[Fkbp38 deletion induces premature ovarian insufficiency in mice by activating mTOR signaling and inducing granulosa cell apoptosis].

OBJECTIVE: To investigate the role of tacrolimus-binding protein 38 (FKBP38) in follicle development and the mechanism by which Fkbp38 gene deletion causes premature ovarian insufficiency (POI). METHODS: The Cre-loxp system was used to construct oocyte-specific Fkbp38 knockout transgenic mice. The genotype of the transgenic mice was identified using PCR, and the expression of FKBP38 in the oocytes was verified. The numbers of primordial follicles, primary follicles, secondary follicles and antral follicles in Fkbp38 knockout mice and non-transgenic littermate control mice were counted with HE staining under a microscope for analyzing the effect of Fkbp38 deletion on follicular development. The fertility and serum sex hormone levels of the mice were determined by reproduction experiments and ELISA to assess ovarian function. Ovarian granulosa cell apoptosis of the mice was assessed using TUNEL assay. The activity of the downstream target protein of phosphorylated ribosomal S6 (PS6) of mTOR signaling pathway was detected, and the expressions of BCL-2 and BAX proteins were determined using immunofluorescence assay for assessing oocyte development in the mice. RESULTS: The oocyte-specific Fkbp38 knockout transgenic mouse model was successfully constructed, which showed decreased fertility, disordered sex hormone levels, and significantly reduced primordial follicles, primary follicles and secondary follicles in the ovary (P < 0.05), demonstrating POI-like changes. Compared with the control mice, oocyte-specific Fkbp38 knockout caused activation of the mTOR signaling pathway, significantly increased apoptosis of the granulosa cells, and obviously increased the BAX/BCL- 2 ratio by increasing BAX expression and reducing BCL-2 expression in the oocytes (P < 0.05). CONCLUSION: FKBP38 plays an important role in follicle development, and Fkbp38 gene deletion in mice causes POI possibly by activating the mTOR signaling pathway and inducing granulosa cell apoptosis.

[Analysis of gene variation and clinical characteristics of Wiedemann-Steiner syndrome].

Objective: To summarize and analyze the clinical characteristics and gene mutations of 6 patients with Wiedemann-Steiner syndrome (WDSTS). Methods: To review and analyze the clinical data, including general conditions, clinical manifestations, growth hormone, cranial or pituitary gland magnetic resonance imaging (MRI),gene results and other data, 6 cases with WDSTS admitted to the Department of Endocrinology, Genetics and Metabolism of Jiangxi Provincial Children's Hospital and the Department of Child Care of Pingxiang Maternity and Child Care from April 2017 to February 2021 were recruited. Results: Of the 6 patients, 2 were male and 4 were female. The age of the first visit ranged from 1.0 to 11.2 years. All the 6 children presented with growth retardation and mental retardation and they all had typical facial dysmorphism and hypertrichosis (mainly on the back and limbs). Among them, case 5 had a growth hormone deficiency, and case 2 and 4 had abnormalities revealed by cranial MRI. Variations in KMT2A gene were identified in these 6 patients: c.10900+2T>C,c.10837C>T(p.Gln3613*), c.4332G>A(p.E1444E), c.2508dupC(p.W838Lfs*9), c.11695_11696delinsT(p.T3899Sfs*73), c.9915dupA (p.P3306Tfs*22).Among these variations, c.4332G>A, c.11695_11696delinsT and c.9915dupA were novel mutations. Therefore, the final diagnosis of these patients was WDSTS. Conclusions: Patients presented with short stature and mental retardation, typical facial dysmorphism and hypertrichosis should be considered WDSTS. Whole-exome sequencing plays an important role in disease diagnosis and genetic counseling.

[miR-483-5p aggravates cisplatin-induced premature ovarian insufficiency in rats by targeting FKBP4].

OBJECTIVE: To investigate the role of FKBP4 protein in cisplatin-induced premature ovarian insufficiency (POI). OBJECTIVE: We performed ITRAQ assay of the ovarian tissues from 4 mice with cisplatin-induced POI and 4 control mice, and identified FKBP4 as a significantly down-regulated protein in the oocytes and granulosa cells following cisplatin treatment. TargetScan software was used for target analysis of FKBP4, and qRT-PCR and Western blotting were used to verify the expression levels of miR-483-5p and FKBP4 in the mouse models. Serum samples were collected from patients with POI and healthy women for detecting miR-483-5p level with qRT-PCR. Cell transfection and dual-luciferase assay were performed to determine the relationship between miR-483-5p and FKBP4. In primary granulosa cells and KGN cells, we examined the effect of miR-483-5p alone, miR-483-5p and cisplatin, and miR-483-5p combined with both cisplatin and FKBP4 on cell apoptosis. We also assessed ovarian function in a transgenic mouse model with ovarian miR-483-5p overexpression in comparison wigh wildtype mice using immunofluorescence assay, in situ hybridization and ELISA. OBJECTIVE: Ovarian FKBP4 expression was significantly decreased in mice with cisplatin-induced POI. Analysis using TargetScan software indicated that FKBP4 was the potential target of miR-483-5p, which was highly expressed in the ovaries and serum of POI mice and in the serum of patients with POI. In vitro experiments further confirmed that FKBP4 was the target of miR-483-5p. In KGN and primary granulosa cells, FKBP4 overexpression significantly reduced cell apoptosis induced by both cisplatin and miR-483-5p overexpression (P= 0.0045 and 0.0177, respectively). In the transgenic mice with miR-483-5p overexpression in the oocytes, cisplatin induced more severe ovarian damages as compared with those in the wild-type mice. OBJECTIVE: miR-483-5p/FKBP4 is a new and important pathway in cisplatin-induced POI, in which cisplatin increases ovarian miR- 483-5p expression to result in targeted downregulation of FKBP4. Up-regulation of miR-483-5p may increase ovarian sensitivity to cisplatin and cause severe ovarian dysfunction. Detection of serum miR-483-5p level may help to predict the occurrence and development of POI.

