A quantitative assay system for protein C activity, the regulator of blood coagulation, based on a chromogenic method mimicking the blood coagulation cascade.

Background and aims: Protein C is a plasma protein, and its active form regulates blood coagulation. The recommended unit of protein C activity is IU/mL; however, some laboratories use percentage. Some deficiencies cannot be detected owing to measurement principles. This study sought to quantify protein C activity levels and overcome the limitations of the current measurements. Materials and methods: Our protein C activity measurement method mimicked the blood coagulation cascade and used a thrombin-specific chromogenic reagent. The control was prepared by adding protein C to the protein C deficient plasma. The calibration curve was plotted as the increase in the absorbance per minute and the concentration of protein C in the control. Statistical tests were performed to compare our method with the current chromogenic method. Results: A calibration curve was constructed (y = -0.0132x + 0.14, R2 = 0.9987, n = 10). The statistical results of our method suggested non-inferiority when compared to the current chromogenic method (α = 0.05). Conclusion: The quantitative measurement was performed using plasma samples. Our method provides the possibility of expressing protein C activity quantitatively and detecting deficiencies that cannot be detected using the current chromogenic method.

Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression.

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health.

Ovaries are responsible for forming the eggs humans and other mammals need to reproduce. Once mature, the egg cell is released into the fallopian tube where it can be potentially fertilized by a sperm. Despite their crucial role, how eggs are made in the ovary is poorly understood. This is because ovaries are hard to access, making it difficult to conduct experiments on them. To overcome this, researchers have built artificial ovaries in the laboratory using stem cells from the embryos of mice which can develop into all cell types in the adult body. By culturing these embryonic stem cells under special conditions, researchers can convert them in to the two main cell types of the developing ovary: germ cells which go on to form eggs, and granulosa cells which help eggs grow and mature. The resulting lab-grown ovary can make eggs that produce live mice when fertilized. This approach has also been applied to human induced pluripotent stem cells (iPSCs), adult human cells which have been reprogrammed to a stem-like state. While this has produced human germ cells, generating human granulosa cells has been more challenging. Here, Pierson Smela, Kramme et al. show that activating a specific set of transcription factors (proteins that switch genes on or off) in iPSCs can make them transition to granulosa cells. First, the team tested random combinations of 35 transcription factors which, based on previous literature and genetic data, were likely to play a role in the formation of granulosa cells. This led to the identification of a small number of factors that caused the human iPSCs to develop features and carry out roles seen in mature granulosa cells; this includes producing an important reproductive hormone and supporting the maturation of germ cells. Pierson Smela, Kramme et al. found that growing these granulosa-like cells together with germ cells (also generated via iPSCs) resulted in structures similar to ovarian follicles which help eggs develop. These findings could help researchers build stable systems for studying how granulosa cells behave in human ovaries. This could lead to new insights about reproductive health.

Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development.

Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast toward the amniotic ectoderm, primitive streak/mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.

Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers.

In vitro expansion of human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived PGC model, has proved challenging due to rapid loss of primordial germ cell (PGC)-like identity and limited cell survival/proliferation. Here, we describe long-term culture hPGCLCs (LTC-hPGCLCs), which actively proliferate in a serum-free, feeder-free condition without apparent limit as highly homogeneous diploid cell populations maintaining transcriptomic and epigenomic characteristics of hPGCLCs. Histone proteomics confirmed reduced H3K9me2 and increased H3K27me3 marks in LTC-hPGCLCs compared with induced pluripotent stem cells (iPSCs). LTC-hPGCLCs established from multiple human iPSC clones of both sexes were telomerase positive, senescence-free cells readily passaged with minimal cell death or deviation from the PGC-like identity. LTC-hPGCLCs are capable of differentiating to DAZL-positive M-spermatogonia-like cells in the xenogeneic reconstituted testis (xrTestis) organ culture milieu as well as efficiently producing fully pluripotent embryonic germ cell-like cells in the presence of stem cell factor and fibroblast growth factor 2. Thus, LTC-hPGCLCs provide convenient access to unlimited amounts of high-quality and homogeneous hPGCLCs.

Transcriptomic and Epigenetic Preservation of Genetic Sex Identity in Estrogen-feminized Male Chicken Embryonic Gonads.

