Presence of immune factors in freshwater mussel (Hyriopsis cumingii) entails autologous serum an essential component in the culture of mantle cells.

Mussel cell culture is a challenging problem and serum serves a crucial biological role in cell culture as an autologous supply and an immunizing agent. In this study, the biology (calcium ions, total protein, pH, and osmotic pressure) of fetal bovine serum (FBS) and Hyriopsis cumingii serum (HCS) was investigated, and the development of Hyriopsis cumingii (H. cumingii) mantle cells in HCS and FBS systems was examined. The results showed that total protein, calcium ions, and osmotic pressure varied significantly (p<0.05). The activity of mantle cells was superior in the HCS culture system to that in the FBS culture system. The label-free technique was used to distinguish the two serum proteins to investigate the supportive effect of autologous serum on cell culture. These were examined for 109 unique proteins and 35 particular HCS proteins. Most differentially expressed proteins (DEPs) were involved in immune response, cell differentiation, and calcium ion binding. Furthermore, immune factors such as HSP, CALR, APOB, C3 were identified with significant differences. HSP was significantly more present in HCS than in FBS as an endogenous protective protein that regulates immune system function, cell differentiation, transport, and activity regulation. Parallel reaction monitoring (PRM) analysis was carried out to validate the expression levels of 19 DEPs, indicating high reliability of the proteomic results. This study reveals the important role of immune factors in mussel cell culture, providing a theoretical basis for explaining the applicability of autologous serum in cell culture. It is also helpful in improving the cell culture conditions of mussels.

Correction: Li et al. Identification and Functional Analysis of the Cell Proliferation Regulator, Insulin-like Growth Factor 1 (IGF1) in Freshwater Pearl Mussel (Hyriopsis cumingii). Biology 2022, 11, 1369.

In the original publication [...].

Identification and Functional Analysis of the Cell Proliferation Regulator, Insulin-like Growth Factor 1 (IGF1) in Freshwater Pearl Mussel (Hyriopsis cumingii).

Insulin-like growth factor 1 (IGF1) plays an important regulatory role in the regulation of growth, differentiation, and anabolism in a variety of cells. In this study, the full-length cDNA of the IGF1 gene was cloned from Hyriopsis cumingii, named HcIGF1. The expression level of HcIGF1 in six tissues (adductor muscle, foot, hepatopancreas, gill, mantle, and gonad) was determined. In addition, the localization of HcIGF1 in the mantle was analyzed by in situ hybridization, and finally the function of HcIGF1 was explored by RNA interference and prokaryotic expression. The results showed that the amino acid sequence contained a typical IIGF structural domain. The phylogenetic tree showed that HcIGF1 clustered with other marine bivalve sequences. Quantitative real-time PCR and in situ hybridization analysis showed that HcIGF1 was expressed in all tissues. The highest expression was in the foot and the lowest was in the mantle. In the mantle tissue, the hybridization signal was mainly concentrated in the outer mantle. After RNA interference, the expression of IGF1 was found to be significantly decreased (p < 0.05), and its related genes IGF1R, AKT1, and cyclin D2 were downregulated, while MAPK1 were upregulated. The recombinant HcIGF1 protein was purified and its growth-promoting effect was investigated. The results showed that the recombinant HcIGF1 protein could significantly promote the proliferative activity of the mantle cells of mussels, with the best proliferative effect at 12.5 µg/mL. The results of this study provide a new method to solve the problem of weak proliferation of shellfish cells in vitro and lay the foundation for further understanding of the growth regulation mechanism of H. cumingii, as well as a better understanding of the physiological function of IGF1 in mollusks.

A proteomic approach reveals biomineralization and immune response for mantle to pearl sac in the freshwater pearl mussel (Hyriopsis cumingii).

In the process of production of freshwater pearl, implanted mantle pieces undergo a series of complex physiological and biochemical processes to form pearl sac, which produce pearl. This is a very important site of occurrence due to immune-induced biomineralization, while its molecular regulatory mechanism is still unclear. Here, we use proteomics to identify differentially expressed proteins (DEPs) of the mantle and pearl sac and examine the biomineralization and immune response of the pearl sac formation process in Hyriopsis cumingii. Using iTRAQ technology and bioinformatics analysis, we obtained DEP profiles between the mantle and pearl sac. A total of 1871 proteins were identified. Of these, 74 DEPs were found between the pearl sac and outer mantle, 112 DEPs between the pearl sac and inner mantle, and 124 DEPs between the outer and inner mantles. Bioinformatics analysis revealed that the screened biomineralization-related DEPs were mainly enriched in signaling pathways associated with calcium signaling, regulation of the actin cytoskeleton and protein processing in the endoplasmic reticulum, while the immune-related DEPs were mainly enriched in the Notch, Hippo, nuclear factor kappa-B (NF-κB), and transforming growth factor-ß (TGF-ß) signaling pathways. In addition, the expression of six biomineralization-related and four immune-related proteins were verified at the transcriptional level using quantitative real-time PCR. Our findings contribute to furthering the understanding of the mechanisms of pearl formation and immune response, and have long-term implications for future studies on the production of high-quality freshwater pearls and development of the freshwater pearl industry.

[Relationship between vitamin D receptor gene polymorphism and preterm birth].

OBJECTIVE: To explore the relationship between vitamin D receptor (VDR) gene pol-ymorphisms at Fok I site and the risk of preterm birth for potential intervention of of preterm birth or threatened premature delivery. METHODS: Fifty-seven women with preterm birth and 84 with full-term birth were included in this analysis. Polymerase chain reaction-restriction frag-ment length polymorphism (PCR-RFLP) was performed to identify VDR gene Fok I geno-types. RESULTS: No significant difference was found in age, D-dimer (DDI), fibrinogen (Fg), serum calcium (Ca2+), leukocyte count or glycosylated hemoglobin (HbA1c) level between the women in the preterm and full-term birth groups (P>0.05). The two groups differed signifi-cantly in the distribution of VDR gene Fok I site genotypes and allele frequency of F/F (P<0.05).The frequency of FF genotype was significantly higher in the preterm group than in the full-term group. Compared with Ff and ff genotypes, FF genotype was associated with an in-creased risk of preterm delivery (χ2=9.701, P=9.701, OR=3.320, 95% CI [1.560, 7.066]). In the preterm group, the maternal age, DDI, Fg, serum Ca2+, leukocyte count or HbA1c did not differ significantly between the genotypes (P>0.05). CONCLUSION: VDR gene Fok I site geno-types are related with preterm birth, and the FF genotype may serve as a potential risk factor for preterm birth.