Derivation of Arbas Cashmere Goat Induced Pluripotent Stem Cells in LCDM with Trophectoderm Lineage Differentiation and Interspecies Chimeric Abilities.

The Arbas cashmere goat is a unique biological resource that plays a vital role in livestock husbandry in China. LCDM is a medium with special small molecules (consisting of human LIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride) for generation pluripotent stem cells (PSCs) with bidirectional developmental potential in mice, humans, pigs, and bovines. However, there is no report on whether LCDM can support for generation of PSCs with the same ability in Arbas cashmere goats. In this study, we applied LCDM to generate goat induced PSCs (giPSCs) from goat fetal fibroblasts (GFFs) by reprogramming. The derived giPSCs exhibited stem cell morphology, expressing pluripotent markers, and could differentiate into three germ layers. Moreover, the giPSCs differentiated into the trophectoderm lineage by spontaneous and directed differentiation in vitro. The giPSCs contributed to embryonic and extraembryonic tissue in preimplantation blastocysts and postimplantation chimeric embryos. RNA-sequencing analysis showed that the giPSCs were very close to goat embryos at the blastocyst stage and giPSCs have similar properties to typical extended PSCs (EPSCs). The establishment of giPSCs with LCDM provides a new way to generate PSCs from domestic animals and lays the foundation for basic and applied research in biology and agriculture.

Mixed-Lineage Leukemia 1 Inhibition Enhances the Differentiation Potential of Bovine Embryonic Stem Cells by Increasing H3K4 Mono-Methylation at Active Promoters.

Mixed-lineage leukemia 1 (MLL1) introduces 1-, 2- and 3-methylation into histone H3K4 through the evolutionarily conserved set domain. In this study, bovine embryonic stem cells (bESCs, known as bESCs-F7) were established from in vitro-fertilized (IVF) embryos via Wnt signaling inhibition; however, their contribution to the endoderm in vivo is limited. To improve the quality of bESCs, MM-102, an inhibitor of MLL1, was applied to the culture. The results showed that MLL1 inhibition along with GSK3 and MAP2K inhibition (3i) at the embryonic stage did not affect bESCs' establishment and pluripotency. MLL1 inhibition improved the pluripotency and differentiation potential of bESCs via the up-regulation of stem cell signaling pathways such as PI3K-Akt and WNT. MLL1 inhibition decreased H3K4me1 modification at the promoters and altered the distribution of DNA methylation in bESCs. In summary, MLL1 inhibition gives bESCs better pluripotency, and its application may provide high-quality pluripotent stem cells for domestic animals.

Dynamics and hydrodynamic efficiency of diving beetle while swimming.

Diving beetle, an excellent biological prototype for bionic underwater vehicles, can achieve forward swimming, backward swimming, and flexible cornering by swinging its two powerful hind legs. An in-depth study of the propulsion performance of them will contribute to the micro underwater vehicles. In this paper, the kinematic and dynamic parameters, and the hydrodynamic efficiency of the diving beetle are studied by analysis of swimming videos using Motion Capture Technology, combined with CFD simulations. The results show that the hind legs of diving beetle can achieve high propulsion force and low return resistance during one propulsion cycle at both forward and backward swimming modes. The propulsion efficiencies of forward and backward swimming are 0.47 and 0.30, respectively. Although the efficiency of backward swimming is lower, the diving beetle can reach a higher speed in a short time at this mode, which can help it avoid natural enemies. At backward swimming mode, there is a long period of passive swing of hind legs, larger drag exists at higher speed during the recovery stroke, which reduces the propulsion efficiency to a certain extent. Reasonable planning of the swing speed of the hind legs during the power stroke and the recovery stroke can obtain the highest propulsion efficiency of this propulsion method. This work will be useful for the development of a bionic propulsion system of micro underwater vehicle.

The adaptation of bovine embryonic stem cells to the changes of feeder layers.

