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Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe disease with substantial economic consequences for the swine industry. The DEAD-box helicase 3 (DDX3X) is an RNA helicase that plays a crucial role in regulating RNA metabolism, immunological response, and even RNA virus infection. However, it is unclear whether it contributes to PRRSV infection. Recent studies have found that the expression of DDX3X considerably increases in Marc-145 cells when infected with live PRRSV strains Ch-1R and SD16; however, it was observed that inactivated viruses did not lead to any changes. By using the RK-33 inhibitor or DDX3X-specific siRNAs to reduce DDX3X expression, there was a significant decrease in the production of PRRSV progenies. In contrast, the overexpression of DDX3X in host cells substantially increased the proliferation of PRRSV. A combination of transcriptomics and metabolomics investigations revealed that in PRRSV-infected cells, DDX3X gene silencing severely affected biological processes such as ferroptosis, the FoxO signalling pathway, and glutathione metabolism. The subsequent transmission electron microscopy (TEM) imaging displayed the typical ferroptosis features in PRRSV-infected cells, such as mitochondrial shrinkage, reduction or disappearance of mitochondrial cristae, and cytoplasmic membrane rupture. Conversely, the mitochondrial morphology was unchanged in DDX3X-inhibited cells. Furthermore, silencing of the DDX3X gene changed the expression of ferroptosis-related genes and inhibited the virus proliferation, while the drug-induced ferroptosis inversely promoted PRRSV replication. In summary, these results present an updated perspective of how PRRSV infection uses DDX3X for self-replication, potentially leading to ferroptosis via various mechanisms that promote PRRSV replication.

With the continuous improvement of shale oil and gas recovery technologies and achievements, a large amount of geological information and data have been accumulated for the description of shale reservoirs, and it has become possible to use machine learning methods for “sweet spots” prediction in shale oil and gas areas. Taking the Duvernay shale oil and gas field in Canada as an example, this paper attempts to build recoverable shale oil and gas reserve prediction models using machine learning methods and geological and development big data, to predict the distribution of recoverable shale oil and gas reserves and provide a basis for well location deployment and engineering modifications. The research results of the machine learning model in this study are as follows: ① Three machine learning methods were applied to build a prediction model and random forest showed the best performance. The R2 values of the built recoverable shale oil and gas reserves prediction models are 0.7894 and 0.8210, respectively, with an accuracy that meets the requirements of production applications; ② The geological main controlling factors for recoverable shale oil and gas reserves in this area are organic matter maturity and total organic carbon (TOC), followed by porosity and effective thickness; the main controlling factor for engineering modifications is the total proppant volume, followed by total stages and horizontal lateral length; ③ The abundance of recoverable shale oil and gas reserves in the central part of the study area is predicted to be relatively high, which makes it a favorable area for future well location deployment.

BackgroundLaryngeal squamous cell carcinoma (LSCC) is one of the most frequent head and neck cancers worldwide. Long non-coding RNAs (lncRNAs) play a critical role in tumorigenesis. However, the clinical significance of lncRNAs in LSCC remains largely unknown.MethodsIn this study, transcriptome sequencing was performed on 107 LSCC and paired adjacent normal mucosa (ANM) tissues. Furthermore, RNA expression and clinical data of 111 LSCC samples were obtained from The Cancer Genome Atlas (TCGA) database. Bioinformatics analysis were performed to construct a model for predicting the overall survival (OS) of LSCC patients. Moreover, we investigated the roles of lncRNAs in LSCC cells through loss-of-function experiments.ResultsA seven-lncRNAs panel including ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and LINC02893 was identified. The Kaplan–Meier analysis demonstrated that the seven-lncRNAs panel was significantly associated with OS (HR:6.21 [3.27-11.81], p-value

