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Background Heterogeneity of leukemia-initiating cells (LICs) is a major obstacle in acute myeloid leukemia (AML) therapy. Accumulated evidence indicates that the coexistence of multiple types of LICs with different pathogenicity in the same individual is a common feature in AML. However, the functional heterogeneity including the drug response of coexistent LICs remains unclear. Therefore, this study aimed to clarify the intra-heterogeneity in LICs that can help predict leukemia behavior and develop more effective treatments. Methods Spleen cells from the primary Setd2-/- -AML mouse were transplanted into C57BL/6 recipient mice to generate a transplantable model. Flow cytometry was used to analyze the immunophenotype of the leukemic mice. Whole-genome sequencing was conducted to detect secondary hits responsible for leukemia transformation. A serial transplantation assay was used to determine the self-renewal potential of Setd2-/- -AML cells. A limiting-dilution assay was performed to identify the LIC frequency in different subsets of leukemia cells. Bulk and single-cell RNA sequencing were performed to analyze the transcriptional heterogeneity of LICs. Small molecular inhibitor screening and in vivo drug treatment were employed to clarify the difference in drug response between the different subsets of LICs. Results In this study, we observed an aged Setd2-/- mouse developing AML with co-mutation of NrasG12S and BrafK520E . Further investigation identified two types of LICs residing in the c-Kit+ B220+ Mac-1- and c-Kit+ B220+ Mac-1+ subsets, respectively. In vivo transplantation assay disclosed the heterogeneity in differentiation between the coexistent LICs. Besides, an intrinsic doxorubicin-resistant transcriptional signature was uncovered in c-Kit+ B220+ Mac-1+ cells. Indeed, doxorubicin plus cytarabine (DA), the standard chemotherapeutic regimen used in AML treatment, could specifically kill c-Kit+ B220+ Mac-1- cells, but it hardly affected c-Kit+ B220+ Mac-1+ cells. Transcriptome analysis unveiled a higher activation of RAS downstream signaling pathways in c-Kit+ B220+ Mac-1+ cells than in c-Kit+ B220+ Mac-1- cells. Combined treatment with DA and RAS pathway inhibitors killed both c-Kit+ B220+ Mac-1- and c-Kit+ B220+ Mac-1+ cells and attenuated disease progression. Conclusions This study identified two cell subsets enriched for LICs in murine Setd2-/- -AML and disclosed the transcriptional and functional heterogeneity of LICs, revealing that the coexistence of different types of LICs in this model brings about diverse drug response.

The outbreak of coronavirus disease 2019 (COVID-19) is a global health emergency. Various omics results have been reported for COVID-19, but the molecular hallmarks of COVID-19, especially in those patients without comorbidities, have not been fully investigated. Here we collect blood samples from 231 COVID-19 patients, prefiltered to exclude those with selected comorbidities, yet with symptoms ranging from asymptomatic to critically ill. Using integrative analysis of genomic, transcriptomic, proteomic, metabolomic and lipidomic profiles, we report a trans-omics landscape for COVID-19. Our analyses find neutrophils heterogeneity between asymptomatic and critically ill patients. Meanwhile, neutrophils over-activation, arginine depletion and tryptophan metabolites accumulation correlate with T cell dysfunction in critical patients. Our multi-omics data and characterization of peripheral blood from COVID-19 patients may thus help provide clues regarding pathophysiology of and potential therapeutic strategies for COVID-19., COVID-19 is a critical public health threat, but molecular characterizations of patients’ immunity is still lacking. Here the authors collected blood from patients with various disease severity, and prefiltered to exclude selected comorbidity, to obtain genomic, transcriptomic, proteomic, metabolomic and lipidomic profiles to report a trans-omics landscape.

Since the Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2010, many techniques have been adopted for the discovery of missing proteins (MPs). Because of these efforts, only 1481 MPs remained as of July 2020; however, by relying only on technique optimization, researchers have reached a bottleneck in MP discovery. Protein expression is tissue- or cell-type-dependent. The tissues of the human testis and brain have been reported to harbor a large number of tissue-specific genes and proteins; however, few studies have been performed on human brain tissue or cells to identify MPs. Herein a metastatic cell line derived from brain cancer, D283 Med, was used to search for MPs. With a traditional and simple shotgun workflow to separate the peptides into 20 fractions, 12 MPs containing at least two unique non-nested peptides (amino acid length ≥9) were identified in this cell line with a protein false discovery rate of

