PABPN1 functions as a hub in the assembly of nuclear poly(A) domains that are essential for mouse oocyte development.

Growing oocytes store a large amount of maternal mRNA to support the subsequent "maternal-zygotic transition" process. At present, it is not clear how the growing oocytes store and process the newly transcribed mRNA under physiological conditions. In this study, we report non-membrane-bound compartments, nuclear poly(A) domains (NPADs), as the hub for newly transcribed mRNA, in developing mouse oocytes. The RNA binding protein PABPN1 promotes the formation of NPAD through its N-terminal disordered domain and RNA-recognized motif by means of liquid phase separation. Pabpn1-null growing oocytes cannot form NPAD normally in vivo and have defects in stability of oocyte growing-related transcripts and formation of long 3' untranslated region isoform transcripts. Ultimately, Pabpn1fl/fl;Gdf9-Cre mice are completely sterile with primary ovarian insufficiency. These results demonstrate that NPAD formed by the phase separation properties of PABPN1-mRNA are the hub of the newly transcribed mRNA and essential for the development of oocytes and female reproduction.

Maternal DCAF13 Regulates Chromatin Tightness to Contribute to Embryonic Development.

Maternal-zygotic transition (MZT) is critical for the developmental control handed from maternal products to newly synthesized zygotic genome in the earliest stage of embryogenesis. However, the spatiotemporal dynamic regulation of MZT by maternal factors is largely unknown. Here, we reported a novel maternal factor, DCAF13, which was highly expressed in growing oocyte nucleolus and had key maternal effects on oocyte and zygotic chromatin tightness during maternal to zygotic transition. DCAF13 specifically deleted in oocytes resulted in loose chromatin structure in fully grown germinal vesicle oocytes. Despite normal nuclear maturation in maternal DCAF13-deleted oocytes, the chromosomes at MII stage were not properly condensed. Consequently, the nuclear and nucleolar structure reorganized abnormally, and transcription was inactive in zygotic embryos. RNA-seq analysis of MII oocytes and 2-cell embryos demonstrated that the transcriptomes between knockout and control oocyte were similar, but the maternal DCAF13 deleted two-cell embryos showed a significant decrease in transcription. In addition, the maternal DCAF13-deleted embryos displayed arrest at the two-cell stage, which could not be rescued by injecting flag-Dcaf13 mRNA in the zygote. This revealed that DCAF13 was a unique maternal effect factor regulating the nucleolus.

Methyl-CpG-Binding Protein 2 Improves the Development of Mouse Somatic Cell Nuclear Transfer Embryos.

Methyl-CpG-binding domain proteins (MBPs) connect DNA methylation and histone modification, which are the key changes of somatic cell reprogramming. Methyl-CpG-binding protein 2 (MeCP2) was the first discovered MBP that has been extensively studied in the neurodevelopmental disorder Rett syndrome. However, a role for MeCP2 during cellular reprogramming associated with somatic cell nuclear transfer (SCNT) has not been examined. In this study, we discovered that MeCP2 expression was significantly lower in embryos generated by SCNT compared with those generated by intracytoplasmic sperm injection (ICSI). We genetically modified mouse embryonic fibroblasts (MEFs) to overexpress MeCP2 and serve as donor cells for nuclear transfer (NT) to investigate the effects of MeCP2 on preimplantation development of SCNT embryos. The blastocyst rate (35.71%) of MeCP2 overexpressed embryos (NT(+)) was significantly greater than in nontransgenic embryos (NT(-), 24.29%). Furthermore, immunofluorescence experiments revealed that 5-methylcytosine (5mC) was transferred to 5-hydroxymethylcytosine (5hmC) to a greater extent in NT(+) embryos than in NT(-) embryos. Real-time PCR evaluation of gene expression also showed that embryonic development-associated genes, such as Oct4 and Nanog, were significantly higher in the NT(+) group compared to the NT(-) group. Collectively, these results suggested that MeCP2 facilitated Tet3 activity, enhanced expression of pluripotency-related genes, and eventually improved the development of NT embryos. Finally, we performed chromatin immunoprecipitation to identify direct targets of MeCP2 and constructed a protein interaction network to elucidate several putative MeCP2 targets.

[Identification of Six Isomers of Dimethylbenzoic Acid by Using Terahertz Time-Domain Spectroscopy Technique].

