Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes.

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement.

DLin-MC3-Containing mRNA Lipid Nanoparticles Induce an Antibody Th2-Biased Immune Response Polarization in a Delivery Route-Dependent Manner in Mice.

mRNA-based vaccines have made a leap forward since the SARS-CoV-2 pandemic and are currently used to develop anti-infectious therapies. If the selection of a delivery system and an optimized mRNA sequence are two key factors to reach in vivo efficacy, the optimal administration route for those vaccines remains unclear. We investigated the influence of lipid components and immunization route regarding the intensity and quality of humoral immune responses in mice. The immunogenicity of HIV-p55Gag encoded mRNA encapsulated into D-Lin-MC3-DMA or GenVoy-ionizable lipid-based LNPs was compared after intramuscular or subcutaneous routes. Three sequential mRNA vaccines were administrated followed by a heterologous boost composed of p24-HIV protein antigen. Despite equivalent IgG kinetic profiles of general humoral responses, IgG1/IgG2a ratio analysis showed a Th2/Th1 balance toward a Th1-biased cellular immune response when both LNPs were administrated via the intramuscular route. Surprisingly, a Th2-biased antibody immunity was observed when DLin-containing vaccine was injected subcutaneously. A protein-based vaccine boost appeared to reverse this balance to a cellular-biased response correlated to an increase in antibody avidity. Our finding suggests that the intrinsic adjuvant effect of ionizable lipids appears to be dependent on the delivery route used, which could be relevant to reach potent and long-lasting immunity after mRNA-based immunization.

Comparison of Physicochemical Properties of LipoParticles as mRNA Carrier Prepared by Automated Microfluidic System and Bulk Method.

Polymeric and/or lipid platforms are promising tools for nucleic acid delivery into cells. We previously reported a lipid-polymer nanocarrier, named LipoParticles, consisting of polylactic acid nanoparticles surrounded by cationic lipids, and allowing the addition of mRNA and cationic LAH4-1 peptide at their surface. Although this mRNA platform has shown promising results in vitro in terms of mRNA delivery and translation, the bulk method used to prepare LipoParticles relies on a multistep and time-consuming procedure. Here, we developed an automated process using a microfluidic system to prepare LipoParticles, and we compared it to the bulk method in terms of morphology, physicochemical properties, and ability to vectorize and deliver mRNA in vitro. LipoParticles prepared by microfluidic presented a smaller size and more regular spherical shape than bulk method ones. In addition, we showed that the total lipid content in LipoParticles was dependent on the method of preparation, influencing their ability to complex mRNA. LipoParticles decorated with two mRNA/LAHA-L1 ratios (1/20, 1/5) could efficiently transfect mouse DC2.4 cells except for the automated 1/5 assay. Moreover, the 1/5 mRNA/LAHA-L1 ratio drastically reduced cell toxicity observed in 1/20 ratio assays. Altogether, this study showed that homogeneous LipoParticles can be produced by microfluidics, which represents a promising platform to transport functional mRNA into cells.

Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA.

mRNA is a blooming technology for vaccination and has gained global attention during the SARS-CoV-2 pandemic. However, the recent clinical trials have highlighted increased reactogenicity when using high mRNA doses. Intending to increase the potency of mRNA therapeutics and to decrease the therapeutic dose, we designed a mRNA backbone and optimized the mRNA purification process. We used the enhanced green fluorescent protein (eGFP) reporter gene flanked by one 5' untranslated region (UTR) and two 3' UTRs of the human ß-globin as a reference mRNA and identified the most promising mRNA sequence using in vitro and in vivo models. First, we assessed the impact of different poly(A) sizes on translation and selected the most optimal sequence. Then, we selected the best 5' UTR among synthetic sequences displaying a high ribosome loading. Finally, we evaluated the transfection efficiency of our standard mRNA template after two capping strategies and purification using either double-stranded RNA (dsRNA) depletion or dephosphorylation of 5'PPP RNA or both combined. Double purification was shown to give the best results. Altogether, the use of a newly defined 5' UTR coupled to post-transcriptional treatments will be of great interest in the mRNA vaccine field, by limiting the amount of the antigen-coding transcript and subsequently the formulation components needed for an efficient vaccination.

Tailoring mRNA Vaccine to Balance Innate/Adaptive Immune Response.

mRNA vaccine platforms present numerous advantages, such as versatility, rapid production, and induction of cellular and humoral responses. Moreover, mRNAs have inherent adjuvant properties due to their complex interaction with pattern recognition receptors (PRRs). This recognition can be either beneficial in activating antigen-presenting cells (APCs) or detrimental by indirectly blocking mRNA translation. To decipher this Janus effect, we describe the different innate response mechanisms triggered by mRNA molecules and how each element from the 5' cap to the poly-A tail interferes with innate/adaptive immune responses. Then, we emphasize the importance of some critical steps such as production, purification, and formulation as key events to further improve the quality of immune responses and balance innate and adaptive immunity.

