Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the Nautiloids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautiloid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we conducted transcriptomics of the mantle, and proteomics of the shell of N. pompilius simultaneously. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet, and the Euhadra snail) revealed that three proteins and six domains of the shell proteins are conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Although further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be done in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the basally diverging Nautilids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautilid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we performed a multiomics study on the shell of N. pompilius, by conducting transcriptomics of its mantle tissue and proteomics of its shell matrix. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet and the Euhadra snail) revealed that three proteins and six protein domains were conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six protein domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were probably present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Even though further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be carried out in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

Background Cachexia is a life-threatening condition observed in several pathologies, such as cancer or chronic diseases. Interleukin 10 (Il10) gene transfer is known to improve cachexia by downregulating Il6. Here, we used an IL10-knockout mouse model to simulate cachexia and investigate the effects of eggshell membrane (ESM), a resistant protein, on general pre-cachexia symptoms, which is particularly important for the development of cachexia therapeutics. Methods Five-week-old male C57BL6/J mice were fed an AIN-93G powdered diet (WT), and 5-week-old male B6.129P2-Il10 < tm1Cgn>/J (IL10 À/À ) mice were fed either the AIN-93G diet (KO) or an 8% ESM-containing diet (KOE) for 28 weeks. The tissue weight and levels of anaemia-, blood glucose-, lipid metabolism-, and muscular and colonic inflammation-related biochemical markers were measured. Transcriptomic analysis on liver and colon mucus and proteomic analysis on skeletal muscle were performed. Ingenuity Pathway Analysis was used to identify molecular pathways and networks. Caecal short-chain fatty acids (SCFAs) were identified using HPLC, and caecal bacteria DNA were subjected to metagenomic analysis. Flow cytometry analysis was performed to measure the CD4 + IL17 + T cells in mesenteric lymph nodes. ResultsThe body weight, weight of gastrocnemius muscle and fat tissues, colon weight/length ratio, plasma HDL and NEFA, muscular PECAM-1 levels (P < 0.01), plasma glucose and colonic mucosal myeloperoxidase activity (P < 0.05) and T helper (Th) 17 cell abundance (P = 0.071) were improved in KOE mice over KO mice. Proteomic analysis indicated the protective role of ESM in muscle weakness and maintenance of muscle formation (>1.5-fold). Transcriptomic analysis revealed that ESM supplementation suppressed the LPS/IL1-mediated inhibition of RXR function pathway in the liver and downregulated the colonic mucosal expression of chemokines and Th cell differentiation-related markers (P < 0.01) by suppressing the upstream BATF pathway. Analysis of the intestinal microenvironment revealed that ESM supplementation ameliorated the microbial alpha diversity and the abundance of microbiota associated with the degree of inflammation (P < 0.05) and increased the level of total organic acids, particularly of SCFAs such as butyrate (2.3fold), which could inhibit Th1 and Th17 production. Conclusions ESM supplementation ameliorated the chief symptoms of cachexia, including anorexia, lean fat tissue mass, skeletal muscle wasting and reduced physical function. ESM also improved colon and skeletal muscle inflammation, lipid metabolism and microbial dysbiosis. These results along with the suppressed differentiation of Th cells could be associated with the beneficial intestinal microenvironment and, subsequently, attenuation of pre-cachexia. Our findings provide insights into the potential of ESM in complementary interventions for pre-cachexia prevention.