In this paper, the absorption spectra of 6 isomers of dimethylbenzoic acid, which were widely used in chemical and pharmaceutical production as intermediate substance, were measured by using the terahertz time-domain spectroscopy (THz-TDS) system in the range 0.2-2.2 THz at room temperature. The experimental results show that the six measured isomers present apparent different spectral response. However, the results of using infrared spectroscopy indicates that different isomers show high similarity in absorption spectra in the range 1450-1700 cm⁻¹. The vibrational frequencies are calculated by using the density functional theory (DFT), and identification of vibrational modes are given. It is clear that the absorption peaks of the 6 isomers in the range 1450-1700 cm⁻¹ come from the stretching vibration of benzene ring and C==O, while the absorption peaks in the terahertz range are caused by the relative wagging of benzene ring and all the chains out of plane, which lead to the different absorption characteristics of the 6 isomers in the range 0.2-2.2 THz. The results suggest that the difference and similarity of the absorption spectra observed in the two different frequency range are resulted from the difference and similarity of the molecular structures of the six isomers. By using the different absorption characteristics, we can identify the six isomers of dimethylbenzoic acid effectively. Our study indicates that it is feasible to distinguish the isomers by using terahertz and infrared spectroscopy technique. It provides an effective way to identify different isomers and test the purity of the intermediate substance in the process of production quickly and accurately.

[A Double Split Ring Terahertz Filter on Ploymide Substrate].

Metamaterials are artificial composites that acquire their electromagnetic properties from embeded subwavelength metalic structure. With proper design of metamaterials, numerrous intriguing phenomena that not exhibited naturally can be realized, such as invisible cloaking, perfect absorption, negative refractive index and so on. In recent years, With the development of THz technology, the extensive research onTHz metamaterials devices areattracting more and more attentions. Since silicon (Si) has a higher transmittance for THz wave, it is usually selected as substrate in metamaterials structure. However, Si has the shortcomings of hardness, not easy to bend, and fragile, which limit the application of THz metamaterials. In this work, we use polyimide as the substrate to overcome the shortcomings of the Si substrate. Polyimide is flexible, smooth, suitable for conventional lithography process and the THz transmittance can compete with that of the Si. Frist, we test the THz optical properties of polymide, and get the refractive index of 1.9, and the transmittance of 80%. Second, we design a double splits ring resonators (DSRRs), and study the properties of transmission by changing the THz incidence angle and curvature of the sample. We find the resonant amplitude and resonant frequencies are unchanged. Fabricating metamaterials structures on a thin plastic substrate is a possible way to extend plane surface filtering to curved surface filtering. Third, we try to make a broadband filter by stacking two samples, and the 181GHz bandwidth at 50% has been achieved. By stacking several plane plastic metamaterial layers with different resonance responses into a multi-layer structure, a broadband THz filter can be built. The broadband filter has the advantages of simple manufacture, obvious filtering effect, which provides a new idea for the production of terahertz band filter.

Continuous passages accelerate the reprogramming of mouse induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are usually generated by reprogramming somatic cells through transduction with a transcription factor cocktail. However, the low efficiency of this procedure has kept iPSCs away from the study of the clinical application of stem cell biology. Our research shows that continuous passage increases the efficiency of reprogramming. Compared with conventional method of establishment of iPSCs, more embryonic stem cell (ESC)-like clones are generated by continuous passage during early reprogramming. These inchoate clones, indistinguishable from genuine ESC clones, are closer to fully reprogrammed cells compared with those derived from classical iPSC induction, which increased the expression of pluripotent gene markers and the levels of demethylation of Oct4 and Nanog. These results suggested that full reprogramming is a gradual process that does not merely end at the point of the activation of endogenous pluripotency-associated genes. Continuous passage could increase the pluripotency of induced cells and accelerate the process of reprogramming by epigenetic modification. In brief, we have provided an advanced strategy to accelerate the reprogramming and generate more nearly fully reprogrammed iPSCs efficiently and rapidly.

Generation of neural progenitors from induced Bama miniature pig pluripotent cells.

Pig pluripotent cells may represent an advantageous experimental tool for developing therapeutic application in the human biomedical field. However, it has previously been proven to be difficult to establish from the early embryo and its pluripotency has not been distinctly documented. In recent years, induced pluripotent stem (iPS) cell technology provides a new method of reprogramming somatic cells to pluripotent state. The generation of iPS cells together with or without certain small molecules has become a routine technique. However, the generation of iPS cells from pig embryonic tissues using viral infections together with small molecules has not been reported. Here, we reported the generation of induced pig pluripotent cells (iPPCs) using the iPS technology in combination with valproic acid (VPA). VPA treatment significantly increased the expression of pluripotent genes and played an important role in early reprogramming. We showed that iPPCs resembled pig epiblast cells in their morphology and pluripotent markers, such as OCT4, NANOG, and SSEA1. It had a normal karyotype and could form embryoid bodies, which express three germ layer markers in vitro. In addition, the iPPCs might directly differentiate into neural progenitors after being induced with the retinoic acid and extracellular matrix. Our study established a reasonable method to generate pig pluripotent cells, which might be a new donor cell source for human neural disease therapy.

