Pillar[6]arene acts as a biosensor for quantitative detection of a vitamin metabolite in crude biological samples

ビタミン代謝物を迅速定量できる超分子バイオセンサーを開発. 京都大学プレスリリース. 2020-12-09.Metabolic syndrome is associated with obesity, hypertension, and dyslipidemia, and increased cardiovascular risk. Therefore, quick and accurate measurements of specific metabolites are critical for diagnosis; however, detection methods are limited. Here we describe the synthesis of pillar[n]arenes to target 1-methylnicotinamide (1-MNA), which is one metabolite of vitamin B3 (nicotinamide) produced by the cancer-associated nicotinamide N-methyltransferase (NNMT). We found that water-soluble pillar[5]arene (P5A) forms host–guest complexes with both 1-MNA and nicotinamide, and water-soluble pillar[6]arene (P6A) selectively binds to 1-MNA at the micromolar level. P6A can be used as a “turn-off sensor” by photoinduced electron transfer (detection limit is 4.38 × 10−6 M). In our cell-free reaction, P6A is used to quantitatively monitor the activity of NNMT. Moreover, studies using NNMT-deficient mice reveal that P6A exclusively binds to 1-MNA in crude urinary samples. Our findings demonstrate that P6A can be used as a biosensor to quantify 1-MNA in crude biological samples

p53とFoxp4発現を指標とした子宮体部類内膜癌の新しい術前悪性度診断法の開発

金沢大学学際科学実験センター子宮体癌において、癌進展に伴う扁平上皮化生の誘導が、PTEN欠損を有するヒト臨床検体とマウス病態モデルの両者に共通して認められることから、PTENを欠損した癌細胞では癌の進展を抑制する異方向への分化・成熟誘導機構が存在するのではないかと考え、 扁平上皮化生誘導の機序の解明を目指した。子宮体癌を自然発症する子宮特異的PTEN欠損マウスでは、濃度依存的なエストロゲンの作用によって扁平上皮化生が強く誘導されることが明らかになった。またこの変化は、本来重層扁平上皮には分化しない子宮内膜上皮において、PTEN欠損細胞の存在下で重層扁平上皮に分化可能な細胞集団が出現し、増殖した結果であると考えられた。Squamous metaplasia is found in both endometrial cancer patients and the mouse model which carries PTEN inactivation. We found that squamous metaplasia is developed in estrogen-dependent manner by the investigation of animal models. We further demonstrated that a small number of precursor cells which can differentiate into the stratified epithelium arises under the environment with existence of PTEN inactivated epithelial cells.研究課題/領域番号:19K18691, 研究期間(年度):2019-04-01 – 2021-03-31出典：「エストロゲンによる子宮体癌細胞の異方向分化誘導とその分子生物学的機序の解明」研究成果報告書　課題番号19K18691（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-19K18691/19K18691seika/）を加工して作

Ovarian LGR5 is critical for successful pregnancy

Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is expressed in many organs, including female reproductive organs, and is a stem cell marker in the stomach and intestinal epithelium, hair follicles, and ovarian surface epithelium. Despite ongoing studies, the definitive physiological functions of Lgr5 remain unclear. We utilized mice with conditional deletion of Lgr5 (Lgr5(d/d)) in the female reproductive organs by progesterone receptor-Cre (Pgr(Cre)) to determine Lgr5's functions during pregnancy. Only 30% of plugged Lgr5(d/d) females delivered live pups, and their litter sizes were lower. We found that pregnancy failure in Lgr5(d/d) females was due to insufficient ovarian progesterone (P4) secretion that compromised decidualization, terminating pregnancy. The drop in P4 levels was reflected in elevated levels of P4-metabolizing enzyme 20α-hydroxysteroid dehydrogenase in corpora lutea (CL) inactivated of Lgr5. Of interest, P4 supplementation rescued decidualization failure and supported pregnancy to full term in Lgr5(d/d) females. These results provide strong evidence that Lgr5 is critical to normal CL function, unveiling a new role of LGR5 in the ovary

Dual expression of immunoreactive estrogen receptor β and p53 is a potential predictor of regional lymph node metastasis and postoperative recurrence in endometrial endometrioid carcinoma.

Although histological grade and muscular invasion are related to the malignant behaviors of endometrial endometrioid carcinoma, lymphatic and/or distant metastases are unexpectedly encountered, even in patients in the low-risk group. To re-evaluate additional reliable parameters to predict the risk of progression, we examined the immunohistochemical expression profiles of p53 and estrogen receptor (ER) β proteins. Patients with endometrial endometrioid carcinoma who underwent surgical treatment at our hospital (n = 154) were recruited to this study, and the significance of the relationships between the incidence of regional lymph node metastasis and/or postoperative recurrence and clinical or experimental parameters was evaluated. By multivariate analysis, we found that histological grades, detection of immunoreactive p53 (positive rates more than 10%, p53-stained), and high expression of ERβ (high-ERβ) were independently associated with metastasis and/or recurrence. Among these parameters, the sensitivity and negative predictive values of high-ERβ were very high (up to 100%). In the population with high-ERβ, the positive rates of metastasis and/or recurrence were 61.1% in the p53-stained group and 21.9% in the p53-non-stained (negative) group. Furthermore, the positive rate in the group showing myometrial invasion of more than 1/2 and showing both p53-stained and high-ERβ was 80%. The disease-free survival of patients who were double-positive for p53-stained and high-ERβ was significantly shorter than that in other patients. In summary, our findings showed that increases in ERβ and p53 immunoreactivity were significantly correlated with the incidence of metastasis and/or recurrence in endometrial endometrioid carcinoma, suggesting that double-positivity for p53-stained and high-ERβ may provide a promising clinical indicator to predict the risk of progression

The uterine epithelial loss of Pten is inefficient to induce endometrial cancer with intact stromal Pten.

