Maternal and embryonic signals cause functional differentiation of luminal epithelial cells and receptivity establishment.

Embryo implantation requires temporospatial maternal-embryonic dialog. Using single-cell RNA sequencing for the uterus from 2.5 to 4.5 days post-coitum (DPC) and bulk sequencing for the corresponding embryos of 3.5 and 4.0 DPC pregnant mice, we found that estrogen-responsive luminal epithelial cells (EECs) functionally differentiated into adhesive epithelial cells (AECs) and supporting epithelial cells (SECs), promoted by progesterone. Along with maternal signals, embryonic Pdgfa and Efna3/4 signaling activated AECs and SECs, respectively, enhancing the attachment of embryos to the endometrium and furthering embryo development. This differentiation process was largely conserved between humans and mice. Notably, the developmental defects of SOX9-positive human endometrial epithelial cells (similar to mouse EEC) were related to thin endometrium, whereas functional defects of SEC-similar unciliated epithelial cells were related to recurrent implantation failure (RIF). Our findings provide insights into endometrial luminal epithelial cell development directed by maternal and embryonic signaling, which is crucial for endometrial receptivity.

Transcriptome-based analyses reveal venom diversity in two araneomorph spiders, Psechrus triangulus and Hippasa lycosina.

The spiders Psechrus triangulus and Hippasa lycosina are widely distributed in Yunnan Province, China, and are important natural enemies of agricultural pests, yet studies regarding the composition of their venom are lacking. In this study, cDNA libraries were constructed from venom gland tissue of P. triangulus and H. lycosina and used for transcriptomic analysis. From the analysis, 39 and 31 toxin-like sequences were predicted for P. triangulus and H. lycosina, respectively. The predicted neurotoxin sequences were categorized according to cysteine sequence motifs, and the predicted neurotoxin sequences of P. triangulus and H. lycosina could be classified into 9 and 6 toxin families, respectively. In addition, potential acetylcholinesterase, hyaluronidase, and astaxanthin-like metalloproteinases were identified through annotation. In summary, transcriptomic techniques were invaluable in mining the gene expression information from these two spider species to explore the toxin composition of their venom and determine how they differ. Studies of this type provide essential baseline data for studying the evolution and physiological activities of spider toxins and for the potential development of medicinal compounds.

Toxin diversity revealed by de novo transcriptome assembly for venom gland in two species of spiders (Trichonephila clavata and Sinopoda pengi).

During long-term predator-prey coevolution, spiders have generated a vast diversity of toxins. Trichonephila clavata is a web-spinning spider whose large, well-constructed webs and venomous arsenal facilitate prey capture. In contrast, Sinopoda pengi is an ambush predator with agile locomotion and strong chelicerae for hunting. In this study, transcriptomic analysis was performed to describe the predicted toxins of S. pengi and T. clavata. A total of 43 and 47 of these unigenes from S. pengi and T. clavata, respectively, were predicted to have toxin activity. Putative neurotoxins were classified to the family level according to cysteine arrangement; 4 and 6 toxin families were produced by S. pengi and T. clavata, respectively. In addition, potential metalloproteases, acetylcholinesterases, serine proteases, hyaluronidases and phospholipases were found by annotation in databases. In summary, molecular templates with potential application value for medical and biological fields were obtained by classifying and characterizing presumed venom components, which established a foundation for further study of venom.

Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors.

Porcupine is an O-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported.

Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.

Discovery and Optimization of a Porcupine Inhibitor.

Wnt proteins regulate various cellular functions and serve distinct roles in normal development throughout life. Wnt signaling is dysregulated in various diseases including cancers. Porcupine (PORCN) is a membrane-bound O-acyltransferase that palmitoleates the Wnts and hence is essential for their secretion and function. The inhibition of PORCN could serve as a therapeutic approach for the treatment of a number of Wnt-dependent cancers. Herein, we describe the identification of a Wnt secretion inhibitor from cellular high throughput screening. Classical SAR based cellular optimization provided us with a PORCN inhibitor with nanomolar activity and excellent bioavailability that demonstrated efficacy in a Wnt-driven murine tumor model. Finally, we also discovered that enantiomeric PORCN inhibitors show very different activity in our reporter assay, suggesting that such compounds may be useful for mode of action studies on the PORCN O-acyltransferase.

MicroRNA miR-302 inhibits the tumorigenicity of endometrial cancer cells by suppression of Cyclin D1 and CDK1.

MicroRNA miR-302 has been found to induce some tumor cell lines to "transdifferentiate" into miRNA-induced pluripotent stem cells (mirPS), thereby inhibiting tumor cell proliferation and reducing tumorigenicity. This study firstly found that miR-302 inhibited the proliferation and migration of endometrial cell line, Ishikawa and HEC-1-B, and arrested cell cycle at the G2/M phase. In addition, miR-302 inhibited tumorigenicity in immunodeficient mice transplanted with Ishikawa cells. Microarray and Western blotting results showed that miR-302 significantly inhibited CDK1 and Cyclin D1 gene expression in Ishikawa cells. MiR-302 directly targeted Cyclin D1, but indirectly regulated CDK1 gene expression.

FSH modulates the expression of inhibin-alpha and the secretion of inhibins via orphan nuclear receptor NUR77 in ovarian granulosa cells.