Effects of Birinapant on Proliferation and Invasion of MGC-803 Gastric Cancer Cells and Mechanism Underlying These Effects.

We studied the effects of birinapant, a mimetic of the second mitochondria-derived activator of caspase (SMAC), on invasion and proliferation of MGC-803 gastric cancer cells and the molecular mechanisms underlying these processes. The expression of cellular inhibitor of apoptosis 1 (cIAP1) and TNF receptor-associated factor 3 (TRAF3) in gastric cancer cell line MGC-803 and normal gastric mucosa GES-1 cells were analyzed by Western blotting and cell immunofluorescence assay. After pretreatment of MGC-803 cells with birinapant, a Transwell invasion assay was used to evaluate the cell invasion ability. MGC-803 cells were implanted under the skin of BALB/c nude mice. The tumors were removed 10 days later and its size was measured. Protein expression of proliferating cell nuclear antigen (PCNA) in the subcutaneous tumors was analyzed by immunohistochemical method. In addition, the expression of cIAP1, TRAF3, pNF-κB, and NF-κB in control and birinapant-treated cells was compared by Western blotting and the rate of cell apoptosis was evaluated by flow cytometry. In untreated MGC-803 gastric cancer cells, the expression of cIAP1 was higher and the expression of TRAF3 was lower than in normal gastric mucosa cell line GES-1. Pretreatment with birinapant inhibited the invasion and proliferation of MGC-803 cells and promoted cell apoptosis. Birinapant also promoted the expression of TRAF3 and inhibited the expression of cIAP1 and pNF-κB in MGC-803 cells. Thus, birinapant inhibited the expression of cIAP1, prevented degradation of TRAF3, and suppressed invasion and proliferation of MGC-803 cells by promoting cell apoptosis.

An instrument for measuring scintillators efficiently based on silicon photomultipliers.

An instrument used for measuring multiple scintillators' light output and energy resolution was developed. The instrument consisted of a light sensor array which was composed of 64 discrete SiPMs (Silicon Photomultipliers), a corresponding individual channel readout electronics system, and a data processing algorithm. A Teflon grid and a large interval between adjacent SiPMs were employed to eliminate the optical cross talk among scintillators. The scintillators' light output was obtained by comparing with a reference sample with known light output. Given the SiPM temperature dependency and the difference among each SiPM, a temperature offset correction algorithm and a non-uniformity correction algorithm were added to the instrument. A positioning algorithm, based on nine points, was designed to evaluate the performance of a scintillator array. Tests were performed to evaluate the instrument's performance. The uniformity of 64 channels for light output measurement was better than 98%, the stability was better than 98% when temperature varied from 15 °C to 40 °C, and the nonlinearity under 511 keV was better than 2%. This instrument was capable of selecting scintillators and evaluating the packaging technology of scintillator arrays with high efficiency and accuracy.

TRAIL-secreting mesenchymal stem cells promote apoptosis in heat-shock-treated liver cancer cells and inhibit tumor growth in nude mice.

Liver cancer is one of the top six leading causes of cancer-related death. Radiofrequency ablation (RFA) is an important means of treating liver cancer. Residual cancer after RFA is the most frequent cause of recurrence in cases of liver cancer. The main difference between residual cancer cells and ordinary liver cancer cells is that residual cancer cells experience heat shock. The secretable form of trimeric human tumor necrosis factor-related apoptosis-inducing ligand (stTRAIL) induces apoptosis in a variety of human cancers but not in normal tissues. It has shown potent cancer-selective killing activity and has drawn considerable attention as a possible cancer therapy. In the present work, the therapeutic potential of this stTRAIL-based gene therapy was evaluated in hepatocellular carcinoma subjected to RFA. Rat bone marrow mesenchymal stem cells (BM-MSCs) were isolated and transduced with a lentiviral vector encoding stTRAIL (stTRAIL-MSCs, T-MSCs). Cells treated with heat treatment at 43 °C for 45 min served as simulated residual cancer cells. After treatment with T-MSCs, apoptosis in heat-shock-treated liver cancer cells increased significantly, and caspase-3 was upregulated. When T-MSCs were subcutaneously injected into nude mice, they localized to the tumors and inhibited tumor growth, significantly increasing survival. Collectively, the results of the present study indicate that BM-MSC can provide a steady source of stTRAIL and may be suitable for use in the prevention of the recurrence of hepatocellular carcinoma after RFA with secretable trimeric TRAIL.

Comparative study of SPORL and dilute-acid pretreatments of spruce for cellulosic ethanol production.

The performance of two pretreatment methods, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA), was compared in pretreating softwood (spruce) for fuel ethanol production at 180 degrees Celsius for 30 min with a sulfuric acid loading of 5% on oven-dry wood and a 5:1 liquor-to-wood ratio. SPORL was supplemented with 9% sodium sulfite (w/w of wood). The recoveries of total saccharides (hexoses and pentoses) were 87.9% (SPORL) and 56.7% (DA), while those of cellulose were 92.5% (SPORL) and 77.7% (DA). The total of known inhibitors (furfural, 5-hydroxymethylfurfural, and formic, acetic and levulinic acids) formed in SPORL were only 35% of those formed in DA pretreatment. SPORL pretreatment dissolved approximately 32% of the lignin as lignosulfonate, which is a potential high-value co-product. With an enzyme loading of 15 FPU (filter paper units) per gram of cellulose, the cellulose-to-glucose conversion yields were 91% at 24h for the SPORL substrate and 55% at 48 h for the DA substrate, respectively.