Metformin Promotes Proliferation of Mouse Female Germline Stem Cells by Histone Acetylation Modification of Traf2.

Female germline stem cells (FGSCs) are adult stem cells that can both self-renew and differentiate into mature oocytes. Although small-molecule compounds are capable of regulating the development of FGSCs, the effects and mechanisms of action of metformin, a commonly used drug for diabetes, on FGSCs are largely unknown. Here, we found that metformin promoted the viability and proliferation of FGSCs through H3K27ac modification. To elucidate the mechanism by which metformin promoted FGSCs proliferation, Chromatin Immunoprecipitation Sequencing of histone 3 lysine 27 acetylation (H3K27ac) in FGSCs was performed with or without metformin-treatment. The results indicate that metformin modulates FGSCs via the mitogen-activated protein kinase (MAPK) signaling pathway, and tumor necrosis factor receptor associated factor 2 (Traf2) was identified as an important target gene for H3K27ac modification during FGSCs proliferation. Subsequent experiments showed metformin promoted FGSCs proliferation by H3K27ac modification of Traf2 to regulate MAPK signaling. Our findings deepen understanding of how H3K27ac modifications regulate FGSCs development and provide a theoretical basis for the prevention and treatment of premature ovarian failure, polycystic ovary syndrome, infertility, and related diseases.

Metformin promotes female germline stem cell proliferation by upregulating Gata-binding protein 2 with histone ß-hydroxybutyrylation.

BACKGROUND: Metformin as a first-line clinical anti-diabetic agent prolongs the lifespan of model animals and promotes cell proliferation. However, the molecular mechanisms underlying the proliferative phenotype, especially in epigenetics, have rarely been reported. The aim of this study was to investigate the physiological effects of metformin on female germline stem cells (FGSCs) in vivo and in vitro, uncover ß-hydroxybutyrylation epigenetic modification roles of metformin and identify the mechanism of histone H2B Lys5 ß-hydroxybutyrylation (H2BK5bhb) in Gata-binding protein 2 (Gata2)-mediated proliferation promotion of FGSCs. METHODS: The physiological effects of metformin were evaluated by intraperitoneal injection and histomorphology. The phenotype and mechanism studies were explored by cell counting, cell viability, cell proliferation assay and protein modification omics, transcriptomics, chromatin immunoprecipitation sequencing in FGSCs in vitro. RESULTS: We found that metformin treatment increased the number of FGSCs, promoted follicular development in mouse ovaries and enhanced the proliferative activity of FGSCs in vitro. Quantitative omics analysis of protein modifications revealed that H2BK5bhb was increased after metformin treatment of FGSCs. In combination with H2BK5bhb chromatin immunoprecipitation and transcriptome sequencing, we found that Gata2 might be a target gene for metformin to regulate FGSC development. Subsequent experiments showed that Gata2 promoted FGSC proliferation. CONCLUSION: Our results provide novel mechanistic understanding of metformin in FGSCs by combining histone epigenetics and phenotypic analyses, which highlight the role of the metformin-H2BK5bhb-Gata2 pathway in cell fate determination and regulation.

YTHDF1 phase separation triggers the fate transition of spermatogonial stem cells by activating the IκB-NF-κB-CCND1 axis.

N6-methyladenosine (m6A) modification controls cell fate determination. Here, we show that liquid-liquid phase separation (LLPS) of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), a pivotal m6A "reader" protein, promotes the transdifferentiation of spermatogonial stem cells (SSCs) into neural stem cell-like cells by activating the IκB-nuclear factor κB (NF-κB)-CCND1 axis. The inhibition of IκBα/ß mRNA translation mediated by YTHDF1 LLPS is the key to the activation of the IκB-NF-κB-CCND1 axis. Disrupting either YTHDF1 LLPS or NF-κB activation inhibits transdifferentiation efficiency. Moreover, overexpression of the YTH domain of YTHDF1 inhibits the activation of the IκB-NF-κB-CCND1 axis by promoting IκBα/ß mRNA translation. Overexpression of the tau-YTH fusion protein reactivates IκB-NF-κB-CCND1 axis by inhibiting the translation of IκBα/ß mRNAs, and tau LLPS is observed, which can restore transdifferentiation efficiency. Our findings demonstrate that the protein-RNA LLPS plays essential roles in cell fate transition and provide insights into translational medicine and the therapy of neurological diseases.

Mechanistic insights into HuR inhibitor MS-444 arresting embryonic development revealed by low-input RNA-seq and STORM.