[Applications of terahertz spectroscopy in illicit drugs detection].

Abstract The present paper reviewed our recent research on illicit drugs detection by using terahertz (THz) spectroscopy technique. First, we found a THz spectral database which contains 38 types of pure illicit drugs. Second, we employ the artificial neural network (ANN) and support vector machine (SVM) methods as useful automatic tools to identify the illicit drugs. Third, a theoretical and experimental method has been developed to confirm the illicit drug purity and effective component content. Forth, Gaussian Software was used for spectral analysis of some individual samples, such as MA, ketamine, heroin, etc. Fifth, we built a smart portable Terahertz time domain spectroscopy system (THz-TDS). These works provide a solid scientific foundation for illicit drug identification by using THz spectroscopy technique. And also, as a complement detection approach, it will serve public security work.

[Study on cephalosporins with terahertz time-domain spectroscopy technique].

Terahertz Time-Domain Spectroscopy (THz-TDS) technique has a wide range of applications in substances identification and quantitative analysis. In the present paper, we report absorption spectra and index of refraction of 14 kinds of pure cephalosporins in 0.2-2.6 THz, in which eight kinds have apparent absorption peaks, while the others have different index of refraction. Based on these results, different kinds of antibiotics can be identified. Besides, according to THz absorption spectra of both pure sample and real pills we calculated the contents of cefixime in the two pills produced by two manufacturers. Compared with the contents marked on the package, relative errors are 9.38% and 0.92%, respectively. The results manifest that THz-TDS technique is reliable and promising in medicine detection.

Association between the interleukin-4, interleukin-13 polymorphisms and asthma: a meta-analysis.

A numbers studies had been reported that the polymorphisms in the Interleukin 4 (IL-4) and Interleukin 13 (IL-13) genes were associated with susceptibility to asthma. However, the results were inconsistent and inconclusive. We carried out a meta-analysis of case-control genetic association studies to assess whether the combined data showed this association by using a genetic model-free approach. Thirty studies (total 12,781 asthma and 11,500 controls) for the IL-4 C-33T and C-589T, IL-13 C-1112T and G+2044A with asthma were included in the meta-analysis. The results indicated that there were an association between the IL-4 C-33T (P = 0.006) and C-589T (P = 0.04), IL-13 C-1112T (P = 0.002) and G+2044A (P = 0.04) and susceptibility to asthma. And the definition of asthma subgroup meta-analysis demonstrates that the IL-4 C-33T is not associated with nonatopic or atopic, and IL-4 C-589T and IL-13 C-1112T polymorphisms are not associated with atopic. In the ethnicity subgroup meta-analysis, the IL-4 -589T (P = 0.003) and the IL-13 -1112T (P < 0.00001) alleles are associated with asthma among Caucasian, but not on the IL-13 +2044A allele. In conclusion, IL-4 C-33T and C-589T, IL-13 C-1112T and G+2044A could be proposed as asthma susceptible SNPs. Further investigation in larger studies and meta-analysis is required.

rRNA genes are not fully activated in mouse somatic cell nuclear transfer embryos.

The well known and most important function of nucleoli is ribosome biogenesis. However, the nucleolus showed delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Previous studies indicated that nearly half rRNA genes (rDNA) in somatic cells were inactive and not transcribed. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. The results showed embryonic stem cells (ESCs) had the most active NORs and lowest rDNA methylation level (7.66 and 6.76%), whereas mouse embryonic fibroblasts (MEFs) were the opposite (4.70 and 22.57%). After the donor cells were injected into enucleated MII oocytes, cumulus cells and MEFs nuclei lost B23 and UBF signals in 20 min, whereas in ESC-NT embryos, B23 and UBF signals could still be detected at 60 min post-NT. The embryos derived from ESCs, cumulus cells, and MEFs showed the same trend in active NORs numbers (7.19 versus 6.68 versus 5.77, p < 0.05) and rDNA methylation levels (6.36 versus 9.67% versus 15.52%) at the 4-cell stage as that in donor cells. However, the MEF-NT embryos displayed low rRNA synthesis/processing potential at morula stage and had an obvious decrease in blastocyst developmental rate. The results presented clear evidences that the rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived.

Aggregation of pre-implantation embryos improves establishment of parthenogenetic stem cells and expression of imprinted genes.