Although the feeder-free culture system has been established, the microenvironment provided by the feeder cells still possesses a unique advantage in maintaining the long-term stability and the rapid proliferation of pluripotent stem cells (PSCs). The aim of this study is to discover the adaptive ability of PSCs upon changes of feeder layers. In this study, the morphology, pluripotent marker expression, differentiation ability of bovine embryonic stem cells (bESCs) cultured on low-density, or methanol fixed mouse embryonic fibroblasts were examined by immunofluorescent staining, Western blotting, real-time reverse transcription polymerase chain reaction, and RNA-seq. The results showed that the changes of feeder layers did not induce the rapid differentiation of bESCs, while resulting in the differentiation initiation and alteration of pluripotent state of bESCs. More importantly, the expression of endogenous growth factors and extracellular matrix were increased, and the expression of cell adhesion molecules was altered, which indicated that bESCs may compensate some functions of the feeder layers upon its changes. This study shows the PSCs have the self-adaptive ability responded to the feeder layer alteration.

Isolation, synthesis, identification of new process-related impurities in evocalcet.

Evocalcet is a calcimimetic for the treatment of secondary hyperparathyroidism. In the preparation of evocalcet, eleven process-related impurities were detected in the reaction solution of the last step at levels of 0.05%- 2.50% by a new HPLC method. Ten impurities were separated from the enriched mother liquor and synthesized directly. These impurities were characterized and identified by HRMS and NMR spectra and then coinjected with commercial products to confirm their retention times in HPLC. The possible formation pathways and synthetic methods of ten process-related impurities ware discussed in detail.

Observation and analysis of diving beetle movements while swimming.

The fast swimming speed, flexible cornering, and high propulsion efficiency of diving beetles are primarily achieved by their two powerful hind legs. Unlike other aquatic organisms, such as turtle, jellyfish, fish and frog et al., the diving beetle could complete retreating motion without turning around, and the turning radius is small for this kind of propulsion mode. However, most bionic vehicles have not contained these advantages, the study about this propulsion method is useful for the design of bionic robots. In this paper, the swimming videos of the diving beetle, including forwarding, turning and retreating, were captured by two synchronized high-speed cameras, and were analyzed via SIMI Motion. The analysis results revealed that the swimming speed initially increased quickly to a maximum at 60% of the power stroke, and then decreased. During the power stroke, the diving beetle stretched its tibias and tarsi, the bristles on both sides of which were shaped like paddles, to maximize the cross-sectional areas against the water to achieve the maximum thrust. During the recovery stroke, the diving beetle rotated its tarsi and folded the bristles to minimize the cross-sectional areas to reduce the drag force. For one turning motion (turn right about 90 degrees), it takes only one motion cycle for the diving beetle to complete it. During the retreating motion, the average acceleration was close to 9.8 m/s2 in the first 25 ms. Finally, based on the diving beetle's hind-leg movement pattern, a kinematic model was constructed, and according to this model and the motion data of the joint angles, the motion trajectories of the hind legs were obtained by using MATLAB. Since the advantages of this propulsion method, it may become a new bionic propulsion method, and the motion data and kinematic model of the hind legs will be helpful in the design of bionic underwater unmanned vehicles.

miR-375 Promotes Pancreatic Differentiation In Vitro by Affecting Different Target Genes at Different Stages.

Human embryonic stem cells (hESCs) possess the ability to differentiate into insulin-producing cells (IPCs), which can be used to treat type I diabetes. miR-375 is an essential transcriptional regulator in the development and maturation of the pancreas. In this study, we optimized a protocol to differentiate hESCs into IPCs and successfully obtained IPCs. Then, we performed overexpression and inhibition experiments of miR-375 on cells at different stages of differentiation and performed RNA-seq. The results showed that the expression of miR-375 fluctuated during hESC differentiation and was affected by miR-375 mimics and inhibitors. miR-375 influences global gene expression and the target genes of miR-375. The overexpression of miR-375 can cause changes in multiple signaling pathways during pancreatic development. miR-375 is a major participant in the differentiation of pancreatic ß-cells through different target genes at different stages. This study provides new ideas for further investigation of how microRNAs affect cell fate and gene transcription.