Oxidative stress-induced dopaminergic neuronal loss and apoptosis play a crucial role in the pathogenesis of Parkinson’s disease (PD), and as a vital antioxidant protein, thioredoxin (Trx) exerts neuroprotection against PD. In this study, we investigated the effect of Schisanhenol (Sal), an active component from a traditional Chinese herb Schisandra rubriflora (Franch.), on MPP+-induced apoptosis and its association with thioredoxin-1 (Trx1) in SH-SY5Y cells. The protein levels of Trx1 and apoptosis-related proteins were detected by Western blot, the expression of Trx1 mRNA by real time qPCR, and apoptosis was detected by fluorescence microscopy and flow cytometry. Pretreatment with Sal (1 µM, 10 µM, and 50 µM) dose-dependently ameliorated MPP+-induced neuronal injury, confirmed by the improvement of the viability and morphological changes. Sal decreased the apoptosis rate of cells, suppressed the production of DNA ladder and sub-G1 peak, inhibited the Caspase-3 activity and the expression of apoptosis-related proteins. Sal enhanced the expression of Trx1 both in the protein and mRNA levels. However, the Trx1 inhibitor PX-12 suppressed the protective effects of Sal. In addition, Sal inhibited NF-κB translocation and activation. These results suggest that Sal has a protective effect against MPP+-induced apoptosis in SH-SY5Y cells via up-regulation of Trx1 expression and suppression of ASK1-P38-NF-κB pathway.

Immune and targeted therapy are becoming the first-line treatment for renal cell carcinoma (RCC). However, therapeutic outcomes are limited due to the low efficiency and side effect. Here, it is found that helicenes are able to exhibit an anticancer capability through changing the molecular structure from planar to nonplanar. Furthermore, the cytotoxicity in vitro and cancer inhibition ability of nonplanar helicenes increase with its aromatic rings' number. It is further demonstrated that benzo[4]helicenium shows the specific killing efficiency against the RCC cancer as compared to normal kidney cells. This is majorly originated from a more selective damage of benzo[4]helicenium for mitochondria and DNA in RCC cancer cells, not the normal kidney. The selective killing ability of benzo[4]helicenium makes it have potential to be used as a targeted drug for the precise treatment of RCC.

BackgroundLeukemic stem cell (LSC) is thought to be responsible for chronic myelogenous leukemia (CML) initiation and relapse. However, the inherent regulation of LSCs remains largely obscure. Herein, we integratedly analyzed miRNA and gene expression alterations in bone marrow (BM) Lin-Sca1+c-Kit+ cells (LSKs) of a tet-off inducible CML mouse model, Scl/tTA-BCR/ABL (BA).MethodsScl/tTA and TRE-BA transgenic mice were crossed in the presence of doxycycline to get double transgenic mice. Both miRNA and mRNA expression profiles were generated from BM LSKs at 0 and 3 weeks after doxycycline withdrawal. The target genes of differentially expressed miRNAs were predicted, followed by the miRNA-mRNA network construction. In vitro and in vivo experiments were further performed to elucidate their regulation and function in CML progression.ResultsAs a result of the integrated analysis and experimental validation, an anti-apoptotic pathway emerged from the fog. miR-142a was identified to be downregulated by enhanced ERK-phosphorylation in BA-harboring cells, thereby relieving its repression on Ciapin1, an apoptosis inhibitor. Moreover, miR-142a overexpression could partially rescue the abnormal anti-apoptotic phenotype and attenuate CML progression.ConclusionTaken together, this study explored the miRNA-mRNA regulatory networks in murine CML LSKs and demonstrated that ERK-miR-142a-Ciapin1 axis played an essential role in CML pathogenesis.

SummarySystem-wide molecular characteristics of COVID-19, especially in those patients without comorbidities, have not been fully investigated. We compared extensive molecular profiles of blood samples from 231 COVID-19 patients, ranging from asymptomatic to critically ill, importantly excluding those with any comorbidities. Amongst the major findings, asymptomatic patients were characterized by highly activated anti-virus interferon, T/natural killer (NK) cell activation, and transcriptional upregulation of inflammatory cytokine mRNAs. However, given very abundant RNA binding proteins (RBPs), these cytokine mRNAs could be effectively destabilized hence preserving normal cytokine levels. In contrast, in critically ill patients, cytokine storm due to RBPs inhibition and tryptophan metabolites accumulation contributed to T/NK cell dysfunction. A machine-learning model was constructed which accurately stratified the COVID-19 severities based on their multi-omics features. Overall, our analysis provides insights into COVID-19 pathogenesis and identifies targets for intervening in treatment.