Poly(lactic acid) nanoparticles and cell-penetrating peptide potentiate mRNA-based vaccine expression in dendritic cells triggering their activation.

Messenger RNA-based vaccines have the potential to trigger robust cytotoxic immune responses, which are essential for fighting cancer and infectious diseases like HIV. Dendritic Cells (DCs) are choice targets for mRNA-based vaccine strategies, as they link innate and adaptive immune responses and are major regulators of cytotoxic and humoral adaptive responses. However, efficient delivery of antigen-coding mRNAs into DC cytosol has been highly challenging. In this study, we developed an alternative to lipid-based mRNA delivery systems, using poly(lactic acid) nanoparticles (PLA-NPs) and cationic cell-penetrating peptides as mRNA condensing agent. The formulations are assembled in two steps: (1) formation of a polyplex between mRNAs and amphipathic cationic peptides (RALA, LAH4 or LAH4-L1), and (2) adsorption of polyplexes onto PLA-NPs. LAH4-L1/mRNA polyplexes and PLA-NP/LAH4-L1/mRNA nanocomplexes are taken up by DCs via phagocytosis and clathrin-dependent endocytosis, and induce strong protein expression in DCs in vitro. They modulate DC innate immune response by activating both endosome and cytosolic Pattern Recognition Receptors (PRRs), and induce markers of adaptive responses in primary human DCs in vitro, with prevalent Th1 signature. Thus, LAH4-L1/mRNA and PLA-NP/LAH4-L1/mRNA represent a promising platform for ex vivo treatment and mRNA vaccine development.

Poly(Lactic Acid) Nanoparticles Targeting α5ß1 Integrin as Vaccine Delivery Vehicle, a Prospective Study.

Biodegradable polymeric nanoparticles are vehicles of choice for drug delivery and have the ability to encapsulate and present at their surface different molecules of interest. Among these bio-nanocarriers, poly(lactic acid) (PLA) nanoparticles have been used as adjuvant and vehicle for enhanced vaccine efficacy. In order to develop an approach to efficient vaccine delivery, we developed nanoparticles to target α5ß1 positive cells. We first overproduced, in bacteria, human fibronectin FNIII9/10 recombinant proteins possessing an integrin α5ß1 binding site, the RGDS sequence, or a mutated form of this site. After having confirmed the integrin binding properties of these recombinant proteins in cell culture assays, we were able to formulate PLA nanoparticles with these FNIII9/10 proteins at their surface. We then confirmed, by fluorescence and confocal microscopy, an enhanced cellular uptake by α5ß1+ cells of RGDS-FNIII9/10 coated PLA nanoparticles, in comparison to KGES-FNIII9/10 coated or non-coated controls. As a first vaccination approach, we prepared PLA nanoparticles co-coated with p24 (an HIV antigen), and RGDS- or KGES-FNIII9/10 proteins, followed by subcutaneous vaccine administration, in mice. Although we did not detect improvements in the apparent humoral response to p24 antigen in the serum of RGDS/p24 nanoparticle-treated mice, the presence of the FNIII proteins increased significantly the avidity index of anti-p24 antibodies compared to p24-nanoparticle-injected control mice. Future developments of this innovative targeted vaccine are discussed.

Evaluation of polylactic acid nanoparticles safety using Drosophila model.

Cytotoxicity of nanoparticles and their sub-lethal effect on cell behavior and cell fate are a high topic of studies in the nanomaterial field. With an explosion of nanoparticle types (size, shape, polarity, stiffness, composition, etc.), Drosophila has become an attractive animal model for high throughput analysis of these nanocarriers in the drug delivery field with applications in cancer therapy, or simply to generate a fast and complete cytotoxic study of a peculiar nanoparticle. In respect to that, we have conducted an in cellulo study of poly(lactic acid) (PLA) nanoparticle cytotoxicity, and determined that near lethal nanoparticle doses, oxidative stress as well as P53 and ATP pathways may lead to cell cycle arrest at G1, and ultimately to cell death. Neither viability nor the development of Drosophila larvae are affected by the ingestion of PLA nanoparticles at sub-lethal concentrations. Drosophila will be a useful model to study PLA and PLA-modified nanoparticle toxicity, and nanoparticle fate after ingestion.

A purified truncated form of yeast Gal4 expressed in Escherichia coli and used to functionalize poly(lactic acid) nanoparticle surface is transcriptionally active in cellulo.

Gal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticles. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models.

Tenascin-X: beyond the architectural function.