MLL1 combined with GSK3 and MAP2K inhibition improves the development of in vitro-fertilized embryos.

The MM-102 compound prevents the interaction between mixed lineage leukemia 1 (MLL1) and WD Trp-Asp repeat domain 5 (WDR5) and results in the inhibition of MLL1 H3K4 histone methyltransferase (HMT) activity. The inhibition of the FGFR signaling pathway and activation of the WNT pathway by small molecule inhibitors (known as 2i) improves blastocyst development. However, studies on the effects of MLL1 combined with GSK3 and MAP2K inhibition (3i) on the development of embryos have not been reported. Our results show that 3i improves bovine and mouse IVF development only when added at the appropriate time point and affects ICM-related gene (OCT4, SOX2 and NANOG) expression in a concentration-dependent manner. 3i increases the expression of blastocyst-related genes such as PRDM14, KLF4 and KLF17 and decreases the expression of the de novo DNA methyltransferase genes DNMT3L and DNMT1 in bovines, but increases Prdm14, Stella, Klf2 and Klf4 expression and significantly decreases Dnmt3l, Dnmt3b, and Dnmt1 expression in mice. The analysis of transcription data showed that the expression of DNMTs increases slightly later than that of PRDM14 during embryo development, which indicates that PRDM14 is the upstream regulator. 3i upregulates PRDM14 and then downregulates DNMTs to affect IVF embryo development. When 3i-treated mouse embryos were transplanted, the morphology and body weight of the offspring were not significantly different from those of the control group. These offspring were as fertile as normal mice. 3i improves the development of bovine and mouse IVF embryos but does not affect the quality of the embryos. The application of 3i provides a new method for improving IVF embryo production in domestic animals.

De novo lipogenesis and desaturation of fatty acids during adipogenesis in bovine adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ADSCs) are useful cell model to study adipogenesis and energy metabolism. However, the biological characteristics of bovine ADSCs (bADSCs) remain unclear. This study aimed to isolate and identify bADSCs and further investigate fatty acid (FA)-related gene expression and composition of FAs during adipogenesis. The growth curve showed the bADSCs of P5 cells had rapid proliferation superior to P10-P50. The colony formation assay showed colony number of P5 cells was higher than that of P50 cells (51.67 ± 3.06 vs 35.67 ± 6.43, P < 0.05). The immunofluorescence showed that bADSCs were positive for CD13, CD44, CD49d, CD90, CD105, and Vimentin while negative for CD34. The multipotential towards adipocyte, osteocyte, and chondrocyte was confirmed by specific histological staining and lineage gene expression. During adipogenic induction, the genes related to lipogenesis and lipolysis were assessed by real-time PCR and the FA composition was detected by GC-MS. Expression of lipogenesis-related genes showed coordinated regulation as peaking on day 7 and declining until induction ended, including PPARγ, SREBP1, ACC1, FAS, ELOVL6, SCD1, and FABP4. FA deposition-related genes (DGAT1 and ACAT1) increased until day 14. Lipolysis genes (CPT-1A, VLCAD, and ACO) showed a variant expression pattern. The profile of FAs showed that proportion of the FAs (C4-C15, ≥ C22) increased, but proportion of long-chain fatty acids (C16-C20) reduced after induction. And saturated FAs (SFA) decreased while monounsaturated FAs (MUFA) and polyunsaturated FAs (PUFA) increased during adipogenesis. These data suggest that bADSCs possess the characteristics of mesenchymal stem cells and have active de novo lipogenesis (DNL) and desaturation of FAs during adipogenesis.

Epigenetic reprogramming of human lung cancer cells with the extract of bovine parthenogenetic oocytes.