For the automatic target recognition (ATR) based on synthetic aperture radar (SAR) images, enough training data are required to effectively characterize target features and obtain good recognition performance. However, in practical applications, it is difficult to collect sufficient training data. To tackle the limitation, a novel end-to-end expansion method, called conditional Wasserstein deep convolutional generative adversarial network with gradient penalty (CWDCGAN), is proposed to achieve SAR image expansion with specified category. To be specific, the CWDCGAN innovatively designed a generative adversarial network architecture based on convolutional and deconvolution networks to improve the quality of generated images. At the same time, conditional information is introduced to control the categories of generated images, and Wasserstein distance and gradient penalty are used to modify the loss function, which makes the network training more stable. Besides, feature extraction and classifier design in a typical ATR system often rely heavily on subjective expert knowledge, which seriously affects its generalization performance. Therefore, a joint recognition method of Resnet18 and support vector machine (Renset18-SVM) is adopted to improve the generalization capacity and the recognition performance. Experimental results with public measured data show that the CWDCGAN can generate higher quality SAR images, and by feeding expanded data to Renset18-SVM, the recognition accuracy is improved under different proportions of training samples.

Due to the complex shield construction characteristics and the complex effects of geological environment, it is difficult to control the direction of shield tunneling and to determine the reasonable tunneling parameters such as tunneling speed and so on. During the tunneling, shield tunneling machine may rise, shift and snake advance, which are not conducive to control tunnel axis. Aiming at the problem that it is difficult to accurately predict and correct the axis attitude deviation in the shield tunneling process, a prediction of axis attitude deviation and deviation correction method based on data driven during shield tunneling was put forward in this paper. Under certain geological conditions, the relationship between the attitude deviation of the construction axis and tunneling parameters during shield tunneling process with different tunneling mileages is established. Based on the tunneling historical data, the XGBoost prediction model is constructed, and the axis deviation variation is predicted and analyzed with the shield construction parameters. The multi-ring deviation correction parameter calculation model based on the fusion of geometric model and association rules is designed to obtain the internal correlation of the deviation amount, the number of deviation correction rings and the deviation correction parameters of each ring under the maximum deviation of different deviation correction sections, so as to realize the accurate prediction and deviation correction of shield axis deviation in the complex construction process. Under the verification of 155 ring data in a certain subway construction section, the method proposed in this paper has higher prediction accuracy, which is important for improving the safety and quality of shield tunneling. Results from the measured shield construction ring data verify the reasonability of the proposed axis attitude deviation prediction and multi-ring deviation correction method.

Because of the arid climate and fragile ecological environment in Xinjiang, China, land reclamation should be carried out after mining. The core of land reclamation is the water content of the surface covering soil. In this paper, the law of water distribution in reclamation reconstructed soil of nonmetal mines in Xinjiang was studied. In order to obtain the law of water distribution in reconstructed soil, we set up an observation system of the neutron probe and tensiometer. The neutron probe was used to monitor the soil water content. The tensiometers were used to obtain the matrix potential of soil for verifying the water distribution in reconstructed soil. Volumetric water content and matrix potential of reconstructed soil during 1-year period of management and irrigation were obtained by long-term monitoring. After one year’s field in situ test, 2424 sets of neutron probe data and 1368 sets of tensiometer data were obtained. By studying the above parameters, we summarized the law of water distribution in reconstructed soil of variable thickness and degree of compaction with nonmetallic waste rock filling. The results showed that covering soil was helpful to retain water content. Whether the soil was compacted or uncompacted, the soil water content at the depth of 10 cm was less than that at other depth of reconstructed soil because it was greatly affected by meteorological factors. The water content of reconstructed soil at 30 cm depth was greater than that at other depths. Under the influence of factors such as the thickness and compaction of the soil, the response time of soil water content and matrix potential to each irrigation infiltration was different. According to the characteristics of reclamation-vegetation such as alfalfa growth in Xinjiang, the thickness of surface reconstructed soil should be not less than 50 cm. Over time, soil that was compacted once was better for the vegetation. The research results could provide a reference for the land reclamation of nonmetallic mines in Xinjiang, China.

Background/Aims: Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. Methods: In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. Results: SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. Conclusion: SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1.

