Identification and expression analysis of fetuin B (FETUB) in turbot (Scophthalmus maximus L.) mucosal barriers following bacterial challenge.

Fetuin B (FETUB), a recently described cysteine proteinase inhibitor, has numerous conserved N-glycosylation sites, species-specific O-glycosylation sites, and two cystatin (CY) domains. FETUB is likely to play regulatory roles in acute inflammation, female infertility, fish organogenesis and tumor suppression. In the present study, transcript of turbot FETUB gene was captured, its protein structure and expression patterns in different tissues with emphasis on mucosal barriers following different bacterial infection were characterized. Turbot FETUB gene showed the closest relationship with Takifugu rubripes in phylogenetic analysis. In addition, FETUB was ubiquitously expressed in all examined tissues with the highest expression level in skin. Finally, FETUB gene showed different expression patterns following both bacterial challenge. The rapidly and significantly differential expression patterns of FETUB in mucosal surfaces against bacterial infections might indicate its key roles to prevent pathogen attachment and entry in turbot mucosal immunity. Functional studies should be carried out to further characterize the FETUB and avail utilization of its function to increase the disease resistance of turbot in maintaining the integrity of the mucosal barriers against infections and to facilitate selection of the fine family/varieties of disease resistance in turbot.

Effects of Tcte1 knockout on energy chain transportation and spermatogenesis: implications for male infertility.

STUDY QUESTION: Is the Tcte1 mutation causative for male infertility? SUMMARY ANSWER: Our collected data underline the complex and devastating effect of the single-gene mutation on the testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADY: Recent data have revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity and is built from the DRC1-DRC7 proteins. DRC5 (TCTE1), one of the N-DRC elements, has already been reported as a candidate for abnormal sperm flagella beating; however, only in a restricted manner with no clear explanation of respective observations. STUDY DESIGN SIZE DURATION: Using the CRISPR/Cas9 genome editing technique, a mouse Tcte1 gene knockout line was created on the basis of the C57Bl/6J strain. The mouse reproductive potential, semen characteristics, testicular gene expression levels, sperm ATP, and testis apoptosis level measurements were then assessed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n = 248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS SETTING METHODS: To check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but for no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild-type (WT, control group), heterozygous Tcte1+/- and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis samples, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) was done for mice testis tissues. STRING interactions were checked for protein-protein interactions, based on changed expression levels of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the amount of ATP in spermatozoa, the luminescence level was measured. In addition, immunofluorescence in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n = 137 with non-obstructive azoospermia or cryptozoospermia, n = 111 samples with a spectrum of oligoasthenoteratozoospermia, including n = 47 with asthenozoospermia) was extracted to perform genomic sequencing (WGS, WES, or Sanger). Protein prediction modeling of human-identified variants and the exon 3 structure deleted in the mouse knockout was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: No progeny at all was found for the homozygous males which were revealed to have oligoasthenoteratozoospermia, while heterozygous animals were fertile but manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing or linked with apoptosis or spermatogenesis. In Tcte1-/- males, the protein was revealed in only residual amounts in the sperm head nucleus and was not transported to the sperm flagella, as were other N-DRC components. Decreased ATP levels (2.4-fold lower) were found in the spermatozoa of homozygous mice, together with disturbed tail:midpiece ratios, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at a similar level in all three mouse genotypes. Mutation screening of human infertile males revealed one novel and five ultra-rare heterogeneous variants (predicted as disease-causing) in 6.05% of the patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus suggesting disrupted interactions of TCTE1 with its binding partners located within the axoneme. LARGE SCALE DATA: All data generated or analyzed during this study are included in this published article and its supplementary information files. RNAseq data are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE207805. The results described in the publication are based on whole-genome or exome sequencing data which includes sensitive information in the form of patient-specific germline variants. Information regarding such variants must not be shared publicly following European Union legislation, therefore access to raw data that support the findings of this study are available from the corresponding author upon reasonable request. LIMITATIONS REASONS FOR CAUTION: In the study, the in vitro fertilization performance of sperm from homozygous male mice was not checked. WIDER IMPLICATIONS OF THE FINDINGS: This study contains novel and comprehensive data concerning the role of TCTE1 in male infertility. The TCTE1 gene is the next one that should be added to the 'male infertility list' because of its crucial role in spermatogenesis and proper sperm functioning. STUDY FUNDING/COMPETING INTERESTS: This work was supported by National Science Centre in Poland, grants no.: 2015/17/B/NZ2/01157 and 2020/37/B/NZ5/00549 (to M.K.), 2017/26/D/NZ5/00789 (to A.M.), and HD096723, GM127569-03, NIH SAP #4100085736 PA DoH (to A.N.Y.). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Combined proteomics and transcriptomics identifies serpin family C member 1 associated protein as a biomarker of endometriosis.