The tumour suppressor gene silencing and proto-oncogene activation caused by epigenetic alterations plays an important role in the initiation and progression of cancer. Re-establishing the balance between the expression of tumour suppressor genes and proto-oncogenes by epigenetic modulation is a promising strategy for cancer treatment. In this study, we investigated whether cancer cells can be epigenetically reprogrammed by oocyte extract. H460 human lung cancer cells were reversibly permeabilized and incubated with the extract of bovine parthenogenetic oocytes. Bisulphite sequencing showed that bovine parthenogenetic oocyte extract induced significant demethylation at the promoters of the tumour suppressor genes RUNX3 and CDH1, but not at the promoter of the oncogenic pluripotency gene SOX2. Chromatin immunoprecipitation showed that the histone modifications at RUNX3 and CDH1 promoters were modulated towards a transcriptionally activating state, while those at SOX2 promoter towards a transcriptionally repressive state. Correspondingly, bovine parthenogenetic oocyte extract reversed the epigenetic silencing of RUNX3 and CDH1, and repressed the expression of SOX2. At the functional level, proliferation, anchorage-independent growth, migration and invasion of H460 cells was strongly inhibited. These results indicate that bovine parthenogenetic oocyte extract changes the expression patterns of tumour suppressor and oncogenic genes in cancer cells by remodelling the epigenetic modifications at their promoters. Bovine parthenogenetic oocyte extract may provide a useful tool for epigenetically reprogramming cancer cells and for dissecting the epigenetic mechanisms involved in tumorigenesis.

The effect of valproic acid on bovine oocyte maturation and early embryonic development in vitro.

Our objective is to investigate the effect of valproic acid (VPA), a histone deacetylase inhibitor, on early embryonic development. We studied the effect of VPA on the in vitro maturation of bovine oocytes, and on the development of bovine embryos derived from in vitro fertilization (IVF) or parthenogenesis. Germinal vesicle stage bovine oocytes were cultured with different concentrations of VPA for 24 h; low dose VPA treatment (0.03 and 0.3 mM) had no effect on oocyte maturation, but 3 and 6 mM VPA significantly decreased maturation rate; when used for IVF or parthenogenesis, VPA-treated oocytes generated significantly lowered blastocyst rate. Oocytes matured in vitro were fertilized or underwent parthenogenetic activation; 6 h later, they were exposed to VPA for 48 h, and then the cleavage rate, blastocyst rate and mRNA expression levels of transcription factors (Oct4, Nanog, and Cdx2) were assessed. For embryos cultured in 0.3 mM VPA, there was no remarkable change in cleavage rate or blastocyst rate, but the expression of Oct4 and Nanog in blastocysts was significantly increased. For embryos treated with 3.0 mM VPA, the cleavage rate and blastocyst rate were significantly decreased. In conclusion, low dose VPA has no effect on oocyte maturation but affects subsequent embryonic development. Low dose VPA administration to IVF embryos had no effect on embryonic development, but the expression of several important transcription factors was increased. Treatment of IVF embryos with low dose VPA may improve their development potential.

Remodeling epigenetic modifications at tumor suppressor gene promoters with bovine oocyte extract.

BACKGROUND AIMS: Epigenetic silencing of tumor suppressor genes by aberrant DNA methylation and histone modifications at their promoter regions plays an important role in the initiation and progression of cancer. The therapeutic effect of the widely used epigenetic drugs, including DNA methyltransferase inhibitors and histone deacetylase inhibitors, remains unsatisfactory. One important underlying factor in the ineffectiveness of these drugs is that their actions lack specificity. METHODS: To investigate whether oocyte extract can be used for epigenetic re-programming of cancer cells, H460 human lung cancer cells were reversibly permeabilized and incubated with bovine oocyte extract. RESULTS: Bisulfite sequencing showed that bovine oocyte extract induced significant demethylation at hypermethylated promoter CpG islands of the tumor suppressor genes RUNX3 and CDH1; however, the DNA methylation levels of repetitive sequences were not affected. Chromatin immunoprecipitation showed that bovine oocyte extract significantly reduced transcriptionally repressive histone modifications and increased transcriptionally activating histone modifications at the promoter regions of RUNX3 and CDH1. Bovine oocyte extract reactivated the expression of RUNX3 and CDH1 at both the messenger RNA and the protein levels without up-regulating the transcription of pluripotency-associated genes. At the functional level, anchorage-independent proliferation, migration and invasion of H460 cells was strongly inhibited. CONCLUSIONS: These results demonstrate that bovine oocyte extract reactivates epigenetically silenced tumor suppressor genes by remodeling the epigenetic modifications at their promoter regions. Bovine oocyte extract may provide a useful tool for investigating epigenetic mechanisms in cancer and a valuable source for developing novel safe therapeutic approaches that target epigenetic alterations.