The production of a single gas well is influenced by many geological and completion factors. The aim of this paper is to build a production prediction model based on machine learning technique and identify the most important factor for production. Firstly, around 159 horizontal wells were collected, targeting the Duvernay Formation with detailed geological and completion records. Secondly, the key factors were selected using grey relation analysis and Pearson correlation. Then, three statistical models were built through multiple linear regression (MLR), support vector regression (SVR), gaussian process regression (GPR). The model inputs include fluid volume, proppant amount, cluster counts, stage counts, total horizontal lateral length, gas saturation, total organic carbon content, condensate-gas ratio. The model performance was assessed by root mean squared errors (RMSE) and R-squared value. Finally, sensitivity analysis was applied based on best performance model. The analysis shows following conclusions: (1) GPR model shows the best performance with the highest R-squared value and the lowest RMSE. In the testing set, the model shows a R-squared of 0.8 with a RMSE of 280.54 × 104 m3 in the prediction of cumulative gas production within 1st 6 producing months and gives a R-squared of 0.83 with a RMSE of 1884.3 t in the prediction of cumulative oil production within 1st 6 producing months (2) Sensitivity analysis based on GPR model indicates that condensate-gas ratio, fluid volume, and total organic carbon content are the most important features to cumulative oil production within 1st 6 producing months. Fluid volume, Stages, and total organic carbon content are the most significant factors to cumulative gas production within 1st 6 producing months. The analysis progress and results developed in this study will assist companies to build prediction models and figure out which factors control well performance.

Light emitting diode (LED) lamps are now an established lighting technology, which is becoming prevalent in all load sectors. However, LED lamps are non-linear electrical loads, and their impact on distribution system voltage quality must be evaluated. This paper provides a detailed analysis of time domain and frequency domain approaches for developing and evaluating models suitable for use in large scale steady-state harmonic power flow analysis of the low frequency (LF) emission of LED lamps. The considered approaches are illustrated using four general categories of LED lamps, which have been shown to cover the vast majority of LED lamps currently available on the market. The aim is an in-depth assessment of the ability of commonly applied models to represent the specific design characteristics of different categories of LED lamps. The accuracy of the models is quantitatively evaluated by means of laboratory tests, numerical simulations, and statistical analyses. This provides an example, for each LED lamp category, of comprehensive information about the overall accuracy that can be achieved in the general framework of large scale LF harmonic penetration studies, particularly in the assessment of voltage quality in low voltage networks and their future evolution.

Background: Colony morphology of embryonic stem (ES) cells contributes to the maintenance of undifferentiated ES cells. Small molecule 3,4-dihydropy-rimido[4,5-d]pyrimidine (SC1), an inhibitor of ERK1- and RasGAP-dependent signaling pathways, can maintain the compact colony morphology of ES cells. However, information on the influence of SC1 on cell morphological change remains lacking. Methods: In this study, mouse ES cells J1 and embryonic carcinoma (EC) cells F9 were cultured in SC1-containing medium to determine the effect of SC1 on cell morphology. Results: SC1 promotes a more compact morphology of J1 mouse ES cells and induces colony growth of F9 EC cells. Furthermore, the cell adhesion protein E-cadherin is a downstream target of SC1, and E-cadherin is critical for SC1-mediated colony growth of F9 EC cells. Conclusions: SC1 maintains and induces compact colony morphology of pluripotent cells, and its downstream target, E-cadherin, is involved in the colony phenotype of F9 EC cells. These results explored the potential role of SC1 in morphological change and gene expression in pluripotent cells.