OBJECTIVE: To explore potential biomarkers indicating endometriosis (EM). MATERIALS AND METHODS: A proteomics method and combined quantitative transcriptomics were adopted to highlight markers in the EM. Venn analysis was used to integrate the ribonucleic acid sequencing (RNA-seq) and protein profiles. Promising candidate markers were tested by enzyme-related immunosorbent assay. RESULTS: A sum of 979 mRNAs and 39 proteins were tested to be significantly differentially expression in the standard cluster compared with the EM cluster. Venn analysis showed a filtered signature of only two down-regulated molecules in the EM group, i.e. fetuin B (FETUB) and serpin family C member 1 (SERPINC1); the latter showed a big variance between the control category and the EM set in the authentication test. CONCLUSION: SERPINC1 may be a useful possible biomarker for the analysis of EM.

Fetuin B overexpression suppresses proliferation, migration, and invasion in prostate cancer by inhibiting the PI3K/AKT signaling pathway.

Fetuin B (FETUB) is a glycoprotein that is a member of the cysteine protease inhibitor family, and it is associated with cancer. However, the role of FETUB in prostate carcinogenesis is unknown. In this study, we overexpressed FETUB in prostate cancer cells by using lentivirus and then studied the impacts on cell apoptosis, migration and invasion. We found that apoptosis was increased and the migration and invasion of prostate cancer cells were significantly inhibited after overexpression. Then, we performed experiments in vivo and found that there were fewer tumors in the overexpression groups than in the control groups. In addition, we demonstrated that overexpression of FETUB inactivates the PI3K/AKT signaling pathway. Rescue assays revealed that intervention of 740Y-P reversed the anti-tumor effect of FETUB on prostate cancer cells. Taken together, our results revealed that FETUB may act as a novel regulator to promote apoptosis and inhibit the migration and invasion of prostate cancer cells and that FETUB is related to the inactivation of the PI3K/AKT signaling pathway.

The rs4686434 variant in the fetuin B (FETUB) locus is associated with intrahepatic triglyceride content in obese Chinese adults.

BACKGROUND: This study explored associations of genetic variants in the fetuin B (FETUB) locus with intrahepatic triglyceride (IHTG) content. METHODS: Four tagging single-nucleotide polymorphisms (SNPs) of the FETUB locus and patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 and transmembrane 6 super family member 2 (TM6SF2) rs58542926 were genotyped in 418 obese Chinese adults in whom serum FETUB and IHTG were measured. RESULTS: Subjects carrying the minor G allele for FETUB rs4686434 (AG/GG) had lower serum FETUB levels (mean [±SD] 3.89 ± 1.36 vs 4.22 ± 1.46 µg/mL; P = 0.021) and IHTG content (12.7 ± 9.4% vs 14.6 ± 9.8%; P = 0.045) than their controls (AA), whereas IHTG content was higher in those carrying the minor G allele for PNPLA3 rs738409 (CG/GG) than in their controls (CC; 14.5 ± 10.1% vs 12.0 ± 8.6%; P = 0.012). After adjusting for potential confounders, IHTG content was lower in carriers of the minor G allele for FETUB rs4686434 (AG/GG vs AA, ß -2.27 ± 0.91, P = 0.012), but was higher in carriers of the minor G allele for PNPLA3 rs738409 (CG/GG vs CC, ß 2.65 ± 0.97, P = 0.006). There was a significant joint effect between FETUB rs4686434 and PNPLA3 rs738409 on IHTG content, with increasing genetic risk score (counting the risk allele of A in rs4686434 and G in rs738409) being associated with higher IHTG content (ß 1.85 ± 0.48, P <0.001). CONCLUSIONS: Carrying the minor G allele for FETUB rs4686434 was significantly associated with decreased IHTG content and may affect hepatic triglyceride accumulation in individuals at high risk of non-alcoholic fatty liver disease.