Chemical Investigation of the Calcareous Marine Sponge Pericharax heteroraphis, Clathridine-A Related Derivatives Isolation, Synthesis and Osteogenic Activity.

As a result of screening a panel of marine organisms to identify lead molecules for the stimulation of endochondral bone formation, the calcareous sponge Pericharax heteroraphis was identified to exhibit significant activity during endochondral differentiation. On further molecular networking analysis, dereplication and chemical fractionation yielded the known clathridine A-related metabolites 3-6 and the homodimeric complex (clathridine A)2 Zn2+ (9), together with the new unstable heterodimeric complex (clathridine A-clathridimine)Zn2+ (10). With the presence of the zinc complexes annotated through the LC-MS analysis of the crude extract changing due to the instability of some metabolites and complexes constituting the mixture, we combined the isolation of the predicted molecules with their synthesis in order to confirm their structure and to understand their reactivity. Interestingly, we also found a large quantity of the contaminant benzotriazoles BTZ (7) and its semi-dimer (BTZ)2CH2 (8), which are known to form complexes with transition metals and are used for preventing corrosion in water. All isolated 2-aminoimidazole derivatives and complexes were synthesized not only for structural confirmation and chemical understanding but to further study their bioactivity during endochondral differentiation, particularly the positively screened imidazolone derivatives. Compounds leucettamine B, clathridine A and clathridimine were found to increase type X collagen transcription and stimulate endochondral ossification in the ATDC5 micromass model.

Organic Matrices of Calcium Carbonate Biominerals Improve Osteoblastic Mineralization.

Many organisms incorporate inorganic solids into their tissues to improve functional and mechanical properties. The resulting mineralized tissues are called biominerals. Several studies have shown that nacreous biominerals induce osteoblastic extracellular mineralization. Among them, Pinctada margaritifera is well known for the ability of its organic matrix to stimulate bone cells. In this context, we aimed to study the effects of shell extracts from three other Pinctada species (Pinctada radiata, Pinctada maxima, and Pinctada fucata) on osteoblastic extracellular matrix mineralization, by using an in vitro model of mouse osteoblastic precursor cells (MC3T3-E1). For a better understanding of the Pinctada-bone mineralization relationship, we evaluated the effects of 4 other nacreous mollusks that are phylogenetically distant and distinct from the Pinctada genus. In addition, we tested 12 non-nacreous mollusks and one extra-group. Biomineral shell powders were prepared, and their organic matrix was partially extracted using ethanol. Firstly, the effect of these powders and extracts was assessed on the viability of MC3T3-E1. Our results indicated that neither the powder nor the ethanol-soluble matrix (ESM) affected cell viability at low concentrations. Then, we evaluated osteoblastic mineralization using Alizarin Red staining and we found a prominent MC3T3-E1 mineralization mainly induced by nacreous biominerals, especially those belonging to the Pinctada genus. However, few non-nacreous biominerals were also able to stimulate the extracellular mineralization. Overall, our findings validate the remarkable ability of CaCO3 biomineral extracts to promote bone mineralization. Nevertheless, further in vitro and in vivo studies are needed to uncover the mechanisms of action of biominerals in bone.

Biasing the conformation of ELMO2 reveals that myoblast fusion can be exploited to improve muscle regeneration.

Myoblast fusion is fundamental for the development of multinucleated myofibers. Evolutionarily conserved proteins required for myoblast fusion include RAC1 and its activator DOCK1. In the current study we analyzed the contribution of the DOCK1-interacting ELMO scaffold proteins to myoblast fusion. When Elmo1-/- mice underwent muscle-specific Elmo2 genetic ablation, they exhibited severe myoblast fusion defects. A mutation in the Elmo2 gene that reduced signaling resulted in a decrease in myoblast fusion. Conversely, a mutation in Elmo2 coding for a protein with an open conformation increased myoblast fusion during development and in muscle regeneration. Finally, we showed that the dystrophic features of the Dysferlin-null mice, a model of limb-girdle muscular dystrophy type 2B, were reversed when expressing ELMO2 in an open conformation. These data provide direct evidence that the myoblast fusion process could be exploited for regenerative purposes and improve the outcome of muscle diseases.

Defining the interactomes of proteins involved in cytoskeletal dynamics using high-throughput proximity-dependent biotinylation in cellulo.

Proximity-dependent biotinylation (BioID) screens are excellent tools to capture in cellulo interactomes for a large variety of baits, including transient and weak affinity interactions, as well as localization-specific proximity components, which are much harder to detect with conventional approaches. Here, we describe the major starting steps and a detailed protocol on how to perform BioID in mammalian cells. We also describe the mass spectrometry procedure and the bioinformatics pipeline for the data analysis. For complete details on the use and execution of this profile, please refer to Bagci et al. (2020).

Cardiotrophin-like cytokine (CLCF1) modulates mesenchymal stem cell osteoblastic differentiation.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into adipocytes, chondrocytes, or osteocytes. MSCs secrete an array of cytokines and express the LIFRß (leukemia inhibitory factor receptor) chain on their surface. Mutations in the gene coding for LIFRß lead to a syndrome with altered bone metabolism. LIFRß is one of the signaling receptor chains for cardiotrophin-like cytokine (CLCF1), a neurotrophic factor known to modulate B and myeloid cell functions. We investigated its effect on MSCs induced to differentiate into osteocytes in vitro Our results indicate that CLCF1 binds mouse MSCs, triggers STAT1 and -3 phosphorylation, inhibits the up-regulation of master genes involved in the control of osteogenesis, and markedly prevents osteoblast generation and mineralization. This suggests that CLCF1 could be a target for therapeutic intervention with agents such as cytokine traps or blocking mAbs in bone diseases such as osteoporosis.