Parthenogenetic embryonic stem cells (PgES) might advance cell replacement therapies and provide a valuable in vitro model system to study the genomic imprinting. However, the differential potential of PgES cells was limited. It could result from relative low heterology of PgES cells compared with ES cells from fertilization (fES), which produce different expression of most imprinted genes. Here, we described the establishment of PgES cells by aggregating parthenogenetic embryos at the 8-cell stage (aPgES cells), which may increase heterozygy. We found that derivation of aPgES cells in association with an increased number of inner cell mass cells by aggregating was more efficient than that of PgES cells from a single parthenogenetic blastocyst. The aPgES cells have normal karyotype, stain positive for alkaline phosphatase, express high levels of ES cell markers and can differentiate into teratomas composed of the three germ layers. Moreover, compared with PgES cells, the more highly upregulated paternally expressed imprinted genes were observed in aPgES cells, the same change was not shown in aPg blastocysts. This suggested that the aggregation induced effect could modify the expression of paternally expressed imprinted genes. Our studies showed that aPgES cells, the expression of imprinted genes in which more closely resemble fES cells than PgES cells, would contribute to all organs and avoiding immuno-rejection, which may provide invaluable material for regeneration medicine.

[Terahertz spectroscopic testing of food additive tert-butylhydroquinone].

Terahertz time-domain spectroscopy (THz-TDS) technique has a wide range of applications in nondestructive testing. After many years study, people have found that many materials have characteristic absorptions in terahertz range. This letter studies the THz spectra of tert-butylhydroquinone (TBHQ), a food additive that was reported excessively in Mak chicken of McDonald. The authors applied terahertz nondestructive testing technique in identifying this material, testing the absorption and refractive index. The absorption spectra of TBHQ and flour mixture in 0.2-2.2 THz were also investigated. The simulation of vibration for single molecular was undertaken. The results represent that this method is possible by comparing the difference in absorption lines and this method paves the way for detecting food additives.

Generation of dorsal spinal cord GABAergic neurons from mouse embryonic stem cells.

Developmental signaling molecules involved in dorsal patterning of the spinal cord have been identified in vivo; however, studies have not produced specific functional dorsal spinal cord neurons in vitro. We present here differentiation of R1 embryonic stem (ES) cells into GABAergic dorsal spinal cord neurons by sequential treatment with developmental signaling molecules. We found that retinoic acid, Bmp4 altered the specification of neural progenitors and instructed neural fate when applied at distinct stages of development. High concentration of retinoic acid initiated caudal patterning during early differentiation; Bmp4 induced dorsal development. The combination of retinoic acid and different concentration Bmp4 controlled the differing regional progenitors of spinal cord. Low-concentration Bmp4 and high concentration of retinoic acid-treated embryoid bodies resulted in the differentiation of GABAergic neurons. In summary, we demonstrate this simple treatment paradigm produced simple dorsal spinal cord neurons, which could be utilized for developmental and preclinical studies.

[Study on terahertz spectroscopy of Hotan jade].

Terahertz time-domain spectroscopy (THz-TDS) technique has a wide range of applications including illicit drugs and explosive detection, and organic molecules recognition. In the present paper, the spectral features of three kinds of Hotan jade were studied experimentally by THz-TDS technique and the characteristic absorption spectra and refractive index were obtained in the range of 0.2 to 2.6 THz. The experimental results show that different samples have different absorption characters, and the refractive index is 2.4-2.7 in the range of 0.2-2.6 THz. The results indicate that it is feasible to apply THz-TDS technique to identification of Hotan jade, which provides a new approach to the nondestructive examination of Hotan jade.

Effect of carbamylated erythropoietin on major histocompatibility complex expression and neural differentiation of human neural stem cells.

The expression of major histocompatibility complex (MHC) on human neural stem cells (hNSCs) is tightly related to the fate of these cells in transplantation, therefore strategies to relieve rejection and promote graft survival are necessary to be applied. This study investigated the effect of carbamylated erythropoietin (CEPO) on MHC expression and differentiation of hNSCs with or without IFN-gamma incubation. Results showed that low levels of MHC molecules were expressed on hNSCs and increased by IFN-gamma. CEPO enhanced MHC-I antigens in both proliferative and differentiated hNSCs, but decreased MHC-II antigens in differentiated hNSCs and those cells exposed to IFN-gamma. Furthermore, CEPO promoted neural differentiation of hNSCs and outgrowth of neurites. Western blot analysis revealed activation of Stat3, Stat5 and Akt during these processes. These results suggest that CEPO may have immunoregulatory function in hNSCs besides its neuroprotection.

ES cell extract-induced expression of pluripotent factors in somatic cells.