With improvements in the survival rate of patients with cancer, fertility maintenance has become a major concern in terms of cancer treatment for women of reproductive age. Thus, it is important to examine the impact on fertility of anticancer drugs that are used clinically or are undergoing trials. The HuR small-molecule inhibitor MS-444 has been used in many cancer treatment studies, but its reproductive toxicity in females is unknown. Here, we reported that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization, resulting in the developmental arrest of 2-cell stage embryos in mouse. Combining analysis of low-input RNA-seq for MS-444-treated 2-cell embryos and mapping binding sites of RNA-binding protein, Agbl2 was predicted to be the target gene of MS-444. For further confirmation, RNAi experiment in wild-type zygotes showed that Agbl2 knockdown reduced the proportion of embryos successfully developed to the blastocyst stage: from 71% in controls to 23%. Furthermore, RNA-FISH and luciferase reporter analyses showed that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA and reduced its stability by inhibiting HuR dimerization. In addition, optimized stochastic optical reconstruction microscopy (STORM) imaging showed that MS-444 significantly reduced the HuR dimerization, and HuR mainly existed in cluster form in 2-cell stage embryos. In conclusion, this study provides clinical guidance for maintaining fertility during the treatment of cancer with MS-444 in women of reproductive age. And also, our research provides guidance for the application of STORM in nanometer scale studies of embryonic cells. HuR inhibitor MS-444 arrested embryonic development at 2-cell stage. Low-input RNA-seq revealed that Agbl2 was the target gene of MS-444. MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization and reduced the stability of Agbl2 mRNA. STORM with our optimized protocol showed that HuR tended to form elliptical and dense clusters in 2-cell stage embryos.

Three-dimensional genome structure shapes the recombination landscape of chromatin features during female germline stem cell development.

BACKGROUND: During meiosis of mammalian cells, chromatin undergoes drastic reorganization. However, the dynamics of the three-dimensional (3D) chromatin structure during the development of female germline stem cells (FGSCs) are poorly understood. METHODS: The high-throughput chromosome conformation capture technique was used to probe the 3D structure of chromatin in mouse germ cells at each stage of FGSC development. RESULTS: The global 3D genome was dramatically reorganized during FGSC development. In topologically associating domains, the chromatin structure was weakened in germinal vesicle stage oocytes and still present in meiosis I stage oocytes but had vanished in meiosis II oocytes. This switch between topologically associating domains was related to the biological process of FGSC development. Moreover, we constructed a landscape of chromosome X organization, which showed that the X chromosome occupied a smaller proportion of the active (A) compartment than the autosome during FGSC development. By comparing the high-order chromatin structure between female and male germline development, we found that 3D genome organization was remodelled by two different potential mechanisms during gamete development, in which interchromosomal interactions, compartments, and topologically associating domain were decreased during FGSC development but reorganized and recovered during spermatogenesis. Finally, we identified conserved chromatin structures between FGSC development and early embryonic development. CONCLUSIONS: These results provide a valuable resource to characterize chromatin organization and for further studies of FGSC development.

Retinoic acid induced meiosis initiation in female germline stem cells by remodelling three-dimensional chromatin structure.

OBJECTIVES: This study aimed to clarify the regulation and mechanism of meiotic initiation in FGSC development. MATERIALS AND METHODS: FGSCs were induced to differentiate into meiosis in differentiation medium. RNA sequencing was performed to analysis the difference of transcription level. High-through chromosome conformation capture sequencing (Hi-C) was performed to analysis changes of three-dimensional chromatin structure. Chromosome conformation capture further confirmed a spatial chromatin loop. ChIP-qPCR and dual luciferase reporter were used to test the interaction between Stimulated by retinoic acid gene 8 (STRA8) protein and Trip13 promoter. RESULTS: Compared with FGSCs, the average diameter of STRA8-positive germ cells increased from 13 µm to 16.8 µm. Furthermore, there were 4788 differentially expressed genes between the two cell stages; Meiosis and chromatin structure-associated terms were significantly enriched. Additionally, Hi-C results showed that FGSCs underwent A/B compartment switching (switch rate was 29.81%), the number of topologically associating domains (TADs) increasing, the average size of TADs decreasing, and chromatin loop changes at genome region of Trip13 from undifferentiated stage to meiosis-initiation stage. Furthermore, we validated that Trip13 promoter contacted distal enhancer to form spatial chromatin loop and STRA8 could bind Trip13 promoter to promote gene expression. CONCLUSION: FGSCs underwent chromatin structure remodelling from undifferentiated stage to meiosis-initiation stage, which facilitated STRA8 binding to Trip13 promoter and promoting its expression.

Stella Regulates the Development of Female Germline Stem Cells by Modulating Chromatin Structure and DNA Methylation.

