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Animal models play crucial roles in the rapid development of vaccines/drugs for the prevention and therapy of COVID-19, but current models have some deficits when studying the pathogenesis of SARS-CoV-2 on some special tissues or organs. Here, we generated a human ACE2 and SARS-CoV-2 NF/F knockin mouse line that constitutively expresses human ACE2 and specifically expresses SARS-CoV-2 N gene induced by Cre-recombinase. By crossing with Cre transgenic lines allowing for lung-specific and constitutive expression, we generated lung-specific (Sftpc-hACE2-NF/F) and constitutive SARS-CoV-2 N (EIIa-hACE2-NF/F) expressing mice. Upon intranasal infection with a SARS-CoV-2 GFP/ΔN strain which can only replicate in SARS-CoV-2 N expressed cells, we demonstrated that both the Sftpc-hACE2-NF/F and EIIa-hACE2-NF/F mice support viral replication. Consistent with our design, viral replication was limited to the lung tissues in Sftpc-hACE2-NF/F mice, while the EIIa-hACE2-NF/F mice developed infections in multiple tissues. Furthermore, our model supports different SARS-CoV-2 variants infection, and it can be successfully used to evaluate the effects of therapeutic monoclonal antibodies (Ab1F11) and antiviral drugs (Molnupiravir). Finally, to test the effect of SARS-CoV-2 infection on male reproduction, we generated Sertoli cell-specific SARS-CoV-2 N expressed mice by crossing with AMH-Cre transgenic line. We found that SARS-CoV-2 GFP/ΔN strain could infect Sertoli cells, led to spermatogenic defects due to the destruction of blood-testis barrier. Overall, combining with different tissue-specific Cre transgenic lines, the human ACE2 and SARS-CoV-2 NF/F line enables us to evaluate antivirals in vivo and study the pathogenesis of SARS-CoV-2 on some special tissues or organs. [ABSTRACT FROM AUTHOR]

In this paper, we present a proof of Schauder estimate on Euclidean space and use it to generalize Donaldson's Schauder estimate on space with conical singularities in the following two directions. The first is that we allow the total cone angle to be larger than 2 π and the second is that we discuss higher-order estimates. [ABSTRACT FROM AUTHOR]

Genetic factors are one of the most important causes of non-obstructive azoospermia (NOA). ESX1 is an X-linked testis-biased expressed gene, and a potential biomarker for testicular sperm retrieval in NOA patients, yet few systematic studies have investigated its association with NOA. Here, we performed selected exonic sequencing in a large cohort of Chinese males, and four novel missense mutations (including one compound mutation), one novel synonymous mutation of ESX1 unique to NOA patients were identified. We analyzed the effects of ESX1 mutations on cyclin A degradation and cell cycle progression by immunoprecipitation assay and flow cytometry, and found that the compound mutant p.[P365R; L366V] ESX1 compromised the stabilizing effect of ESX1 on polyubiquitinated cyclin A, thereby causing the failure of M phase arrest in cells. Further studies showed that the deleterious effect of the compound mutations on ESX1 protein function was attributed to p.P365R but not p.L366V alteration. The novel ESX1 mutation p.P365R might confer high risk for NOA in Han Chinese population, probably via affecting cell cycle control. [ABSTRACT FROM AUTHOR]

Although more than 1,000 androgen receptor (AR) mutations have been identified and these mutants are pathologically important, few theoretical studies have investigated the role of AR protein folding stability in disease and its relationship with the phenotype of the patients. Here, we extracted AR variant data from four databases: ARDB, HGMD, Cosmic, and 1,000 genome. 905 androgen insensitivity syndrome (AIS)-associated loss-of-function mutants and 168 prostate cancer-associated gain-of-function mutants in AR were found. We analyzed the effect of single-residue variation on the folding stability of AR by FoldX and guanidine hydrochloride denaturation experiment, and found that genetic disease-associated mutations tend to have a significantly greater effect on protein stability than gene polymorphisms. Moreover, AR mutants in complete androgen insensitivity syndrome (CAIS) tend to have a greater effect on protein stability than in partial androgen insensitive syndrome (PAIS). This study, by linking disease phenotypes to changes in AR stability, demonstrates the importance of protein stability in the pathogenesis of hereditary disease. [ABSTRACT FROM AUTHOR]