Reprogramming of somatic cells was induced by ES cell-free extract. The system relied on the transient uptake of regulatory components from a nuclear and cytoplasmic extract derived from ES cells by the nucleus of a reversibly permeabilized NIH3T3 cell. NIH3T3 cells were permeabilized by streptolysin O (SLO). Reprogramming cell-free extracts were prepared by repeatedly freezing and thawing ES cells in liquid nitrogen. After incubation in the extract for 1 hr, permeabilized NIH3T3 cells were resealed by CaCl(2) and continually cultured for weeks to assess expression of ES cell specific markers. As we observed using FACS and fluorescence microscope, the optimal SLO concentration for permeabilizing NIH3T3 cells was 25 U. After 2 weeks of culture, the treated NIH3T3 cells began to express Nanog, c-Myc, Klf4, and 6 weeks later Oct4 was detectable. However, Sox2 was detected only after 8 weeks of culture. Differentiated somatic cells could be reprogrammed in ES extract in vitro, which provides a new approach to decreasing differentiation levels in somatic cells without disturbing the DNA sequences.

MSCs guide neurite directional extension and promote oligodendrogenesis in NSCs.

Previous studies have shown that mesenchymal stem cells (MSCs) enhance repair following injury or degenerative diseases in the central nervous system, but the underlying mechanisms remain unclear. The present study investigated the functional relationship between MSCs and neural stem cells (NSCs) using co-culture systems. Results demonstrated that MSCs promoted outgrowth and guided directional extension of NSC-derived neurites. The majority of neurites were oriented parallel along the MSC axis. Stripe assay results indicated that cell adhesion molecule and extracellular matrix, such as N-cadherin, fibronectin, and laminin, contributed to this effect. Furthermore, Western blot analysis revealed that phosphorylation of cAMP response element-binding protein (CREB) increased during this process. In addition, MSCs promoted differentiation of NSCs into oligodendrocytes via secreted soluble factors. The oligodendrocytes were distributed along the MSC surface in a regular pattern. This study demonstrated that MSC transplantation could be a potential strategy for treating central nervous system injuries.

Developmental pattern of hexaploid mouse embryos produced by blastomere fusion of diploid and tetraploid embryos at the 2-cell stage.

Polyploid mouse embryos are important models for understanding the mechanisms of cleavage and preimplantation development in mammals. In this study, hexaploid (6n) mouse embryos were produced by the electrofusion of blastomeres from diploid (2n) and tetraploid (4n) embryos at the 2-cell stage. Furthermore, the developmental pattern of hexaploid embryos was evaluated by blastocyst rate, cell number, karyotype analysis, cytoskeleton staining and Oct-4 immunofluorescence. The results showed that 72.7% of the hexaploid embryos were able to develop to the blastocyst stage, which is a lower number than that found with normal diploid embryos (98.0%, p < 0.05). The cell number in hexaploid blastocyst was 12.3 +/- 2.0, which was less than that found in diploid or tetraploid blastocysts (41.2 +/- 7.2; 18.4 +/- 3.5). Karyotype analysis confirmed that the number of chromosomes in hexaploid embryos was 120. beta-Tubulin and Oct-4 immunofluorescence indicated that the hexaploid blastocysts were nearly lacking inner cell mass (ICM), but some blastomeres did show Oct-4-positive expression.

[Programmed application of extracellular matrix promotes neural differentiation of mouse embryonic stem cells].

OBJECTIVE: To study the role of extracellular matrix (ECM) in neural differentiation of mouse embryonic stem cells (ESCs). METHODS: Mouse ESCs were incubated in the ESC conditioned medium, and the formation of embryonic bodies (EBs) were induced in bacteriological dishes using high-concentration all-trans retinoic acid (RA). The EBs were seeded on different matrixes (gelatin, fibronectin, and laminin/poly-L-ornithine) to test their impact on neural differentiation of the ESCs using immunofluorescence assay. The effect of laminin/poly-L-ornithine on the growth of neurites was evaluated with fluorescence microscopy. RESULTS: High-concentration RA activated and accelerated the differentiation of ESCs toward nestin-positive neural progenitor cells. Fibronectin supplement in the matrix dose-dependently promoted ESC differentiation into neural progenitor cells, while laminin/poly-L-ornithine increased the growth of the neurites and induced the maturation of the differentiated neural cells. CONCLUSION: ECM plays an important role in neural differentiation of mouse ESCs, and application of FN produces the most conspicuous effect during the differentiation of the ESCs into the neural progenitor cells;laminin/poly-L-ornithine is the most effective during their differentiation into neural cells.