Inorganic boron-based nanostructures have great potential for field emission (FE), flexible displays, superconductors, and energy storage because of their high melting point, low density, extreme hardness, and good chemical stability. Until now, most researchers have been focused on one-dimensional (1D) boron-based nanostructures (rare-earth boride (REB6) nanowires, boron nanowires, and nanotubes). Currently, two-dimensional (2D) borophene attracts most of the attention, due to its unique physical and chemical properties, which make it quite different from its corresponding bulk counterpart. Here, we offer a comprehensive review on the synthesis methods and optoelectronics properties of inorganic boron-based nanostructures, which are mainly concentrated on 1D rare-earth boride nanowires, boron monoelement nanowires, and nanotubes, as well as 2D borophene and borophane. This review paper is organized as follows. In Section I, the synthesis methods of inorganic boron-based nanostructures are systematically introduced. In Section II, we classify their optical and electrical transport properties (field emission, optical absorption, and photoconductive properties). In the last section, we evaluate the optoelectronic behaviors of the known inorganic boron-based nanostructures and propose their future applications.

Based on the morphology characteristics, low-dimensional (LD) nanostructures with high aspect ratio can be usually divided into nanowire, nanocone, nanotube, nanorod, nanoribbon, nanobelt and so on. Among numerous LD nanostructures, boron-based nanostructures attracted much interest in recent years because they have high melting-point, large electric and thermal conductivity, and low work function. Compared to traditional thermal emission, field emission (FE) has notable advantages, such as lower power dissipation, longer working life, room-temperature operation, higher brightness and faster switching speed. Most studies reveal they have lower turn-on and threshold fields as well as high current density, which are believed as ideal cold cathode nanomaterials. In this review, we will firstly introduce the growth methods of LD boron-based nanostructures (boron monoelement and rare-earth metal hexaboride). Then, we will discuss their FE properties and applications. At last, the conclusions and outlook will be summarized based on the above studies.

Backgroud/Aims: Embryonic stem cells (ES cells) have the capacity to propagate indefinitely, maintain pluripotency, and differentiate into any cell type under defined conditions. As a result, they are considered to be the best model system for research into early embryonic development. AICA ribonucleotide (AICAR) is an activator of AMP-activated protein kinase (AMPK) that is thought to affect ES cell function, but its role in ES cell fate decision is unclear. Methods: In this study, we performed microarray analysis to investigate AICAR downstream targets and further understand its effect on ES cells. Results: Our microarray data demonstrated that AICAR can significantly up-regulate pluripotency-associated genes and down-regulate differentiation-associated transcription factors. Although AICAR cannot maintain ES cell identity without LIF, it can antagonize the action of RA-induced differentiation. Using those differentially expressed genes identified, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the Database for Annotation, Visualization and Integrated Discovery (DAVID) online system. AICAR was not only shown to influence the AMPK pathway, but also act on other signaling pathways such as BMP, MAPK and TGF-β, to maintain the stemness of J1 ES cells. Furthermore, AICAR modulated ES cell epigenetic modification by altering the expression of epigenetic-associated proteins, including Dnmt3a, Dnmt3b, Smarca2, Mbd3, and Arid1a, or through regulating the transcription of long intervening non-coding RNA (lincRNA). Conclusion: Taken together, our work suggests that AICAR is capable of maintaining ES cell self-renewal and pluripotency, which could be useful in future medical treatment.

Embryonic stem cells (ESCs) have the ability to grow indefinitely and retain their pluripotency in culture, and this self-renewal capacity is governed by several crucial molecular pathways controlled by specific regulatory genes and epigenetic modifications. It is reported that multiple epigenetic regulators, such as miRNA and pluripotency factors, can be tightly integrated into molecular pathways and cooperate to maintain self-renewal of ESCs. However, mouse ESCs in serum-containing medium seem to be heterogeneous due to the self-activating differentiation signal of MEK/ERK. Thus, to seek for the crucial miRNA and key regulatory genes that establish ESC properties in MEK/ERK pathway, we performed microarray analysis and small RNA deep-sequencing of J1 mESCs treated with or without PD0325901 (PD), a well-known inhibitor of MEK/ERK signal pathway, followed by verification of western blot analysis and quantitative real-time PCR verification; we found that PD regulated the transcript expressions related to self-renewal and differentiation and antagonized the action of retinoic acid- (RA-) induced differentiation. Moreover, PD can significantly modulate the expressions of multiple miRNAs that have crucial functions in ESC development. Overall, our results demonstrate that PD could enhance ESC self-renewal capacity both by key regulatory genes and ES cell-specific miRNA, which in turn influences ESC self-renewal and cellular differentiation.