It is acknowledged that the bisecting N-acetylglucosamine (GlcNAc) structure, a GlcNAc linked to the core β-mannose residue via a β1,4 linkage, represents a special type of N-glycosylated modification and has been reported to be involved in various biological processes, such as cell adhesion and fetal development. Clark et al. has found that the majority of N-glycans in human trophoblasts bearing a bisecting GlcNAc. This type of glycan has been reported to help trophoblasts get resistant to natural killer (NK) cell-mediated cytotoxicity, and this would provide a possible explanation for the question how could the mother nourish a fetus within herself without rejection. Herein, we hypothesized that human amniotic membrane which is the last barrier for the fetus may also express bisecting type glycans to protect the fetus. To test this hypothesis, glycomic analysis of human amniotic membrane was performed, and the bisecting N-glycans with high abundance were detected. In addition, we re-analyzed our proteomic data with high fractionation and amino acid sequence coverage from human amniotic membrane, which had been released for the exploration of human missing proteins. The presence of bisecting GlcNAc peptides was revealed and confirmed. A total of 41 glycoproteins with 43 glycopeptides were found to possess a bisecting GlcNAc, 25 of which are for the first time to be reported to have this type of modification. These results provide the profiling of bisecting GlcNAc modification in human amniotic membrane and benefit to the function studies of glycoproteins with bisecting GlcNAc modification and the function studies in immune suppression of human placenta. The mass spectrometry placenta data are available via ProteomeXchange (PXD010630).

Puberty is initiated by increased pulsatile gonadotropin-releasing hormone (GnRH) release from the hypothalamus. Epigenetic repression is thought to play a crucial role in the initiation of puberty, although the existence of analogous changes in methylation patterns across species is unclear. We analysed mRNA expression of DNA methyltransferases (DNMTs) and methyl-binding proteins (MBPs) in goats and rats by quantitative real-time PCR (qRT-PCR). DNA methylation profiles of hypothalamic were determined at the pre-pubertal and pubertal stages by bisulphite sequencing. In this study, expression of DNMTs and MBPs mRNA showed different patterns in goats and rats. Global methylation variation was low in goats and rats, and the profile remained stable during puberty. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis revealed the involvement of 62 pathways in puberty in goats and rats including reproduction, type I diabetes mellitus and GnRH signalling pathways and found that Edn3, PTPRN2 and GRID1 showed different methylation patterns during puberty in goats and rats and similar variation patterns for Edn3 and PTPRN2 were showed. These indicated that Edn3 and PTPRN2 would play a role in the timing of puberty. This study provides evidence of the epigenetic control of puberty.

This paper proposed a new friction mechanical model of joint interface to better describe the tangential stiffness softening. A new probability density function is obtained by analyzing the actual two-dimensional pressure distribution of bolted joint. Then, an improved tangential stiffness model and the corresponding extraction method of the model parameters based on FEA method was introduced, and the effectiveness of the model was verified by the designed slip test. Finally, this paper discussed the influence of various parameters on tangential stiffness, and explained the stiffness nonlinear softening from the evolution of sliding area. Compared with the existing model, the calculation accuracy is improved by 18.2%. The research content has a better application prospect in dynamic analysis of connection structures.

SIN3 transcription regulator family member A (SIN3A) is the central scaffold protein of the SIN3/HDAC (histone deacetylase) transcriptional repressor complex. We previously found that SIN3A participates in the mouse pre-implantation development by finetuning HDAC1 expression. However, it remains unresolved whether this functional significance of SIN3A is conserved in other mammals. The objective of this work was thus to characterise the expression profiles and the functional role of SIN3A in pre-implantation development using non-rodent animal models. RNA sequencing results show that a large amount of SIN3A mRNA is present in oocytes and early embryos before embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in all species examined. Interestingly, immunofluorescence data show that SIN3A protein level peaks at the 4-cell stage in pigs compared with the morula stage in cattle, suggesting a differential role of SIN3A among species. To explore the function of SIN3A in early embryonic development, we used a short interfering (si)RNA-mediated knockdown approach in porcine parthenogenetic activated (PA) embryos. Immunocytochemical analysis showed that SIN3A levels were diminished ∼80% compared with nonspecific siRNA (NC) injected control (n=3). To monitor the developmental potential of embryo depleted of SIN3A, we injected SIN3A-siRNA into MII stage oocytes, followed by parthenogenetic activation, and percent cleavage and blastocyst formation were recorded. We found that SIN3A knockdown (KD) did not affect the cleavage rate (NC vs. KD, 83.63±3.63% vs. 80.08±4.66%, n=5), but significantly reduced blastocyst rate compared with the NC group (NC vs. KD, 36.64±4.28% vs. 6.33±3.12%, n=5). Specifically, SIN3A depletion in early embryos causes developmental arrest at 2-cell stage in pigs but does not affect early embryonic development in bovines. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause 2-cell block but leads to a reduced blastocyst rate, suggesting that the effect of SIN3A depletion on porcine early embryos is independent of HDAC1. RNA-Seq analysis was used to compare the global transcript content between NC and KD 2-cell embryos. A total of 23 genes (14 upregulated and 9 downregulated) had undergone significant changes. Interestingly, cyclin B1 (CCNB1) ranked second among downregulated genes. To test whether knockdown of CCNB1 would display a similar phenotype in porcine early embryos, we injected CCNB1-siRNA into pronuclear stage. CCNB1 KD resulted in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 mRNA into SIN3A-depleted embryos could partly rescue embryonic development. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, probably involving the regulation of CCNB1 expression. This work was funded by National Natural Science Foundation of China, the Anhui Provincial Natural Science Foundation and China Postdoctoral Science Foundation.