Whereas in ovo exposure of genetically male (ZZ) chicken embryos to exogenous estrogens temporarily feminizes gonads at the time of hatching, the morphologically ovarian ZZ-gonads (FemZZs for feminized ZZ gonads) are masculinized back to testes within 1 year. To identify the feminization-resistant "memory" of genetic male sex, FemZZs showing varying degrees of feminization were subjected to transcriptomic, DNA methylome, and immunofluorescence analyses. Protein-coding genes were classified based on their relative mRNA expression across normal ZZ-testes, genetically female (ZW) ovaries, and FemZZs. We identified a group of 25 genes that were strongly expressed in both ZZ-testes and FemZZs but dramatically suppressed in ZW-ovaries. Interestingly, 84% (21/25) of these feminization-resistant testicular marker genes, including the DMRT1 master masculinizing gene, were located in chromosome Z. Expression of representative marker genes of germline cells (eg, DAZL or DDX4/VASA) was stronger in FemZZs than normal ZZ-testes or ZW-ovaries. We also identified 231 repetitive sequences (RSs) that were strongly expressed in both ZZ-testes and FemZZs, but these RSs were not enriched in chromosome Z. Although 94% (165/176) of RSs exclusively expressed in ZW-ovaries were located in chromosome W, no feminization-inducible RS was detected in FemZZs. DNA methylome analysis distinguished FemZZs from normal ZZ- and ZW-gonads. Immunofluorescence analysis of FemZZ gonads revealed expression of DMRT1 protein in medullary SOX9+ somatic cells and apparent germline cell populations in both medulla and cortex. Taken together, our study provides evidence that both somatic and germline cell populations in morphologically feminized FemZZs maintain significant transcriptomic and epigenetic memories of genetic sex.

Relaxin-2 May Suppress Endometriosis by Reducing Fibrosis, Scar Formation, and Inflammation.

BACKGROUND: Relaxin (RLX)-2, produced by the corpus luteum and placenta, is known to be potentially effective in fibrotic diseases of the heart, lungs, kidneys, and bladder; however, its effectiveness in endometriosis has not yet been investigated. In the present study, we conducted a comprehensive study on the effect of RLX-2 on endometriosis. We checked the expressions of LGR-7, a primary receptor of RLX-2, in endometriomas using immunohistochemistry. Endometriotic stromal cells (ESCs) purified from surgical specimens were used in in vitro experiments. The effects of RLX-2 on ESCs were evaluated by quantitative-PCR, ELISA, and Western blotting. Gel contraction assay was used to assess the contraction suppressive effect of RLX-2. The effect of RLX-2 was also examined in the endometriosis mouse model. LGR-7 was expressed in endometriotic lesions. In ESCs, RLX-2 increased the production of cAMP and suppressed the secretion of interleukin-8, an inflammatory cytokine, by 15% and mRNA expression of fibrosis-related molecules, plasminogen activator inhibitor-1 (PAI-1), and collagen-I by approximately 50% (p < 0.05). In the gel contraction assay, RLX-2 significantly suppressed the contraction of ESCs, which was cancelled by removing RLX-2 from the medium or by adding H89, a Protein Kinase A (PKA) inhibitor. In ESCs stimulated with RLX-2, p38 MAPK phosphorylation was significantly suppressed. In the endometriosis mouse model, administration of RLX-2 significantly decreased the area of the endometriotic-like lesion with decreasing fibrotic component compared to non-treated control (p = 0.01). RLX-2 may contribute to the control of endometriotic lesion by suppressing fibrosis, scar formation, and inflammation.

Inhibition of autophagy in theca cells induces CYP17A1 and PAI-1 expression via ROS/p38 and JNK signalling during the development of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a clinical syndrome characterized by hyperandrogenism, oligo/anovulation, and polycystic ovary. Autophagy is an intracellular system that degrades cytosolic proteins and organelles. The relationship between autophagy and PCOS has not been clarified. We found that p62 and ubiquitin were significantly increased in theca cells of women with PCOS using immunohistochemistry. Autophagy inhibition by palmitic acid and chloroquine in bovine theca cells increased p62 and ubiquitin and induced the expression of cytochrome P450 17A1 (CYP17A1) and plasminogen activator inhibitor-1 (PAI-1) mRNA. Furthermore, palmitic acid and chloroquine exposure significantly increased reactive oxygen species (ROS) and activated p38 and c-Jun N-terminal kinase (JNK). Inhibition of p38 and JNK significantly reduced CYP17A1 and PAI-1 mRNA expression. We showed that inhibition of autophagy in theca cells may have contributed to the pathogenesis of PCOS, based on CYP17A1 and PAI-1 mRNA expression via the ROS/p38 and JNK signalling pathways.