Understanding the mechanisms that regulate pluripotency of embryonic stem cells (ESCs) is important to ensure their safe clinical use. CHIR99021 (CHIR)-induced activation of Wnt/β-catenin signaling promotes self-renewal in mouse ESCs (mESCs). β-catenin functions individually or cooperates with transcription factors to activate stemness factors such as c-Myc, Esrrb, Pou5f1, and Nanog. However the relationship between the core pluripotent factor, Kruppel-like factor 4 (also known as GKLF or EZF) and Wnt/β-catenin signaling, remains ambiguous in J1 mESCs. DNA microarray analysis revealed that CHIR-treatment promoted pluripotency-maintaining transcription factors and repressed germ layer specification markers. CHIR also promoted genes related to the development of extracellular regions and the plasma membrane to maintain pluripotency of J1 mESCs. Among the CHIR-regulated genes, Klf4 has not been reported previously. We identified a novel cis element in the Klf4 gene that was activated by β-catenin in J1 mESCs. We determined that β-catenin interacted with this cis element, identifying Klf4 as a β-catenin target gene in this context. Moreover, several microRNAs that targeted the 3'-UTR of Klf4 mRNA were identified, with miR-7a being down-regulated by CHIR in a β-catenin-independent manner in J1 mESCs. These data collectively suggest that CHIR enhances Klf4 expression by repressing miR-7a expression or canonical Wnt pathway activation.

Retinoic acid (RA) is a vitamin A metabolite that is essential for early embryonic development and promotes stem cell neural lineage specification; however, little is known regarding the impact of RA on mRNA transcription and microRNA levels on embryonic stem cell differentiation. Here, we present mRNA microarray and microRNA high-output sequencing to clarify how RA regulates gene expression. Using mRNA microarray analysis, we showed that RA repressed pluripotency-associated genes while activating ectoderm markers in mouse embryonic stem cells (mESCs). Moreover, RA modulated the DNA methylation of mESCs by altering the expression of epigenetic-associated genes such as Dnmt3b and Dnmt3l. Furthermore, H3K4me2, a pluripotent histone modification, was repressed by RA stimulation. From microRNA sequence data, we identified two downregulated microRNAs, namely, miR-200b and miR-200c, which regulated the pluripotency of stem cells. We found that miR-200b or miR-200c deficiency suppressed the expression of pluripotent genes, including Oct4 and Nanog, and activated the expression of the ectodermal marker gene Nestin. These results demonstrate that retinoid induces mESCs to differentiate by regulating miR-200b/200c. Our findings provide the landscapes of mRNA and microRNA gene networks and indicate the crucial role of miR-200b/200c in the RA-induced differentiation of mESCs.

ES cells can propagate indefinitely, maintain self-renewal, and differentiate into almost any cell type of the body. These properties make them valuable in the research of embryonic development, regenerative medicine, and organ transplantation. MicroRNAs (miRNAs) are considered to have essential functions in the maintenance and differentiation of embryonic stem cells (ES cells). It was reported that, strong external stimuli, such as a transient low-pH and hypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells). AICA ribonucleotide (AICAR) is an AMP-activated protein kinase activator, which can let cells in the state of energy stress. We have demonstrated that AICAR can maintain the pluripotency of J1 mouse ES cells through modulating protein expression in our previous research, but its effects on ES cell miRNA expression remain unknown. In this study, we conducted small RNA high-throughput sequencing to investigate AICAR influence on J1 mouse ES cells by comparing the miRNA expression patterns of the AICAR-treated cells and those without treatment. The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development. Some differentially expressed miRNAs were selected and confirmed by real-time PCR. For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets. Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2. Lastly, Myc was significantly down-regulated after AICAR treatment; therefore, we predicted miRNAs that may target Myc and identified that AICAR induced up-regulation of miR-34a, 34b, and 34c can repress Myc expression in J1 mouse ES cells. Taken together, our study provide a new mechanism for AICAR in ES cells pluripotency maintenance and give insight for its usage in iPS cells generation.

