A procedure for producing an anti-AXL nanobody in E. coli.

As one of the receptors of the TAM family, AXL plays a vital role in stem cell maintenance, angiogenesis, immune escape of viruses and drug resistance against tumors. In this study, the truncated extracellular segment containing two immunoglobulin-like domains of human AXL (AXL-IG), which has been confirmed to bind growth arrest specific 6 (GAS6) by structural studies [1], was expressed in a prokaryotic expression system and then purified. Immunizing camelid with the purified AXL-IG as antigen could lead to the production of unique nanobodies composed of only variable domain of heavy chain of heavy-chain antibody (VHH), which are around 15 kD and stable. We screened out a nanobody A-LY01 specific binding to AXL-IG. We further determined the affinity of A-LY01 to AXL-IG and revealed that A-LY01 could specifically recognize full-length AXL on the surface of HEK 293T/17 cells. Our study provides appropriate support for the development of diagnostic reagents and antibody therapeutics targeting AXL.

Effect of Ezrin on regulating trophoblast cell invasion via PKC signaling pathway in unexplained recurrent spontaneous abortion.

Trophoblast cells are the most important cells in early pregnancy and their invasion are essential to the establishment and maintenance of pregnancy. Inadequate trophoblast cell invasion has been closely associated with several pregnancy-associated diseases including recurrent spontaneous abortion (RSA). Ezrin is an actin-associated protein, known as a marker for carcinogenesis and metastasis in solid tumors, has been proposed to play a role in the formation of microvilli in the early embryo. To further characterize its function in early pregnancy, we explored the expression of Ezrin in the trophoblast cells in early pregnancy. In this study, compared with normal pregnant women, we demonstrated that the expression of Ezrin and phosphorylated Ezrin decreased in the trophoblast cells in unexplained RSA (URSA) patients, and knockdown of Ezrin expression could suppress the invasiveness of trophoblast cells significantly. Various studies indicated that the phosphorylation of Ezrin on C-terminal threonine residue (T567) is a key event in the regulation of its activity. Our further exploration indicated that Ezrin was activated via PKC pathway. Furthermore, inhibition of the PKC pathway by a specific inhibitor suppressed invasiveness of Bewo cells. On the other hand, activation of the PKC pathway could increase the relative capacity of trophoblast cell invasion, while Ezrin knockdown reversed PKC activation induced cell invasion. These findings might provide a new fundamental mechanism for successful pregnancy and new diagnostic and therapeutic target for RSA.

Long Non-Coding RNA ZEB2-AS1 Augments Activity of Trophoblast Cells and Prevents the Development of Recurrent Spontaneous Abortion in Mice Through EZH2-Mediated CST3 Inhibition.

Recurrent spontaneous abortion (RSA) is the most common complication of pregnancy where reduced invasion of trophoblasts plays a major role. This work aimed to explore the effect of abnormally expressed long non-coding RNA (lncRNA) ZEB2-AS1 on the occurrence of RSA. Differentially expressed lncRNAs in trophoblast cells between healthy controls and patients with RSA were screened using the GEO database. Female CBA/J mice were allowed to mate with male DBA/2 mice to establish inbred mice with RSA. ZEB2-AS1 was poorly expressed in placental tissues and trophoblast cells in the condition of RSA. ZEB2-AS1 upregulation augmented proliferation, migration, and invasion of trophoblast cells in vitro. ZEB2-AS1 negatively regulated cystatin C (CST3) expression. Further overexpression of CST3 blocked the activity of trophoblast cells. ZEB2-AS1 recruited enhancer of EZH2 to the promoter region of CST3, which increased H3K27me3 modification to suppress CST3 expression. In vivo, overexpression of ZEB2-AS1 reduced embryo resorption rate and increased the weights of fetuses and placentas in mice with RSA. However, the protective roles of ZEB2-AS1 were blocked upon artificial silencing of EZH2 or upregulation of CST3. Taken together, this study demonstrates that ZEB2-AS1 enhances activity of trophoblast cells and prevents RSA development through reducing CST3 expression in an EZH2-dependent manner.

Contrast Induced Nephropathy and 2-Year Outcomes of Iso-Osmolar Compared with Low-Osmolar Contrast Media after Elective Percutaneous Coronary Intervention.

BACKGROUND AND OBJECTIVES: This study investigated the relative incidence of contrast induced nephropathy (CIN) and long-term outcomes between iso-osmolar contrast media (IOCM) and low-osmolar contrast media (LOCM) undergoing elective percutaneous coronary intervention (PCI). METHODS: A total of 9,431 patients receiving elective PCI were enrolled in the cohort. The patients were divided into IOCM group and LOCM group. Propensity score matching (PSM) was applied to minimize the selection bias between groups. RESULTS: The multivariate analysis showed that the use of IOCM compared with LOCM did not affect the CIN incidence (odds ratio [OR], 0.912; 95% confidence interval [CI], 0.576-1.446; p=0.696). After PSM, the incidence of CIN was 1.5% and 4.0% in IOCM group (n=979) and LOCM group (n=979), respectively, p=0.001. IOCM significantly reduced the incidence of CIN compared with LOCM (OR, 0.393; 95% CI, 0.214-0.722; p=0.003). After 2 years of follow-up, the all-cause mortality was higher in IOCM group than LOCM group (2.1% vs. 0.9%, p<0.001). Cox regression analysis showed IOCM was not independent risk factor of 2-years all-cause mortality (OR, 0.849; 95% CI, 0.510-1.412; p=0.528). After PSM, the difference of all-cause death between groups disappeared (1.7% vs. 1.9%, p=0.739). Cox regression analysis showed that the use of IOCM compared with LOCM did not affect the incidence of 2-year all-cause mortality (OR, 1.037; 95% CI, 0.534-2.014; p=0.915). CONCLUSIONS: Compared with LOCM, IOCM significantly reduced the incidence of CIN after elective PCI, but had no significant effect on 2-year all-cause mortality.