Sertoli cells are important for spermatogenesis not only by directly interacting with germ line cells in the seminiferous epithelium but also by constituting the blood–testis barrier (BTB) structure to create a favorable environment for spermatogenesis. Blind sterile (bs) male mice are infertile, with excessive germ cell apoptosis and spermatogenesis arrest. TBC1D20 (TBC1 domain family member 20) deficiency has been identified as the causative mutation in bs mice. However, whether TBC1D20 loss of function also impairs BTB integrity, which further contributes to the failed spermatogenesis of bs male mice, remains unclear. In the present study, biotin tracer assay and transmission electron microscopy showed severely disrupted BTB integrity in bs testes. Compared to the wild-type Sertoli cells, BTB components of cultured bs Sertoli cells in vitro was perturbed with downregulation of E-cadherin, ZO-1, β-catenin, and Claudin 11. The obvious rearrangement of F-actin indicated disrupted epithelial–mesenchymal balance in TBC1D20-deficient Sertoli cells. The ability of bs Sertoli cells to maintain the clone formation of spermatogonia stem cells was also obviously limited. Furthermore, the decreasing of SOX9 (sex-determining region Y box 9) and WT1 (Wilms' tumor 1) and increasing of vimentin in bs Sertoli cells indicated that TBC1D20 loss of function attenuated the differentiation progression of bs Sertoli cells. In summary, TBC1D20 loss of function impedes the maturation of adult Sertoli cells and resulted in impaired BTB integrity, which is further implicated in the infertile phenotype of bs male mice. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to evaluate whether ubiquitin-specific peptidase 26 (USP26) gene variations were associated with nonobstructive azoospermia (NOA). Methods: Seven hundred and seventy-six patients diagnosed with NOA and 709 proven fertile men were included in this study. Genetic variations of infertility-related genes, including USP26, were identified by selected exonic sequencing. The effects of USP26 mutations on androgen receptor (AR) binding, ubiquitination, and transcriptional activity were detected by immunoprecipitation and luciferase assay in Hela and TM4 cells. Results: Six novel missense mutations and 1 novel synonymous mutation of USP26 unique to the patients with NOA were identified. Of these missense mutations, USP26 R344W remarkably reduced the binding affinity and deubiquitinating activity of USP26 to AR, thus eliminated the inhibitory effect of USP26 on transcriptional activity of AR in Hela and TM4 cells. Conclusion: A novel USP26 variant p.R344W is associated with NOA probably through affecting AR function. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are known to mainly target protein-coding genes at post-transcriptional level, resulting in mRNA destabilization and/or translational repression. Long non-coding RNAs (lncRNAs) are emerging as a novel set of targets for miRNAs. Here, we report that downregulated hsa-miR-1 and upregulated lncRNA urothelial cancer associated 1 (UCA1) were inversely expressed in bladder cancer. Hsa-miR-1 decreased the expression of UCA1 in bladder cancer cells in an Ago2-slicer-dependent manner. The binding site between UCA1 and hsa-miR-1 was confirmed. Overexpression of hsa-miR-1 inhibited bladder cancer cell growth, induced apoptosis, and decreased cell motility. Knockdown of UCA1 expression phenocopied the effects of upregulation of hsa-miR-1. Transfection of UCA1 expression vector partly reversed the changes caused by transfection of pre-miR-1 plasmids. This study provides evidence for hsa-miR-1 to play tumor suppressive roles via downregulating lncRNA UCA1 in bladder cancer, which may have potential therapeutic significance. [ABSTRACT FROM AUTHOR]

Numerous attempts for detection of circulating tumor cells (CTC) have been made to develop reliable assays for early diagnosis of cancers. In this study, we validated the application of folate receptor α (FRα) as the tumor marker to detect CTC through tumor-specific ligand PCR (LT-PCR) and assessed its utility for diagnosis of bladder transitional cell carcinoma (TCC). Immunohistochemistry for FRα was performed on ten bladder TCC tissues. Enzyme-linked immunosorbent assay (ELISA) for FRα was performed on both urine and serum specimens from bladder TCC patients ( n = 64 and n = 20, respectively) and healthy volunteers ( n = 20 and n = 23, respectively). Western blot analysis and qRT-PCR were performed to confirm the expression of FRα in bladder TCC cells. CTC values in 3-mL peripheral blood were measured in 57 bladder TCC patients, 48 healthy volunteers, and 15 subjects with benign urologic pathologies by the folate receptor α ligand-targeted PCR. We found that FRα protein was overexpressed in both bladder TCC cells and tissues. The levels of FRα mRNA were also much higher in bladder cancer cell lines 5637 and SW780 than those of leukocyte. Values of FRα were higher in both serum and urine specimens of bladder TCC patients than those of control. CTC values were also higher in 3-mL peripheral blood of bladder TCC patients than those of control (median 26.5 Cu/3 mL vs 14.0 Cu/3 mL). Area under the receiver operating characteristic (ROC) curve for bladder TCC detection was 0.819, 95 % CI (0.738-0.883). At the cutoff value of 15.43 Cu/3 mL, the sensitivity and the specificity for detecting bladder cancer are 82.14 and 61.9 %, respectively. We concluded that quantitation of CTCs through FRα ligand-PCR could be a promising method for noninvasive diagnosis of bladder TCC. [ABSTRACT FROM AUTHOR]

Testicular germ cell tumors are the most frequent malignancies found in men between 15 and 44 years old. Although cellular apoptosis susceptibility (CAS) was demonstrated to be upregulated in breast cancer and colon cancer, the expression of CAS in the human testis and testicular germ cell tumors remained elusive. In the present study, CAS-positive signals were detected in the normal testicular tissues, cancer adjacent normal testicular tissues, seminoma, yolk sac tumor, and teratoma. Interestingly, the expression level of CAS in testicular germ cell tumors (TGCTs) (but not seminoma) was significantly lower than that of human testicular tissues and cancer adjacent normal testicular tissues, suggesting that decreased CAS contributed to the progression of TGCTs. Notably, the expression of CAS in seminoma was significantly higher than that of in the non-seminomas, consistent with the results from TCGA database. Furthermore, the localization of CAS is mainly restricted in the nucleus in the lesions of normal human testicular tissue and cancer adjacent normal testicular tissue. Although the expression of CAS was not significantly different between normal testicular tissue and seminoma, CAS was more enriched in cytoplasm in seminoma compared to the normal, cancer adjacent tissue and other types of TGCTs. The current results demonstrated reduced expression of CAS in the human testicular germ cell tumors and the CAS translocation from the nuclear to cytoplasm in seminoma, thereby supporting a possible role in normal testis function and in the development of seminoma. [ABSTRACT FROM AUTHOR]