The vertebrate glycoprotein hormones (GpHs), gonadotropins and thyrotropin, are heterodimers composed of a common alpha- and specific beta-subunit. The recombinant heterodimer of two additional, structurally related proteins identified in vertebrate and protostome genomes, the glycoproteins-alpha2 (GPA2) and-beta5 (GPB5), was shown to activate the thyrotropin receptor and was therefore named thyrostimulin. However, differences in tissue distribution and expression levels of these proteins suggested that they might act as nonassociated factors, prompting further investigation on these proteins. In this study we show that GPA2 and GPB5 appeared with the emergence of bilateria and were maintained in most groups. These genes are tightly associated at the genomic level, an association, however, lost in tetrapods. Our structural and genomic environment comparison reinforces the hypothesis of their phylogenetic relationships with GpH-alpha and -beta. In contrast, the glycosylation status of GPA2 and GPB5 is highly variable further questioning heterodimer secretory efficiency and activity. As a first step toward understanding their function, we investigated the spatiotemporal expression of GPA2 and GPB5 genes at different developmental stages in a basal chordate, the amphioxus. Expression of GPB5 was essentially ubiquitous with an anteroposterior gradient in embryos. GPA2 embryonic and larvae expression was restricted to specific areas and, interestingly, partially overlapped that of a GpH receptor-related gene. In conclusion, we speculate that GPA2 and GPB5 have nondispensable and coordinated functions related to a novelty appeared with bilateria. These proteins would be active during embryonic development in a manner that does not require their heterodimerization.

The urotensin II (UII) family is currently known to consist of two paralogous peptides, namely UII and UII-related peptide (URP). In contrast to UII, which has been identified in all vertebrate classes so far, URP has only been characterized in tetrapods. We report here the occurrence of two distinct URP genes in teleosts, which we have named URP1 and URP2. Synteny analysis revealed that teleost URP1 and URP2 genes and tetrapod URP genes represent three distinct paralog genes that, together with the UII gene, probably arose from the two rounds of tetraploidization, which took place early in vertebrate evolution. The absence of URP in fish indicates that the corresponding gene has been lost in the teleost lineage, whereas it is likely that both the URP1 and URP2 genes have been lost in the tetrapod lineage. Quantitative RT-PCR analysis revealed that the URP2 gene is mainly expressed in the spinal cord and the brain in adult zebrafish. In situ hybridization experiments showed that in zebrafish embryos, URP2 mRNA-containing cells are located in the floor plate of the neural tube. In adult, URP2-expressing cells occur in close contact with the ventral side of the ependymal canal along the whole spinal cord, whereas in the brain, they are located below the fourth ventricle. These URP-expressing cells may correspond to cerebrospinal fluid-contacting neurons. In conclusion, our study reveals the occurrence of four distinct UII paralogous systems in vertebrates that may exert distinct functions, both in tetrapods and teleosts.

Hollow mesoporous silica microsphere (HMSM) particles are one of the most promising vehicles for efficient drug delivery owing to their large hollow interior cavity for drug loading and the permeable mesoporous shell for controlled drug release. Here, we report an easily controllable aerosol-based approach to produce HMSM particles by continuous spray-drying of colloidal silica nanoparticles and Eudragit/Triton X100 composite (EUT) nanospheres as templates, followed by template removal. Importantly, the internal structure of the hollow cavity and the external morphology and the porosity of the mesoporous shell can be tuned to a certain extent by adjusting the experimental conditions (i.e., silica to EUT mass ratio and particle size of silica nanoparticles) to optimize the drug loading capacity and the controlled-release properties. Then, the application of aerosol-synthesized HMSM particles in controlled drug delivery was investigated by loading amoxicillin as an antibiotic compound with high entrapment efficiency (up to 46%). Furthermore, to improve the biocompatibility of the amoxicillin-loaded HMSM particles, their surfaces were functionalized with poly­(allylamine hydrochloride) and alginate as biocompatible polymers via the layer-by-layer assembly. The resulting particles were evaluated toward Escherichia coli (Gram-negative) bacteria and indicated the bacterial inhibition up to 90% in less than 2 h. Finally, we explored the versatility of HMSMs as drug carriers for pancreatic cancer treatment. Because the pH value of the extracellular medium in pancreatic tumors is lower than that of the healthy tissue, chitosan as a pH-sensitive gatekeeper was grafted to the HMSM surface and then loaded with a pro-apoptotic NCL antagonist agent (N6L) as an anticancer drug. The obtained particles exhibited pH-responsive drug releases and excellent anticancer activities with inhibition of cancer cell growth up to 60%.