Disabled homolog 2 (DAB2) is frequently deleted or epigenetically silenced in many human cancer cells. Therefore, DAB2 has always been regarded as a tumor suppressor gene. However, the role of DAB2 in tumor progression and metastasis remains unclear. In this study, DAB2 expression was upregulated along with human prostate cancer (PCa) progression. DAB2 overexpression or knockdown effects in LNCaP and PC3 cell lines were verified to address the biological functions of DAB2 in PCa progression and metastasis. LNCaP and PC3 cell lines were generated from human PCa cells with low and high metastatic potentials, respectively. The results showed that DAB2 shRNA knockdown can inhibit the migratory and invasive abilities of PC3 cells, as well as the tumorigenicity, whereas DAB2 overexpression enhanced LNCaP cell migration and invasion. Further investigation showed that DAB2 regulated the cell migration associated genes in PC3 cells, and the differential DAB2 expression between LNCaP and PC3 cells was partly regulated by histone 4 acetylation. Therefore, DAB2 may play an important role in PCa progression and metastasis.

Summary The objective of this study was to investigate the effects of epigallocatechin gallate (EGCG) on fat metabolism and to establish the molecular mechanism of these effects in broilers. Seventy-two 28-day-old male Ross 308 broiler chickens were divided into three groups with different levels of EGCG supplementation for 4 weeks: normal control (NC) group, L-EGCG (a low-level supplement of EGCG, 40 mg/kg body weight daily) and H-EGCG (a high-level supplement of EGCG, 80 mg/kg body weight daily). After 4 weeks of oral administration, EGCG significantly reduced the level of abdominal fat deposition in broilers. The serum triglycerides and low-density lipoprotein cholesterol of chickens in H-EGCG group were also significantly decreased compared with the NC group, and the high-density lipoprotein cholesterol was notably increased at the same time. Moreover, the vital role of the liver and abdominal adipose tissue in lipid metabolism of poultry animals was examined through gene expression and enzyme activities related to fat anabolism and catabolism in these organs. Our data show that EGCG supplementation for 2 weeks significantly downregulated the expression of fatty acid synthesis and fat deposition-related genes, and upregulated the expression of genes involved in fatty acid β-oxidation and lipolysis genes. Simultaneously, the activities of hepatic fatty acid synthesis enzymes (fatty acid synthase and acetyl CoA carboxylase) were significantly decreased, and the activity of carnitine palmitoyl transferase-1 was notably elevated. The results suggest that EGCG could alleviate fat deposition in broilers through inhibiting fat anabolism and stimulating lipid catabolism in broilers.

It was reported that miR-145, which is significantly decreased in a variety of tumor cells, is associated with cell proliferation and metastasis. In this study, using bioinformatics analysis and in vitro assays, we identified DAB2 (Disabled homolog 2) as a downstream target gene of miR-145 during tumor metastasis process. DAB2 has been characterized as an important tumor suppressor, and is usually expressed at low levels in tumor cells. However, in this study, the relative high-level expression of DAB2 gene was observed in the highly invasive prostate cancer PC3 cells. Moreover, enforced expression of miR-145 could significantly down-regulate the expression level of DAB2 in PC3 cells, and inhibit the migration and invasion of PC3 cells. Notably, dysfunction of PC3 cells induced by miR-145 overexpression can be rescued by co-overexpression of DAB2. These results demonstrate that miR-145 regulates the migration and invasion of highly invasive prostate cancer cells through targeting DAB2 gene.