Antibiotic selectable marker genes have been widely used to generate transgenic animals. Once transgenic animals have been obtained, the selectable marker is no longer necessary but raises public concerns regarding biological safety. The aim of this study was to prepare competent antibiotic selectable marker free transgenic cells for somatic cell nuclear transfer (SCNT). PhiC31 intergrase was used to insert a transgene cassette into a "safe harbor" in the bovine genome. Then, Cre recombinase was employed to excise the selectable marker under the monitoring of a fluorescent double reporter. By visually tracking the phenotypic switch from red to green fluorescence, antibiotic selectable marker free cells were easily detected and sorted by fluorescence-activated cell sorting. For safety, we used phiC31 mRNA and cell-permeant Cre protein in this study. When used as donor nuclei for SCNT, these safe harbor integrated marker-free transgenic cells supported a similar developmental competence of SCNT embryos compared with that of non-transgenic cells. After embryo transfer, antibiotic selectable marker free transgenic cattle were generated and anti-bacterial recombinant human β-defensin-3 in milk was detected during their lactation period. Thus, this approach offers a rapid and safe alternative to produce antibiotic selectable marker free transgenic farm animals, thereby making it a valuable tool to promote the healthy development and welfare of transgenic farm animals.

Maternal effect genes encode proteins that are produced during oogenesis and play an essential role during early embryogenesis. Genetic ablation of such genes in oocytes can result in female subfertility or infertility. Here we report a newly identified maternal effect gene, Nlrp2, which plays a role in early embryogenesis in the mouse. Nlrp2 mRNAs and their proteins (∼118 KDa) are expressed in oocytes and granulosa cells during folliculogenesis. The transcripts show a striking decline in early preimplantation embryos before zygotic genome activation, but the proteins remain present through to the blastocyst stage. Immunogold electron microscopy revealed that the NLRP2 protein is located in the cytoplasm, nucleus and close to nuclear pores in the oocytes, as well as in the surrounding granulosa cells. Using RNA interference, we knocked down Nlrp2 transcription specifically in mouse germinal vesicle oocytes. The knockdown oocytes could progress through the metaphase of meiosis I and emit the first polar body. However, the development of parthenogenetic embryos derived from Nlrp2 knockdown oocytes mainly blocked at the 2-cell stage. The maternal depletion of Nlrp2 in zygotes led to early embryonic arrest. In addition, overexpression of Nlrp2 in zygotes appears to lead to normal development, but increases blastomere apoptosis in blastocysts. These results provide the first evidence that Nlrp2 is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse.

Nanog is a pivotal transcription factor in embryonic stem (ES) cells and is essential for maintaining the pluripotency and self-renewal of ES cells. SUMOylation has been proved to regulate several stem cell markers' function, such as Oct4 and Sox2. Nanog is strictly regulated by Oct4/Sox2 heterodimer. However, the direct effects of SUMOylation on Nanog expression remain unclear. In this study, we reported that SUMOylation repressed Nanog expression. Depletion of Sumo1 or its conjugating enzyme Ubc9 increased the expression of Nanog, while high SUMOylation reduced its expression. Interestingly, we found that SUMOylation of Oct4 and Sox2 regulated Nanog in an opposing manner. SUMOylation of Oct4 enhanced Nanog expression, while SUMOylated Sox2 inhibited its expression. Moreover, SUMOylation of Oct4 by Pias2 or Sox2 by Pias3 impaired the interaction between Oct4 and Sox2. Taken together, these results indicate that SUMOylation has a negative effect on Nanog expression and provides new insights into the mechanism of SUMO modification involved in ES cells regulation.