Female germline stem cells (FGSCs) have the ability to self-renew and differentiate into oocytes. Stella, encoded by a maternal effect gene, plays an important role in oogenesis and early embryonic development. However, its function in FGSCs remains unclear. In this study, we showed that CRISPR/Cas9-mediated knockout of Stella promoted FGSC proliferation and reduced the level of genome-wide DNA methylation of FGSCs. Conversely, Stella overexpression led to the opposite results, and enhanced FGSC differentiation. We also performed an integrative analysis of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), high-throughput genome-wide chromosome conformation capture (Hi-C), and use of our published epigenetic data. Results indicated that the binding sites of STELLA and active histones H3K4me3 and H3K27ac were enriched near the TAD boundaries. Hi-C analysis showed that Stella overexpression attenuated the interaction within TADs, and interestingly enhanced the TAD boundary strength in STELLA-associated regions. Taking these findings together, our study not only reveals the role of Stella in regulating DNA methylation and chromatin structure, but also provides a better understanding of FGSC development.

Integrative analysis of the 3D genome structure reveals that CTCF maintains the properties of mouse female germline stem cells.

The three-dimensional configuration of the genome ensures cell type-specific gene expression profiles by placing genes and regulatory elements in close spatial proximity. Here, we used in situ high-throughput chromosome conformation (in situ Hi-C), RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to characterize the high-order chromatin structure signature of female germline stem cells (FGSCs) and identify its regulating key factor based on the data-driven of multiple omics data. By comparison with pluripotent stem cells (PSCs), adult stem cells (ASCs), and somatic cells at three major levels of chromatin architecture, A/B compartments, topologically associating domains, and chromatin loops, the chromatin architecture of FGSCs was most similar to that of other ASCs and largely different from that of PSCs and somatic cells. After integrative analysis of the three-dimensional chromatin structure, active compartment-associating loops (aCALs) were identified as a signature of high-order chromatin organization in FGSCs, which revealed that CCCTC-binding factor was a major factor to maintain the properties of FGSCs through regulation of aCALs. We found FGSCs belong to ASCs at chromatin structure level and characterized aCALs as the high-order chromatin structure signature of FGSCs. Furthermore, CTCF was identified to play a key role in regulating aCALS to maintain the biological functions of FGSCs. These data provide a valuable resource for future studies of the features of chromatin organization in mammalian stem cells and further understanding of the fundamental characteristics of FGSCs.

Offspring production of haploid spermatid-like cells derived from mouse female germline stem cells with chromatin condensation.

BACKGROUND: During male meiosis, the Y chromosome can form perfect pairing with the X chromosome. However, it is unclear whether mammalian Female germline stem cells (FGSCs) without a Y chromosome can transdifferentiate into functional haploid spermatid-like cells (SLCs). RESULTS: We found that spermatogenesis was restarted by transplanting FGSCs into Kitw/wv mutant testes. Complete meiosis and formation of SLCs was induced in vitro by testicular cells of Kitw/wv mutant mice, cytokines and retinoic acid. Healthy offspring were produced by sperm and SLCs derived from the in vivo and in vitro transdifferentiation of FGSCs, respectively. Furthermore, high-throughput chromosome conformation capture sequencing(Hi-C-seq) and "bivalent" (H3K4me3-H3K27me3) micro chromatin immunoprecipitation sequencing (µChIP-seq) experiments showed that stimulated by retinoic acid gene 8 (STRA8)/protamine 1 (PRM1)-positive transdifferentiated germ cells (tGCs) and male germ cells (mGCs) display similar chromatin dynamics and chromatin condensation during in vitro spermatogenesis. CONCLUSION: This study demonstrates that sperm can be produced from FGSCs without a Y chromosome. This suggests a strategy for dairy cattle breeding to produce only female offspring with a high-quality genetic background.

[A preliminary investigation on a deep learning convolutional neural networks based pulmonary tuberculosis CT diagnostic model].

Objective: To evaluate the clinical value of a pulmonary tuberculosis CT diagnostic model based on deep learning convolutional neural networks (CNN). Methods: From March 2017 to March 2018,a total of 1 764 patients with positive sputum for tuberculous bacterium and had received high-resolution chest CT scan in radiology department of Hebei province chest hospital were enrolled. Among them, 937 were male, and 827 were female, aging from 17-73 years (average 38.4). A total of 20 139 CT images (17 kinds of image features) classified by 4 radiologists were used as training dataset to create a tuberculosis CT CNN diagnostic model. The top 5 image features in training set were: infiltrative pulmonary tuberculosis, cavitary pulmonary tuberculosis, pleural thickening, caseous pneumonia and pleural effusion. A total of 302 images were randomly selected from the marked images as testing dataset. The diagnosis of 2 senior radiologists was taken as "golden standard". The differences of sensitivity and accuracy in CT diagnosis between the CNN diagnostic model and the radiologists were compared. The classification error types and numbers of the CNN diagnostic model were recorded. FROC(free response operating characteristic curve)curve was drawn and the highest diagnostic efficiency of the model was measured. Results: The diagnostic accuracy of infiltrative pulmonary tuberculosis, cavitary pulmonary tuberculosis, pleural thickening, caseous pneumonia and pleural effusion by the CNN diagnostic model were 95.33%(10 982/11 520), 73.68%(2 151/2 920), 73.07%(1 128/1544), 83.33%(1 020/1225)and 94.11%(814/865), respectively. The overall diagnostic sensitivity and accuracy of the CNN model were 95.49%(339/355)and 90.40%(339/375), respectively, and the corresponding values ​​of radiologists were 93.80%(348/371)and 92.80%(348/375), respectively, and there was no statistical difference between the CNN model and the radiologists(sensitivity χ2=1.022,P=0.312;accuracy χ2=1.404,P=0.236). FROC curve showed that when sensitivity of the CNN model was 78% and FPI value was 2.48, it reached the highest diagnostic efficiency. The classification error of CNN diagnostic models was mainly confusion of fiber stripe components, cavitary pulmonary tuberculosis, caseous pneumonia and infiltrative pulmonary tuberculosis. Conclusions: The CNN-based pulmonary tuberculosis CT diagnostic model exhibited high sensitivity and accuracy (95.49% and 90.40% respectively). It could assist radiologists in CT diagnosis of pulmonary tuberculosis and deserve further clinical application.