Human bactericidal/permeability-increasing protein (hBPI) is the only antibacterial peptide which acts against both gram-negative bacteria and neutralizes endotoxins in human polymorphonuclear neutrophils; therefore, hBPI is of great value in clinical applications. In the study, we constructed a hBPI expression vector (pBC1-Loxp-Neo-Loxp-hBPI) containing the full-length hBPI coding sequence which could be specifically expressed in the mammary gland. To validate the function of the vector, in vitro cultured C127 (mouse mammary Carcinoma Cells) were transfected with the vector, and the transgenic cell clones were selected to express hBPI by hormone induction. The mRNA and protein expression of hBPI showed that the constructed vector was effective and suitable for future application in producing mammary gland bioreactor. Then, female and male goat fibroblasts were transfected with the vector, and two male and two female transgenic clonal cell lines were obtained. Using the transgenic cell lines as nuclear donors for somatic cell nuclear transfer, the reconstructed goat embryos produced from all four clones could develop to blastocysts in vitro. In conclusion, we constructed and validated an efficient mammary gland-specific hBPI expression vector, pBC1-Loxp-Neo-Loxp-hBPI, and transgenic hBPI goat embryos were successfully produced, laying foundations for future production of recombinant hBPI in goat mammary gland.

Cumulus cells (CCs) are of great importance in oocyte development and maturation in many species, but detailed influence of CCs has not been extensively examined, especially on rabbit. The present study was designed to investigate the effects of CCs and the elongation of in vitro maturation (IVM) time on rabbit oocyte nuclear and ooplasmic maturation and survival. Cumulus oocyte complexes (COCs) and naked oocytes (NOs) were recovered directly from rabbits super-ovulated with eCG. Corona-enclosed oocytes (COs) and denuded oocytes (DOs) were obtained from COCs after removing a part or whole of CCs. The oocytes were cultured in the following seven groups. (i) Cumulus cell enclosed oocytes (CEOs) were cultured alone (CEOs); (ii) COs were cultured alone (COs); (iii) DOs were cultured alone (DOs); (iv) NOs were cultured alone; (v) DOs were co-cultured with COCs [DOs(COCs)]; (vi) DOs were co-cultured with CCs [DOs(CCs)]; (vii) NOs were co-cultured with CCs [NOs(CCs)]. After the oocytes were cultured for 24 and 30 h, the nuclear maturation was evaluated by first polar body (PB1) extrusion while the ooplasmic maturation was evaluated by the cleavage rate after parthenogenetic activation. The results showed that the nuclear maturation rate of CEOs, COs, DOs(COCs) and DOs(CCs) after 24 h incubation were significantly different from each other (por = 0.05), the rate of DOs(CCs) was similar to that of DOs (por = 0.05). The cleavage rates in the first two groups were significantly higher than those of the others (p0.05). For oocytes cultured for 30 h, the nuclear maturation rates were significantly different for each culture model (p0.05). The cleavage rates in first two groups were significantly higher than those of others (p0.05). Both the nuclear and cleavage rates significantly increased when the culture time of DOs(COCs) was prolonged from 24 to 30 h. DOs(CCs) nuclear maturation was significantly improved when the culture time was prolonged from 24 to 30 h, but the ooplasmic maturation was not. Few NOs incubated with or without CCs accomplished nuclear maturation (approximately 2% both), even when the culture time was prolonged from 24 to 30 h. The oocyte degeneration rates were significantly different for each culture model after both 24 and 30 h incubation (por = 0.05). There was no significant difference in oocyte degeneration in the same groups between 24 and 30 h incubation (p0.05). The results suggest that rabbit CCs affect oocyte nuclear and ooplasmic maturation, and their survival. The prolongation of the culture time of rabbit oocyte from 24 to 30 h improves the nuclear and ooplasmic maturation differently in the present system. Rabbit oocytes free of CCs, especially NOs, show weak meiotic resumption potential and compromised viability, which cannot be improved by co-culture with dispersed CCs. The degeneration mostly happens at early time of IVM.