The selection of good quality oocytes is crucial for in vitro fertilization and somatic cloning. Brilliant cresyl blue (BCB) staining has been used for selection of oocytes from several mammalian species. However, the effects of differential oocyte selection by BCB staining on nuclear reprogramming and in vivo development of SCNT embryos are not well understood. Immature compact cumulus-oocyte complexes (COCs) were divided into control (not exposed to BCB), BCB+ (blue cytoplasm) and BCB- (colorless cytoplasm) groups. We found that BCB+ oocytes yielded a significantly higher somatic cell nuclear transfer (SCNT) blastocyst rate and full term development rate of bovine SCNT embryos than the BCB- and control oocytes. BCB+ embryos (embryos developed from BCB+ oocytes) showed increased acetylation levels of histone H3 at K9 and K18 (AcH3K9, AcH3K18), and methylation levels of histone H3 at K4 (H3K4me2) than BCB- embryos (embryos developed from BCB- oocytes) at the two-cell stage. Furthermore, BCB+ embryos generated more total cells, trophectoderm (TE) cells, and inner cell mass (ICM) cells, and fewer apoptotic cells than BCB- embryos. The expression of SOX2, CDX2, and anti-apoptotic microRNA-21 were up-regulated in the BCB+ blastocysts compared with BCB- blastocysts, whereas the expression of pro-apoptotic gene Bax was down-regulated in BCB+ blastocysts. These results strongly suggest that BCB+ oocytes have a higher nuclear reprogramming capacity, and that BCB staining can be used to select developmentally competent oocytes for nuclear transfer.

Aberrant epigenetic nuclear reprogramming results in low somatic cloning efficiency. Altering epigenetic status by applying histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos. The present study was carried out to examine the effects of Oxamflatin, a novel HDACi, on the nuclear reprogramming and development of bovine SCNT embryos in vitro. We found that Oxamflatin modified the acetylation status on H3K9 and H3K18, increased total and inner cell mass (ICM) cell numbers and the ratio of ICM∶trophectoderm (TE) cells, reduced the rate of apoptosis in SCNT blastocysts, and significantly enhanced the development of bovine SCNT embryos in vitro. Furthermore, Oxamflatin treatment suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-XL and the pluripotency-related genes OCT4 and SOX2 in SCNT blastocysts. Additionally, the treatment also reduced the DNA methylation level of satellite I in SCNT blastocysts. In conclusion, Oxamflatin modifies epigenetic status and gene expression, increases blastocyst quality, and subsequently enhances the nuclear reprogramming and developmental potential of SCNT embryos.

Primordial germ cell 7 (PGC7)(Dppa3 or Stella) is a small inherently disordered protein that is mainly expressed in oocytes and plays a vital role in the regulation of DNA methylation reprogramming in imprinted loci through interaction with other proteins. Most of PGC7-deficient zygotes are blocked at two-cell stage with an increased tri-methylation at lysine 27 of histone H3 (H3K27me3) level in the nucleus. Our previous work has indicated that PGC7 interacts with yin-yang1 (YY1) that is essential for the recruitment of enhancer of zeste homolog 2 (EZH2)-containing Polycomb repressive complex 2 (PRC2) to H3K27me3 modification sites. Here, we found that the presence of PGC7 weakened the interaction between YY1 and PRC2 without disrupting the assembly of core subunits of the PRC2 complex. In addition, PGC7 promoted AKT to phosphorylate serine 21 of EZH2, resulting in inhibition of EZH2 activity and the dissociation of EZH2 from YY1, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 both promoted EZH2 to enter the pronuclei but without disturbing the subcellular localization of YY1 and caused an increase in the level of H3K27me3 in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos. In summary, PGC7 could affect zygotic genome activation during early embryonic development by regulating the level of H3K27me3 through regulation of PRC2 recruitment, EZH2 activity, and subcellular localization. [ABSTRACT FROM AUTHOR]