Multiple histone site epigenetic modifications in nuclear transfer and in vitro fertilized bovine embryos.

During mammalian embryonic development, DNA methylation and histone modifications are important in gene expression regulation and epigenetic reprogramming. In cloned embryos, high levels of DNA methylation and abnormal demethylation were widely observed during the preimplantation period. Little is known whether there is a difference in histone modifications between in vitro fertilization (IVF) and cloned embryos during preimplantation development. In the present study, the distributions and intensity patterns of acetylations in H3 lysine 9, 18 and H4 lysine 8, 5 and tri-methyl lysine 4 and dimethyl-lysine 9 in histone H3 were compared in cloned and IVF bovine preimplantation embryos by using indirect immunofluorescence and scanning confocal microscopy. The results showed that the acetylation and methylation levels of H3K9ac, H3K18ac, H4K5ac, H4K8ac, H3K4me3 and H3K9me2 were abnormally high in the cloned embryos from the pronuclear to the 8-cell stage. H4K8ac and H4K5ac in the cloned embryos were particularly abnormal when compared with the IVF controls. At the blastocyst stage differences dissipated between cloned and IVF embryos and the distribution and intensity patterns of all histone modifications showed no obvious difference. These results suggest that somatic cells in recipient oocytes produced aberrant histone modifications at multiple sites before the donor cell genome is activated. After zygotic genome activation, distributions and intensity patterns of histone modifications were comparable with both cloned and IVF embryos.

One-pot three-component tandem reaction of diazo compounds with anilines and unsaturated ketoesters: a novel synthesis of 2,3-dihydropyrrole derivatives.

A facile synthesis of 2,3-dihydropyrrole derivatives has been achieved through the one-pot, three-component tandem reaction of diazo compounds with anilines and beta,gamma-unsaturated alpha-ketoesters in moderate to good yields.

Trichostatin A improved epigenetic modifications of transfected cells but did not improve subsequent cloned embryo development.

Reprogramming impairment of DNA methylation may be partly responsible for the low efficiency in somatic cell nuclear transfer. In this study, bovine fibroblast cells were transfected with enhancer green fluorescence protein (eGFP), and then treated with a histone-deacetylase inhibitor, trichostatin A (TSA). The results showed that the effect of TSA on transfected cells was dose dependent. When the TSA concentration was over 5 ng/ml, cell proliferation was significantly inhibited. The majority of the cells died when TSA reached 100 ng/ml (P < 0.01). The number of cells in the S phase was significantly decreased in the 5- to 50-ng/ml TSA-treated groups, while the majority of the cells were at the G0/G1 phases. The number of eGFP-expressed cells were approximately twofold higher in 25-ng/ml (30.5%) and 50-ng/ml (29.5%) TSA groups than the control (15.0%). Reduced DNA methylation and improved histone acetylation were observed when the cells were treated with 10 to 50 ng/ml of TSA. Transfer of the TSA-treated cells to enucleated recipient oocytes resulted in similar cleavage rates among the experimental groups and the control. Cells treated with 50 ng/ml of TSA resulted in significantly lower blastocyst development (9.9%) than the other experimental and the control groups (around 20%). Analysis of the putative blastocysts showed that 86.7% of the embryos derived from TSA-treated cells were eGFP positive, which was higher than that from untreated cells (68.8%). In conclusion, treatment of transfected cells with TSA decreased the genome DNA methylation level, increased histone acetylation, and eGFP gene expression was activated. Donor cells with reduced DNA methylation did not improve subsequent cloned embryo development; however, transgene expression was improved in cloned embryos.