Vandetanib, a multikinase inhibitor, is a target of drug treatments for non-small cell lung cancer (NSCLC). However, phase II and III clinical trials have not conclusively demonstrated the curative effects of vandetanib for NSCLC and the reasons for this are unknown. In the present study, we use the NSCLC cell line Calu-6 as a model to determine the cellular and biological effects of vandetanib. Our results demonstrate that vandetanib impairs Calu-6 cell migration and invasion. We find that vandetanib can directly inhibit RET activity, which influences the Rho-JNK pathway. Overexpression of a constitutively active Rho GTPase antagonizes the inhibitory effects of vandetanib on Calu-6 cells invasion and JNK pathway activation. In addition, vandetanib induces autophagy by increasing the level of reactive oxygen species (ROS) in Calu-6 cells and blockade of autophagy or ROS effectively enhances the cell death effect of vandetanib. In this study, we find vandetanib is of a double effect in some NSCLC cells, presenting new possibilities for the pharmacological treatment of NSCLC and introducing a novel role for vandetanib in treatment options.

The tumor microenvironment, including ischemia, has been increasingly recognized as a critical factor in the process of tumor development. Hypoxia and nutrient deficiency resulting from ischemia widely exist in solid tumors. Recent studies have shown that hypoxia and nutrient deficiency contribute to chemoresistance by inducing autophagy, but the underlying mechanism remains unknown. This study aimed to explore the role of autophagy induced by low glucose and hypoxia (LH) in the chemoresistance of hepatocellular carcinoma cells. Our results demonstrated that LH induced autophagy and downregulated Bad and Bim in hepatocellular carcinoma cells. The inhibition of autophagy reversed the reduction of these pro-apoptotic factors during the LH treatment. Furthermore, Bad and Bim were also significantly downregulated by autophagy during the process that LH promoted the chemoresistance of hepatocellular carcinoma cells. In addition, RNAi or the overexpression of Bad and Bim can significantly reduce or increase chemotherapy-induced cell death, respectively. Taken together, these data indicate that the downregulation of Bad and Bim plays a significant role in the autophagy-induced chemoresistance of hepatocellular carcinoma cells.

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.

The maturation and developmental potential on cumulus-cell-free oocytes is of great importance theoretically and practically. The present study was to investigate the effects of l-ascorbic acid, alpha-tocopherol and co-culture on in vitro developmental potential of porcine denuded oocytes (DOs). Porcine DOs were cultured in maturation medium supplemented with vitamin C (0, 50, 100, 250, 500, 750 microM) and vitamin E (0, 10, 20, 50, 100, 250 microm), respectively. And they were also co-cultured with dispersed cumulus cells (group CCscoculture), intact cumulus cells oocyte complexes (COCs) (group COCscoculture), and COCs whose oocytes were removed (group OOXcoculture), respectively. After 44 h incubation, the maturation rates, cleavage rates and blastocyst rates after parthenogenetic activation in three experiments mentioned above were collected and analysed, respectively. L-Ascorbic acid promoted porcine DOs in vitro maturation and blastocyt development after parthenogenetic activation while alpha-tocopherol did not increase the in vitro maturation rates, but improved the blastocyst rate. None of the three co-culture manner promoted the in vitro maturation and the cleavage of porcine DOs after parthenogenetic activation, but all the co-culture manners improved the blastocyst rates. Both Vitamin C and E enhance the in vitro developmental potential of porcine DOs. Co-culture increases the developmental potential of porcine DOs.

The Mof gene (males absent on the first) is crucial to X-chromosome dosage compensation in the fly. It acts specifically to catalyse acetylation of histone H4 lysine 16 (H4K16ac) as one histone acetyltransferase of the MYST family, playing essential roles during mammalian development. However, little is known about Mof gene in pigs. The present study was designed to explore effects of Mof on pre-implantation development of pig parthenogenetic embryos obtained as reported by Cao et al. (2012 Zygote 20, 229–236). Immunofluorescent staining was performed to examine protein expression level of porcine Mof (pMof) and H4K16ac, and fluorescent intensity was measured by Image J software (NIH, Bethesda, MD, USA). Data are presented as mean ± standard error, and statistical analyses of the fluorescent intensity value, embryo development rate, and quality were performed using ANOVA with SPSS software (version 15.0, SPSS Inc., Chicago, IL, USA), and P-value

Summary Setd2 is known as a histone-H3K36-specific methyltransferase. However, its role in physiological function remains unclear. In this study, we show that Setd2 mainly regulates differentiation of murine embryonic stem cells (mESCs) toward primitive endoderm. Furthermore, we show that downregulated endoderm-related genes in Setd2 −/− mESCs are associated with an aberrantly low level of Erk activity and that enforced expression of Fgfr3 can rescue the defective Erk pathway in Setd2 −/− mESCs. Interestingly, the transcriptional initiation of Fgfr3 is directly regulated through histone H3K36me3 modification in its distal promoter region by Setd2. These results indicate that Setd2 controls the primitive endoderm differentiation of mESCs by regulating the Fgfr3-Erk signaling.