It has been previously reported that follicle-stimulating hormone (FSH) regulates the expression of inhibin-alpha in human granulosa cells, but the precise molecular pathway remains unknown. In the present study, we investigated the role of the orphan nuclear receptor, NUR77, in both the transcriptional regulation of the inhibin α-subunit gene and the secretion of inhibins. Our results showed that in a human granulosa cell tumor-derived cell line (KGN) and in human granulosa-lutein cells (hGL), FSH induced the expression of NUR77 and inhibin-alpha, although inhibin-alpha expression did not increased following FSH treatment if NUR77 was knocked down. Furthermore, simply overexpressing or reducing NUR77 levels affected inhibin-alpha expression, while NUR77 overexpression improved the secretion of inhibin A and B from human granulosa cells. In addition, chromatin immunoprecipitation-PCR, avidin-biotin-conjugated DNA precipitation, and luciferase reporter assays confirmed that NUR77 directly regulated the transcription of the inhibin-alpha gene through the specific NGFI-B response element located within its promoter. In the ovarian granulosa cells of the Nur77 knockout mice, the mRNA levels of inhibin-alpha were decreased relative to wild-type mice. These data indicate a role of NUR77 in the regulation of inhibin-alpha in ovarian granulosa cells.

Withdrawal of GnRH agonist decreases oestradiol and VEGF concentrations in high responders.

This study evaluated whether the withdrawal of a gonadotrophin-releasing hormone (GnRH) agonist before triggering ovulation reduces the incidence of ovarian hyperstimulation syndrome (OHSS) in high-risk infertility patients who were treated with gonadotrophins. GnRH agonist was withdrawn for 2 or 3 days when dominant follicles were ≥14 mm in diameter, according to the GnRH agonist long protocol. Non-withdrawal of GnRH agonist was used as control. The serum concentration of oestradiol on the ovulation trigger day was significantly decreased in the GnRH agonist withdrawal group compared with the control group (5750.78 ± 2344.77 pg/ml versus 8076.43 ± 1981.67 pg/ml); however, the number of retrieved oocytes and the fertilization rate were similar between the groups. In addition, the concentrations of vascular endothelial growth factor in plasma on day of human chorionic gonadotrophin administration and follicular fluid on the oocyte retrieval day were decreased following GnRH agonist withdrawal. In fresh embryo transfer cycles, rates of clinical pregnancy, implantation and OHSS were not different between the groups. When GnRH agonist withdrawal was followed by total embryos cryopreserved, the rate of OHSS was decreased compared with the control group (0% versus 8.70%). Clinical pregnancy rates in cryopreserved embryo transfer cycles were comparable between the two groups.

[Diagnostic value of contrast-enhanced ultrasonography in preoperative T-staging of gastric cancer].

OBJECTIVE: To investigate the diagnostic value of contrast-enhanced ultrasonography (CEUS) in preoperative T-staging of gastric cancer. METHODS: A sulfur hexafluoride-filled microbubble ultrasound contrast agent and a continuous real-time imaging technique of contrast pulse sequencing were used. Normal gastric wall was examined by CEUS in 8 healthy volunteers and the results were compared with the findings on multislice computed tomography. Sixty-two patients with gastric cancer proved by biopsies who received preoperative CEUS examination were involved in this study, and the CEUS result was compared with postoperative pathological findings. RESULTS: The normal gastric wall presented a one-layer structure in the portal venous phase and a three-layer structure in the arterial and equilibrium phase including a slightly hyper-enhanced inner layer, a hypo-enhanced intermediate layer, and a markedly hyper-enhanced outer layer, which corresponded histologically to the mucosal, submucosal, and muscular-serosal layer, respectively. The accuracy of transabdominal ultrasonography and CEUS in determining the T stage of gastric cancer was 72.9% and 88.1% respectively, and the difference was statistically significant (chi(2)=4.37, P=0.036). CONCLUSIONS: CEUS shows the normal gastric wall as a one- or a three-layer structure, which provides a theory base for CEUS in preoperative T-staging of gastric cancer. CEUS is a useful diagnostic method for preoperative T-staging of gastric cancer.

Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells.

To study self-renewal, genetic modification, and differentiation of avian spermatogonial stem cells (SSCs), we isolated chicken SSCs from fetal testes on the 16th hatching day via enzyme digestion, and then cultured the SSCs over 2 months after purification in vitro. SSCs were identified by alkaline phosphatase staining and SSEA-1 fluorescence. The EGFP gene was transfected into SSCs by three different methods: electroporation, liposome transfer and calcium acid phosphate precipitation. The transfection rate and cell survival rate using electroporation were higher than when using liposomes or calcium acid phosphate (20.52% vs. 9.75% and 5.61%; 69.86% vs. 65.00% and 51.16%, respectively). After selection with G418 for 8 days, the transgenic SSCs were transplanted into the testes of cocks treated with busulfan. Twenty-five days after transplantation, the recipients' semen was light ivory in color, and the density of spermatozoa was 3.87 (x10(7)/ml), with 4.25% expressing EGFP. By 85 days after transplantation, the number of spermatozoa increased to 32.7 (x10(7)/ml) and the rate of EGFP expression was 16.25%. Frozen sections of the recipients' testes showed that transgenic SSCs were located on the basal membrane of the seminiferous tubules and differentiated into spermatogenic cells at different stages. The EGFP gene was successfully amplified from the DNA of all recipients' semen samples.