IL-33 Exacerbates Endometriotic Lesions via Polarizing Peritoneal Macrophages to M2 Subtype.

In endometriosis, M2 macrophages (MΦ) are dominant and promote the development of endometriosis lesions. However, the factor(s) which induces M2 MΦ are unknown. In the present study, we focused on interleukin (IL)-33, known as an alarmin and investigated its expression and its role in endometriosis, especially from the point of the relevance with MΦ. The expression of IL-33 in endometriosis lesions was examined by immunohistochemistry. The cystic fluid of ovarian cysts/tumors was obtained and used to measure IL-33 concentration. Endometriotic stromal cells (ESC) and MΦ derived from patients were used for in vitro experiments. IL-33 was detected in the epithelium and stromal cells of endometriotic lesions. The mean IL-33 concentration in the cystic fluid of endometriomas was significantly higher than that in non-endometriomas (2.2 ng/ml vs. 0.02 ng/ml, P < 0.01). IL-1ß induced IL-33 mRNA expression in ESC via p38 MAPK activation. With IL-33 stimulation, peritoneal MΦ polarized to M2 MΦ and produced IL-1ß mRNA with a 2.2-fold increase, which was negated with soluble ST2, a decoy receptor of IL-33. IL-33, derived from endometriotic lesions, stimulated MΦ to produce IL-1ß, which results in increasing IL-33 production in ESC. This cycle may continue to exacerbate the endometriotic lesions.

Decreased effector regulatory T cells and increased activated CD4+ T cells in premature ovarian insufficiency.

PROBLEM: Premature ovarian insufficiency (POI) is a clinical syndrome defined by the loss of ovarian activity before 40 years old. An autoimmune mechanism is suggested to be involved in the development of POI. Therefore, we examined the relationship between peripheral blood regulatory T (Treg) cells and autoantibodies in POI. METHOD OF STUDY: Thirty POI patients and 23 control women were enrolled in the study. Using flow cytometry, we measured the abundance of CD4+ T, CD4+ CD69+ T, CD8+ T, CD8+ CD69+ T, naive Treg, effector Treg, and FOXP3+ effector T cells in peripheral blood. Antinuclear and anti-thyroglobulin antibody (Tg-Ab) titers were measured in POI patients. RESULTS: The number of CD4+ T or CD4+ CD69+ T cells was significantly higher in POI patients (P = 0.045, and P = 0.030), and there were significantly fewer effector Treg cells in POI patients (P = 0.016) than in the controls. There were significant negative correlations between effector Treg cells and Tg-Abs (r = -0.584, P = 0.0282), and between effector Treg cells and CD4+ CD69+ T cells (r = -0.415, P = 0.0226) in POI patients. CONCLUSION: This is the first report of decreased numbers of effector Treg cells and increased CD4+ CD69+ activated T cells in peripheral blood in POI, suggesting that POI is an autoimmune disease.

Sphingosine 1 Phosphate (S1P) Increased IL-6 Expression and Cell Growth in Endometriotic Cells.

OBJECTS: There is growing evidence that sphingosine 1-phosphate (S1P) is involved in inflammatory diseases. As endometriosis is known as an inflammatory disease, we investigated the role of S1P system in the development of endometriosis. METHODS: The expression of sphingosine kinase (SphK) 1 in endometriosis lesions was examined by immunohistochemistry. The cystic fluid of ovarian cysts/tumors were obtained to measure S1P concentrations. Endometriotic stromal cells (ESC) derived from endometrioma were used for in vitro experiments. RESULTS: Sphingosine kinase 1 was detected in epithelium and stromal cells of endometriotic lesions. The mean S1P concentration in the cystic fluid of endometriomas was higher than that in nonendometriomas significantly (98.2 nM vs less than 1.5 nM, P < .01). Interleukin-1ß (IL-1ß) or transforming growth factor-ß exhibited 2.7-fold and 11.5-fold increase in SphK1 messenger RNA (mRNA) expression in ESC, respectively (P < .01). Higher dose of S1P (125nM) increased the cell number of ESC by 20%, and low dose of S1P (1.25 nM and 12.5 nM) induced IL-6 mRNA production and IL-6 secretion by ESC dose-dependently. JTE013, an antagonist for S1PR2, partially suppressed IL-6 induction by S1P (P < .05). JTE013 and VPC23019, an antagonist for S1PR1 and S1PR3, suppressed the ESC proliferation induced by S1P. CONCLUSION: The present study for the first time proved that the SphK-S1P-S1PR axis play a role of accelerating inflammation and growth of endometriotic cells.