The present study aimed to investigate the effects of epigallocatechin-3-gallate (EGCG) on the in vitro development of porcine oocytes, parthenogenetic activation embryos (PA), and somatic cell nuclear transfer (SCNT) embryos. In Experiment 1, 0 (control), 10, 30, and 50 μg/mL EGCG were added to in vitro maturation (IVM) medium to explore the effect of EGCG on IVM of pig oocytes. The matured oocytes were then used to produce PA and SCNT embryos. Either for nuclear maturation of oocytes or for the rates of cleavage and blastocyst of PA and SCNT embryos, no significant difference was found among all groups. However, the total cell number per cloned blastocyst was significantly lower in blastocysts derived from oocytes matured in 50 μg/mL EGCG (Pin vitro development of pig cloned embryos.

The objective of the present study was to examine the feasibility of culturing porcine oocytes and embryos in vitro using the human exhaled lung air atmosphere. In Experiment 1, the effects of lung air atmosphere on nuclear maturation of prepubertal gilt oocytes and subsequent development in vitro of parthenogenetic-activated and somatic-cell-cloned embryos were explored. Abattoir-derived prepubertal gilt cumulus–oocyte complexes (COC) were matured in TCM-199 supplemented with 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, 10 ng mL–1 of epidermal growth factor, and 10% porcine follicular fluid (pFF) for 40 to 44 h at 38.5°C, 100% humidity, and 5% CO2+20% O2 (high oxygen tension) or human exhaled air encapsulated in plastic, airtight bags (lung air) or 5% CO2+7% O2 (low oxygen tension) in the incubator. Nuclear maturation was evaluated by the presence of the 1st polar body. For parthenogenetic activation, denuded oocytes with the 1st polar body were selected and stimulated with a single 1.6-kV/cm, 100-μs direct current pulse followed by culture in porcine zygote medium-3. For NT, denuded metaphase II oocytes were enucleated, and then the donor cell was directly injected into the perivitelline space. After NT, reconstructed couplets were fused and activated electrically followed by treatment in 7.5 μg mL–1 of cytochalasin B and 10 μg mL–1 of cycloheximide for 4 to 6 h before culture in porcine zygote medium-3. We found no significant difference among groups in terms of nuclear maturation rate (66.5% v. 60.2%, 63.2%), cleavage rate (94.8% v. 94.2%, 85.2%), blastocyst formation rate (39.5% v. 40.3%, 32.5%), and total cell number (37 v. 38, 32). Moreover, as for porcine cloned embryo, no significant difference between the lung-air and high-oxygen (20% O2) groups was observed in the cleavage rate (88.3% v. 80.3%), blastocyst formation rate (7.3% v. 10.7%), and total cell number (34 v. 36). The above results indicated that porcine oocytes can be matured in vitro safely and efficiently using the human exhaled lung air atmosphere. In Experiment 2, in vitro developmental competence of porcine zona-free parthenogenetically activated embryos cultured in a lung air, low oxygen (5% O2), or high oxygen (20% O2) tension gas environment was studied. We found no obvious difference among the 3 groups regarding the rates of cleavage (83.0%, 83.6%, 82.8%), but blastocyst formation rate (26.8% v. 48.6%, 48.2%) and total cell number (23 v. 34, 29) in lung air were lower than those in the rest of the groups (P

Recently, it has been reported that a new activation method, brief exposure to cycloheximide before electrical activation, could increase development rates and reduce cell death. In our study, we allocated reconstructed SCNT embryos into 3 groups: electrical activation followed by exposure to cycloheximide (10 μg mL–1) for 4 h (ELE+CHX); exposure to cycloheximide (10 μg mL–1) for 10 min followed by electrical activation (CHX+ELE); and electrical pulse treatment alone (ELE). We found the CHX+ELE (10 min) group had a similar blastocyst formation rate and total blastocyst number with the ELE+CHX (4 h) group, and both groups could increase in vitro development compared with the ELE group. Trichostatin A (TSA), an inhibitor of histone deacetylase, has been reported to potentially enhance cloning efficiency. We examine the effect of TSA on nuclear transfer embryos produced by the CHX+ELE activation method. The reconstructed embryos were treated with 50 nM TSA for 0 and 36 h. We found that 50 nM TSA for 36 h after activation had an increased blastocyst rate compared with the control (15.35 v. 8.84%; P

