To investigate the effect of the localization of oligonucleotides decoy (ODNs decoy) on the activation of nuclear factor-kappaB (NF-kappaB) in TNF-alpha induced HeLa cells. The mercapto group-modified nuclear localization signal (NLS) peptide was covalently conjugated to amino group-modified NF-kappaB ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy was transfected into HeLa cells by TransME transfection reagent. The intracellular distribution of fluorescent labeled NLS-ODNs decoy was detected with a microscope. The cell viability was detected by MTT assay, and then the activity of NF-kappaB in cell nuclear extract was assayed by electrophoretic mobility shift assay (EMSA). The results showed that NLS peptide was successfully conjugated to ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy effectively entered into nucleus with high rate of 17.9%. It was observed that the cell viability of HeLa cell was not significantly affected by the transfection of NLS-ODNs decoy, while NLS-ODNs decoy significantly inhibited the activation of NF-kappaB in TNF-alpha induced HeLa cells nuclear extracts. This experiment can provide a new covalent conjugation of NLS peptide to ODNs can effectively drive decoy into nucleus, and thus improve its inhibitory effects on the activation a transcription factor.
Base Sequence ; Cell Nucleus ; metabolism ; HeLa Cells ; Humans ; Molecular Sequence Data ; NF-kappa B ; genetics ; metabolism ; Nuclear Localization Signals ; genetics ; Oligonucleotides ; genetics ; metabolism ; Transfection

To discuss the effect of co-culture on the quality of mouse blastocysts and their epigenetic modification. We divided mouse zygotes into three co-culture experiment groups : with granular cells (group I ), oviduct epithelium cells (group II) and oviduct tissue (group III). Meanwhile, we set up control A (cultured in vitro, only KSOM (KCl+ simplex optimized medium)) and control B (cultured in vivo). Then we compared cleavage rate and blastocyst rate among different groups. After that we evaluated the quality of blastocysts by using ICM/TE (Inner cell mass/Trophectoderm cells) ratio via staining with propidium iodide and Hoechest333258, and analyzed the level of genome methylation and histone acetylation by immunofluorescence. Compared with the control group A, the co-culture groups had increased cleavage rate and blastocyst rate (P < 0.05), blastocyst cells and the ICM/TE ratio of co-culture groups were higher (P < 0.05), the level of genome methylation and histone acetylation had no significant difference between groups in vitro (P > 0.05), but the level of genome methylation in vivo was significantly higher than that of in vitro (P < 0.05). The co-culture methods can successfully promote the development rate of embryos in vitro, and improve the quality of the blastocyst. However, the methods have drawbacks in changing the abnormal genome methylation with in vitro culture.
Animals ; Blastocyst ; cytology ; Coculture Techniques ; DNA Methylation ; Epigenesis, Genetic ; genetics ; Epithelial Cells ; cytology ; Fallopian Tubes ; cytology ; Female ; Male ; Mice

Myostatin, a member of the transforming growth factor beta (TGF-beta) family, is a negative regulator for muscle growth. Loss of the function of this gene is associated with the phenotype described as "double muscling", an extreme form of muscle development characterized by a large increase in muscle mass. Two replacement vectors, pA2T-Mstn4.0 and pA2T-Mstn3.2, were constructed, linearized, and transfected into the bovine fetal fibroblasts through electroporation. 170 drug-resistant cell colonies were obtained in cell culture medium containing 600 microg/mL G418 and 50 nmol/L GCV. Targeted homologous integration occurred in colony No. 58 as identified by PCR, and the targeted colony was further confirmed by sequencing and Southern blotting. This suggested that one allele of myostatin was successfully mutagenized in bovine fetal fibroblasts.
Animals ; Base Sequence ; Cattle ; China ; Electroporation ; Fetus ; Fibroblasts ; cytology ; metabolism ; Gene Targeting ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; veterinary ; Myostatin ; genetics

Extracellular vesicles (EVs) refer to bilayer membrane transport vesicles secreted by cells. EVs can take macromolecules from cells and transfer them to receptor cells. Among these macromolecular substances, the most studied are microRNAs (miRNAs). miRNA is non-coding RNA involved in the regulation of gene expression. It has been confirmed that there are different non-coding RNAs in mammalian follicular fluid EVs. EVs carrying miRNA can act as an alternative mechanism for autocrine and paracrine, affecting follicular development. This paper systematically introduced the kinds, characteristics and methods of isolation and identification of EVs, focusing on the effects of EVs and miRNAs on follicular development, including early follicular development, oocyte maturation, follicular dominance and effects on granulosa cell function. At the same time, the authors prospected the future research of EVs and microRNAs in follicular fluid, and provided ideas and directions for the research and application of EVs and miRNA functions in follicular fluid.
Animals ; Extracellular Vesicles ; metabolism ; Female ; Follicular Fluid ; chemistry ; Granulosa Cells ; drug effects ; MicroRNAs ; pharmacology ; Oogenesis ; drug effects

Extracellular vesicles (EVs) are membrane-bound particles actively released by cells. In prokaryotes and eukaryotes, EVs are effective bridges for communication between cells. EVs carry biological macromolecules, including proteins, lipids and nucleic acid, which affects different physiological functions of parent cells and recipient cells. Among them, the microRNA carried by EVs is the most reported and plays an important role in physiological function of organisms. During the development of follicles, only a few follicles can fully develop and ovulate, whereas most of them undergo atresia at different stages of development. In the whole process of follicular development, the changes at each stage and the regulation mechanism of follicular atresia are not completely understood. In this paper, we introduced the types, characteristics, isolation methods and uses of EVs, and emphasized how microRNA carried by EVs in follicular fluid regulated follicular atresia from the aspects of different cytokines and hormones. Additionally, the application prospect of microRNA carried by EVs in follicular fluid in reproductive regulation and reproductive disease diagnosis was discussed. This paper is significant for studying the regulation of follicular development and the effective utilization of oocytes.
Animals ; Extracellular Vesicles/metabolism* ; Female ; Follicular Atresia ; Follicular Fluid ; MicroRNAs/metabolism* ; Oocytes

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.