10 M of glycine could induce cytotoxicity and cell dissociation. In contrast, micromolar concentrations (10-10 M) increased Trichoplax ciliated locomotion, suggesting that glycine might act as an endogenous signal molecule. However, we showed than glycine (10 M) can also be a chemoattractant (a guiding factor for food sources), and therefore, act as the exogenous signal. These findings provide an evolutionary base for the origin of transmitters as a result of the interplay between exogenous and endogenous signaling systems early in animal evolution.

Molecular beacons in biomedical detection and clinical diagnosis.

Among the diverse nucleic acid probes, molecular beacons (MBs) have shown their excellent potential in a variety of basic researches and practical applications. Their excellent selectivity, sensitivity, and detection without separation have led them to be particularly useful in real-time intracellular monitoring of gene expression, development of biosensors, and clinical diagnostics. This paper will focus on the properties of various MBs and discuss their potential applications.