Tenascin-X is the largest member of the tenascin (TN) family of evolutionary conserved extracellular matrix glycoproteins, which also comprises TN-C, TN-R and TN-W. Among this family, TN-X is the only member described so far to exert a crucial architectural function as evidenced by a connective tissue disorder (a recessive form of Ehlers-Danlos syndrome) resulting from a loss-of-function of this glycoprotein in humans and mice. However, TN-X is more than an architectural protein, as it displays features of a matricellular protein by modulating cell adhesion. However, the cellular functions associated with the anti-adhesive properties of TN-X have not yet been revealed. Recent findings indicate that TN-X is also an extracellular regulator of signaling pathways. Indeed, TN-X has been shown to regulate the bioavailability of the Transforming Growth Factor (TGF)-ß and to modulate epithelial cell plasticity. The next challenges will be to unravel whether the signaling functions of TN-X are functionally linked to its matricellular properties.

Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-ß.

Transforming growth factor ß (TGF-ß) isoforms are secreted as inactive complexes formed through noncovalent interactions between the bioactive TGF-ß entity and its N-terminal latency-associated peptide prodomain. Extracellular activation of the latent TGF-ß complex is a crucial step in the regulation of TGF-ß function for tissue homeostasis. We show that the fibrinogen-like (FBG) domain of the matrix glycoprotein tenascin-X (TNX) interacts physically with the small latent TGF-ß complex in vitro and in vivo, thus regulating the bioavailability of mature TGF-ß to cells by activating the latent cytokine into an active molecule. Activation by the FBG domain most likely occurs through a conformational change in the latent complex and involves a novel cell adhesion-dependent mechanism. We identify α11ß1 integrin as a cell surface receptor for TNX and show that this integrin is crucial to elicit FBG-mediated activation of latent TGF-ß and subsequent epithelial-to-mesenchymal transition in mammary epithelial cells.

Structural basis of fibrillar collagen trimerization and related genetic disorders.

The C propeptides of fibrillar procollagens have crucial roles in tissue growth and repair by controlling both the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils. Mutations in C propeptides are associated with several, often lethal, genetic disorders affecting bone, cartilage, blood vessels and skin. Here we report the crystal structure of a C-propeptide domain from human procollagen III. It reveals an exquisite structural mechanism of chain recognition during intracellular trimerization of the procollagen molecule. It also gives insights into why some types of collagen consist of three identical polypeptide chains, whereas others do not. Finally, the data show striking correlations between the sites of numerous disease-related mutations in different C-propeptide domains and the degree of phenotype severity. The results have broad implications for understanding genetic disorders of connective tissues and designing new therapeutic strategies.

Isolation, characterization and biological evaluation of jellyfish collagen for use in biomedical applications.

Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2-10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications.

The fibrillar collagen family.

Collagens, or more precisely collagen-based extracellular matrices, are often considered as a metazoan hallmark. Among the collagens, fibrillar collagens are present from sponges to humans, and are involved in the formation of the well-known striated fibrils. In this review we discuss the different steps in the evolution of this protein family, from the formation of an ancestral fibrillar collagen gene to the formation of different clades. Genomic data from the choanoflagellate (sister group of Metazoa) Monosiga brevicollis, and from diploblast animals, have suggested that the formation of an ancestral alpha chain occurred before the metazoan radiation. Phylogenetic studies have suggested an early emergence of the three clades that were first described in mammals. Hence the duplication events leading to the formation of the A, B and C clades occurred before the eumetazoan radiation. Another important event has been the two rounds of "whole genome duplication" leading to the amplification of fibrillar collagen gene numbers, and the importance of this diversification in developmental processes. We will also discuss some other aspects of fibrillar collagen evolution such as the development of the molecular mechanisms involved in the formation of procollagen molecules and of striated fibrils.

Tenascin-X increases the stiffness of collagen gels without affecting fibrillogenesis.

Tenascin-X is an extracellular matrix protein whose absence leads to an Ehlers-Danlos Syndrome in humans, mainly characterised by connective tissue defects including the disorganisation of fibrillar networks, a reduced collagen deposition, and modifications in the mechanical properties of dense tissues. Here we tested the effect of tenascin-X on in vitro collagen fibril formation. We observed that the main parameters of fibrillogenesis were unchanged, and that the diameter of fibrils was not significantly different when they were formed in the presence of tenascin-X. Interestingly, mechanical analysis of collagen gels showed an increased compressive resistance of the gels containing tenascin-X, indicating that this protein might be directly involved in determining the mechanical properties of collagen-rich tissues in vivo.

Demosponge and sea anemone fibrillar collagen diversity reveals the early emergence of A/C clades and the maintenance of the modular structure of type V/XI collagens from sponge to human.