Molecular recognition of upper rim functionalized cavitand and its unique dimeric capsule in the solid state.

Cavitand 1 possesses four 2,2'-bipyridyl pillars on its upper rim that encapsulates small guests, such as nitromethane, acetonitrile, methyl acetate, ethyl acetate, and N-methylacetamide, into a deep cavity to form host-guest complexes in a 1:1 ratio. Nitroethane, N,N-dimethylformamide, and N,N-dimethylacetamide were not bound in this manner. A guest-binding study and molecular mechanics calculations revealed that the four 2,2'-bipyridyl pillars of cavitand 1 created a steric boundary that is responsible for selective guest recognition. In the solid state, cavitand 2 formed a unique chiral capsule 2(2) by π-π stacking interactions between the 2,2'-bipyridyl pillars. A nitromethane molecule was unusually placed deep inside the cavity, as directed by the multiple hydrogen bonding interactions between the nitromethane oxygen atoms, the C-H bonds of the bridge methylenes and the pillar phenyl groups.

[A case of drug-induced liver injury caused by entecavir for treatment of hepatitis B virus reactivation during RCHOP in a patient with non-Hodgkin lymphoma].

A 49-year-old-man, a healthy carrier of hepatitis B virus (HBV), received chemotherapy with a rituximab/cyclo- phosphamide/doxorubicin/vincristine/prednisolone (R-CHOP) regimen for non-Hodgkin's lymphoma. At the first course of chemotherapy, not only the liver function but the HBV DNA level was elevated. These symptoms were diagnosed as hepatic injury induced by HBV reactivation, and, therefore, entecavir (ETV) was started. As a result, although the treatment with ETV decreased the HBV DNA level, liver function values were remarkably elevated again (over 3 times the levels before beginning ETV). ETV was discontinued because of suspicion regarding the onset of hepatic injury it caused. After switching to lamivudine (LVD), the liver function quickly improved and no problems were observed with renewal of the R-CHOP regimen. In addition, the HBV DNA level decreased and 3 courses of R-CHOP were performed successfully. In our case, the hepatic injury was induced by ETV, although anti-HBV medicine was used for the treatment of HBV reactivation according to the guideline. Therefore, the medical staff must carefully and consistently observe patients with HBV infection after chemotherapy.

Guest encapsulation and self-assembly of a cavitand-based coordination capsule.

The synthesis and spectroscopic characterization of a cavitand-based coordination capsule 14 BF4 of nanometer dimensions is described. Encapsulation studies of large aromatic guests as well as aliphatic guests were performed by using 1H NMR spectroscopy in [D1]chloroform. In addition to the computational analysis of the shape and geometry of the capsule, an experimental approach to estimate the interior size of the cavity is discussed. The cavity provides a highly rigid binding space in which molecules with lengths of approximately 14 A can be selectively accommodated. The rigid cavity distinguished slight structural differences in the flexible alkyl-chain guests as well as the rigid aromatic guests. The detailed thermodynamic studies revealed that not only CH-pi interactions between the methyl groups on the guest termini and the aromatic cavity walls, but also desolvation of the inner cavity play a key role in the guest encapsulation. The cavity preferentially selected the hydrogen-bonded heterodimers of a mixture of two or three carboxylic acids 18-20. The chiral capsule encapsulated a chiral guest to show diastereoselection.

A new self-assembling capsule via metal coordination.

A new octadentate cavitand forms a stable dimeric molecular capsule via metal-coordination, creating a large and elaborate three-dimensional cavity in which large aromatic guests are accommodated to form supramolecular complexes.