Molecular cloning, sequence analysis, and cadmium stress-rated expression changes of BTG1 in freshwater pearl mussel(Hyriopsis schlegelii)

The B cells translocation gene 1 （BTG1） is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length eDNA sequence of Hyriopsis schlegelii （Hs-BTG1）, an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of eDNA ends （RACE） together with splicing the EST sequence from a haemocyte eDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame （ORF） encoding a 174 amino-acid polypeptide, a 306 bp 5＇ untranslated region （5＇ UTR）, and a 571 bp 3＇ UTR with a Poly（A） tail as well as a transcription termination signal （AATAAA）. Homologne searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas （2.03-fold）, mantle （2.07-fold）, kidney （2.2-fold） and haemocyte （2.5-fold） were enhanced by cadmium （Cd2＋） stress, suggesting that Hs-BTG 1 may have played a significant role in H, schlegelii adaptation to adverse environmental conditions.

Epididymis cell atlas in a patient with a sex development disorder and a novel NR5A1 gene mutation

This study aims to characterize the cell atlas of the epididymis derived from a 46,XY disorders of sex development(DSD)patient with a novel heterozygous mutation of the nuclear receptor subfamily 5 group A member 1(NR5A1)gene.Next-generation sequencing found a heterozygous c.124C>G mutation in NR5A1 that resulted in a p.Q42E missense mutation in the conserved DNA-binding domain of NR5A1.The patient demonstrated feminization of external genitalia and Tanner stage 1 breast development.The surgical procedure revealed a morphologically normal epididymis and vas deferens but a dysplastic testis.Microfluidic-based single-cell RNA sequencing(scRNA-seq)analysis found that the fibroblast cells were significantly increased(approximately 46.5%),whereas the number of main epididymal epithelial cells(approximately 9.2%),such as principal cells and basal cells,was dramatically decreased.Bioinformatics analysis of cell–cell communications and gene regulatory networks at the single-cell level inferred that epididymal epithelial cell loss and fibroblast occupation are associated with the epithelial-to-mesenchymal transition(EMT)process.The present study provides a cell atlas of the epididymis of a patient with 46,XY DSD and serves as an important resource for understanding the pathophysiology of DSD.