Collagens are often considered a metazoan hallmark, with the fibril-forming fibrillar collagens present from sponges to human. From evolutionary studies, three fibrillar collagen clades (named A, B, and C) have been defined and shown to be present in mammals, whereas the emergence of the A and B clades predates the protostome/deuterostome split. Moreover, several C clade fibrillar collagen chains are present in some invertebrate deuterostome genomes but not in protostomes whose genomes have been sequenced. The newly sequenced genomes of the choanoflagellate Monosiga brevicollis, the demosponge Amphimedon queenslandica, and the cnidarians Hydra magnipapillata (Hydra) and Nematostella vectensis (sea anemone) allow us to have a better understanding of the origin and evolution of fibrillar collagens. Analysis of these genomes suggests that an ancestral fibrillar collagen gene arose at the dawn of the Metazoa, before the divergence of sponge and eumetazoan lineages. The duplication events leading to the formation of the three fibrillar collagen clades (A, B, and C) occurred before the eumetazoan radiation. Interestingly, only the B clade fibrillar collagens preserved their characteristic modular structure from sponge to human. This observation is compatible with the suggested primordial function of type V/XI fibrillar collagens in the initiation of the formation of the collagen fibrils.

A model of tenascin-X integration within the collagenous network.

Tenascin-X is an extracellular matrix protein whose absence leads to an Ehlers-Danlos syndrome in humans, characterized mainly by disorganisation of collagen and elastic fibril networks. After producing recombinant full-length tenascin-X in mammalian cells, we find that this protein assembled into disulfide-linked oligomers. Trimers were the predominant form observed using rotary shadowing. By solid phase interaction studies, we demonstrate that tenascin-X interacts with types I, III and V fibrillar collagen molecules when they are in native conformation. The use of tenascin-X variants with large regions deleted indicated that both epidermal growth factor repeats and the fibrinogen-like domain are involved in this interaction. Moreover, we demonstrate that tenascin-X binds to the fibril-associated types XII and XIV collagens. We thus suggest that tenascin-X, via trimerization and multiple interactions with components of collagenous fibrils, plays a crucial role in the organisation of extracellular matrices.

Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates.

Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.

Invertebrate data predict an early emergence of vertebrate fibrillar collagen clades and an anti-incest model.

Fibrillar collagens are involved in the formation of striated fibrils and are present from the first multicellular animals, sponges, to humans. Recently, a new evolutionary model for fibrillar collagens has been suggested (Boot-Handford, R. P., Tuckwell, D. S., Plumb, D. A., Farrington Rock, C., and Poulsom, R. (2003) J. Biol. Chem. 278, 31067-31077). In this model, a rare genomic event leads to the formation of the founder vertebrate fibrillar collagen gene prior to the early vertebrate genome duplications and the radiation of the vertebrate fibrillar collagen clades (A, B, and C). Here, we present the modular structure of the fibrillar collagen chains present in different invertebrates from the protostome Anopheles gambiae to the chordate Ciona intestinalis. From their modular structure and the use of a triple helix instead of C-propeptide sequences in phylogenetic analyses, we were able to show that the divergence of A and B clades arose early during evolution because alpha chains related to these clades are present in protostomes. Moreover, the event leading to the divergence of B and C clades from a founder gene arose before the appearance of vertebrates; altogether these data contradict the Boot-Handford model. Moreover, they indicate that all the key steps required for the formation of fibrils of variable structure and functionality arose step by step during invertebrate evolution.

Distinct maturations of N-propeptide domains in fibrillar procollagen molecules involved in the formation of heterotypic fibrils in adult sea urchin collagenous tissues.

We have characterized the primary structure of a new sea urchin fibrillar collagen, the 5alpha chain, including nine repeats of the sea urchin fibrillar module in its N-propeptide. By Western blot and immunofluorescence analyses, we have shown that 5alpha is co-localized in adult collagenous ligaments with the 2alpha fibrillar collagen chain and fibrosurfin, two other extracellular matrix proteins possessing sea urchin fibrillar modules. At the ultrastructural level, the 5alpha N-propeptide is detected at the surface of fibrils, suggesting the retention of this domain in mature collagen molecules. Biochemical characterization of pepsinized collagen molecules extracted from the test tissue (the endoskeleton) together with a matrix-assisted laser desorption ionization time-of-flight analysis allowed us to determine that 5alpha is a quantitatively minor fibrillar collagen chain in comparison with the 1alpha and 2alpha chains. Moreover, 5alpha forms heterotrimeric molecules with two 1alpha chains. Hence, as in vertebrates, sea urchin collagen fibrils are made up of quantitatively major and minor fibrillar molecules undergoing distinct maturation of their N-propeptide regions and participating in the formation of heterotypic fibrils.