Mutation of the tumor suppressor Pten often leads to tumorigenesis in various organs including the uterus. We previously showed that Pten deletion in the mouse uterus using a Pgr-Cre driver (Ptenf/fPgrCre/+) results in rapid development of endometrial carcinoma (EMC) with full penetration. We also reported that Pten deletion in the stroma and myometrium using Amhr2-Cre failed to initiate EMC. Since the Ptenf/fPgrCre/+ uterine epithelium was primarily affected by tumorigenesis despite its loss in both the epithelium and stroma, we wanted to know if Pten deletion in epithelia alone will induce tumorigenesis. We found that mice with uterine epithelial loss of Pten under a Ltf-iCre driver (Ptenf/f/LtfCre/+) develop uterine complex atypical hyperplasia (CAH), but rarely EMC even at 6 months of age. We observed that Ptenf/fPgrCre/+ uteri exhibit a unique population of cytokeratin 5 (CK5) and transformation related protein 63 (p63)-positive epithelial cells; these cells mark stratified epithelia and squamous differentiation. In contrast, Ptenf/fLtfCre/+ hyperplastic epithelia do not undergo stratification, but extensive epithelial cell apoptosis. This increased apoptosis is associated with elevation of TGFβ levels and activation of downstream effectors, SMAD2/3 in the uterine stroma. Our results suggest that stromal PTEN via TGFβ signaling restrains epithelial cell transformation from hyperplasia to carcinoma. In conclusion, this study, using tissue-specific deletion of Pten, highlights the epithelial-mesenchymal cross-talk in the genesis of endometrial carcinoma

Successful Production of Offspring Derived from Phospholipase C Zeta-Deficient Sperm by Additional Artificial Activation

During mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLCζ) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLCζ in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLCζ (Plcz1−/−) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1−/−-sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1−/− sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1−/− sperm and Plcz1−/− and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1−/−eCS−/−), the rates of pronuclear formation were very low (2.0 ± 2.3% and 6.1 ± 3.7%, respectively) compared to control (92.1 ± 2.6%). However, these rates were dramatically improved by additional procedures of PLCζ-mRNA injection or SrCl2 treatment (Plcz1−/− sperm + PLCζ mRNA, Plcz1−/− sperm + SrCl2 and Plcz1−/−eCS−/− sperm + PLCζ mRNA; 64.2 ± 10.8%, 89.2 ± 2.4% and 72.6 ± 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1−/− sperm + PLCζ mRNA:10.0 ± 2.8%, Plcz1−/− sperm + SrCl2:4.0 ± 4.3% and Plcz1−/−eCS−/− sperm + PLCζ mRNA: 10.0 ± 5.7%). The rate in Plcz1−/− sperm + SrCl2 group was significantly lower than that in control (26.0 ± 2.4%). Taken together, our present results show that additional activation treatment such as SrCl2 and PLCζ mRNA can fully support to develop to term even in oocyte injected Plcz1−/− sperm. In addition, PLCζ-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl2. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species

Uterine epithelial Gp130 orchestrates hormone response and epithelial remodeling for successful embryo attachment in mice

Abstract Leukemia inhibitory factor (LIF) receptor, an interleukin 6 cytokine family signal transducer (Il6st, also known as Gp130) that is expressed in the uterine epithelium and stroma, has been recognized to play an essential role in embryo implantation. However, the molecular mechanism underlying Gp130-mediated LIF signaling in the uterine epithelium during embryo implantation has not been elucidated. In this study, we generated mice with uterine epithelium specific deletion of Gp130 (Gp130 ecKO). Gp130 ecKO females were infertile due to the failure of embryo attachment and decidualization. Histomorphological observation revealed that the endometrial shape and embryo position from Gp130 ecKO were comparable to those of the control, and uterine epithelial cell proliferation, whose attenuation is essential for embryo implantation, was controlled in Gp130 ecKO. Comprehensive gene expression analysis using RNA-seq indicates that epithelial Gp130 regulates the expression of estrogen- and progesterone-responsive genes in conjunction with immune response during embryo implantation. We also found that an epithelial remodeling factor, snail family transcriptional repressor 1 (Snai1), was markedly reduced in the pre-implantation uterus from Gp130 ecKO. These results suggest that not only the suppression of uterine epithelial cell proliferation, but also Gp130-mediated epithelial remodeling is required for successful implantation in mice

Association of p53/ERβ expression and muscular invasion with the incidence of regional lymph node metastasis and/or postoperative recurrence in patients with endometrial endometrioid carcinoma.

<p>Association of p53/ERβ expression and muscular invasion with the incidence of regional lymph node metastasis and/or postoperative recurrence in patients with endometrial endometrioid carcinoma.</p

Association of p53/ERβ expression and muscular invasion with the incidence of regional lymph node metastasis and/or postoperative recurrence in patients with endometrial endometrioid carcinoma.

<p>Association of p53/ERβ expression and muscular invasion with the incidence of regional lymph node metastasis and/or postoperative recurrence in patients with endometrial endometrioid carcinoma.</p