The objectives of the present research were to investigate whether embryo culture media have preferences for oxygen tension, to explore the feasibility of using physical lung air to support the in vitro development of mouse embryos, and to evaluate the effect of well of the well (WOW) culture on in vitro preimplantational development of mouse embryos. The results are as follows. First, cleavage rate and blastocyst rate were not significantly different between medium CZB and mKSOM regardless of using 3 gas phases: 4% CO2 + 16% O2 + 78% N2 + 2% H2O (lung air), 5% CO2 + 5% O2 + 90% N2 (5% O2, low oxygen), and 5% CO2 + 95% air (20% O2, high oxygen; P > 0.05), but mean total cell numbers per blastocyst cultured in CZB medium were higher than those in mKSOM when the lung air was used (P 0.05). Fourth, both the blastocyst rate (74.6 ± 5.1%) and the mean total cell numbers per blastocyst (76 ± 2) cultured in the WOW system were obviously higher than those in the group culture system (38.2 ± 6.6% and 58 ± 4). Taken together, our results indicate that mKSOM and CZB have preferences for oxygen tension during in vitro culture of mouse embryos, the lung air was reaffirmed to be able to effectively support in vitro preimplantation development of mouse embryos, and the WOW culture system can apparently enhance in vitro developmental competence and blastocyst quality of mouse embryos. L. YS, C. ZB: equal contribution; supported by NSFC (30700574), 863 (2008AA101003).

Accumulating evidence suggests that ghrelin plays an important role in female reproduction. The objective of the present study was to investigate the effect of ghrelin on pre-implantation development of porcine in vitro-fertilized (IVF) and parthenogenetic embryos. Cumulus–oocyte complexes were matured for 44 h in BSA-free NCSU23 supplemented with 10 ng mL-1 epidermal growth factor, 10 ng mL-1 leptin, 0.57 mM cysteine, 10 IU mL-1 pregnant mare serum gonadotropin, and 10 IU mL-1 hCG. After removal of the cumulus cells, some oocytes were fertilized with fresh boar semen (1 � 105 sperm mL-1) in modified Tween medium B with milk powder (Abeydeera and Day 1997 Theriogenology 48, 537–544) and some oocytes were activated by a single, 100-�s, direct current pulse of 1.4 kV cm-1. Presumptive zygotes (Experiment 1) and parthenogenetic oocytes (Experiment 2) were subsequently cultured in PZM3 (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) supplemented with ghrelin at 0 (control), 0.5, 5, 50, and 500 ng mL-1 (ghrelin 0.5, 5, 50, 500 groups, respectively) under 5% CO2, 5% O2, 90% N2 and 100% humidity at 39.0�C. Cleavage and blastocyst rates were assessed on Days 2 and 6 (Day 0: the day IVF and activation were conducted). The total cell number in blastocysts was determined by Hoechst 33342 staining on Day 6. All data were analyzed by using SPSS (13.0) with one-way ANOVA. All experiments were done at least 4 times. In Experiment 1, the rate of blasotcyst formation in IVF embryos was significantly (P < 0.05) increased in the ghrelin 500 group compared with that in the control group (26.1 � 1.8 vs. 12.4 � 6.0%, mean � SEM). Furthermore, increased total cell numbers (P < 0.05) were observed in the ghrelin 50 and 500 groups compared with that in the control group (63 � 6.6 and 64 � 5.5 vs. 42 � 6.6). In Experiment 2, we found that the blastocyst rate of parthenogenetic embryos was significantly (P < 0.05) higher in the ghrelin 5 and 500 groups than in the others (24.6 � 4.7 and 25.0 � 3.3 vs. 13.3 � 2.7, 14.9 � 2.4, 18.1 � 2.3% in the control and ghrelin 0.5 and 50 groups, respectively; P < 0.05). The total cell number per blastocyst was significantly increased in the ghrelin 50 group compared with that of the control group (85 � 10.2 vs. 56 � 8.0, P < 0.05). The maximum total cell number in the ghrelin treatment groups of parthenogenetic embryos was higher than in the control group (82, 93, 102, 100 in the ghrelin 0.5, 5, 50, 500 groups, respectively, vs. 69; P < 0.05). We also found that more embryos were developed to the morula stage and fewer embryos died early at the 2- to 4-cell stage in the ghrelin treatment groups than in the control group (data not shown) in both Experiments 1 and 2. The results suggest that supplementation with ghrelin in the embryo culture medium could enhance the pre-implantation development of porcine IVF and parthenogenetic embryos. This study was funded by the Natural Scientific Foundation of Beijing (5030001).