Offspring production of ovarian organoids derived from spermatogonial stem cells by defined factors with chromatin reorganization.

Introduction: Fate determination of germline stem cells remains poorly understood at the chromatin structure level. Objectives: Our research hopes to develop successful offspring production of ovarian organoids derived from spermatogonial stem cells (SSCs) by defined factors. Methods: The offspring production from oocytes transdifferentiated from mouse SSCs with tracking of transplanted SSCs in vivo, single cell whole exome sequencing, and in 3D cell culture reconstitution of the process of oogenesis derived from SSCs. The defined factors were screened with ovarian organoids. We uncovered extensive chromatin reorganization during SSC conversion into induced germline stem cells (iGSCs) using high throughput chromosome conformation. Results: We demonstrate successful production of offspring from oocytes transdifferentiated from mouse spermatogonial stem cells (SSCs). Furthermore, we demonstrate direct induction of germline stem cells (iGSCs) differentiated into functional oocytes by transduction of H19, Stella, and Zfp57 and inactivation of Plzf in SSCs after screening with ovarian organoids. We uncovered extensive chromatin reorganization during SSC conversion into iGSCs, which was highly similar to female germline stem cells. We observed that although topologically associating domains were stable during SSC conversion, chromatin interactions changed in a striking manner, altering 35% of inactive and active chromosomal compartments throughout the genome. Conclusion: We demonstrate successful offspring production of ovarian organoids derived from SSCs by defined factors with chromatin reorganization. These findings have important implications in various areas including mammalian gametogenesis, genetic and epigenetic reprogramming, biotechnology, and medicine.

Spermidine induces cytoprotective autophagy of female germline stem cells in vitro and ameliorates aging caused by oxidative stress through upregulated sequestosome-1/p62 expression.

BACKGROUND: Autophagy is required for oogenesis and plays a critical role in response to aging caused by oxidative stress. However, there have been no reports on regulation of cytoprotective autophagy in female germline stem cells (FGSCs) in response to aging caused by oxidative stress. RESULTS: We found that Spermidine (SPD) significantly increased protein expression of autophagy markers microtubule-associated protein 1 light chain 3 beta-II (MAP1LC3B-II/LC3B-II) and sequestosome-1/p62 (SQSTM1/p62), and evoked autophagic flux in FGSCs. Moreover, SPD increased the number and viability of FGSCs in vitro. Further, we found that SPD significantly reduced basal or hydrogen peroxide (H2O2)-induced up-regulated protein expression of the aging markers, cyclin dependent kinase inhibitor 2A (p16/CDKN2A) and tumor protein 53 (p53). After knockdown of p62 in FGSCs, p16 protein levels were significant higher compared with controls. However, protein p16 levels were not significantly changed in p62 knockdown FGSCs with SPD treatment compared with without SPD. Moreover, SPD significantly changed the expression of autophagy-related genes and pathways in FGSCs, as shown by bioinformatics analysis of RNA sequencing data. Additionally, SPD significantly inhibited AKT/mTOR phosphorylation. CONCLUSIONS: SPD induces cytoprotective autophagy in FGSCs in vitro and ameliorates cellular senescence of FGSCs induced by H2O2. Furthermore, SPD can ameliorate cellular senescence of FGSCs through p62. SPD might induce autophagy in FGSCs via the PI3K/Akt pathway. Our findings could be helpful for delaying aging of female germ cells due to oxidative stress and preserving female fertility.

[Establishment of an auxiliary diagnosis system of newborn screening for inherited metabolic diseases based on artificial intelligence technology and a clinical trial].

Objective: To establish a disease risk prediction model for the newborn screening system of inherited metabolic diseases by artificial intelligence technology. Methods: This was a retrospectively study. Newborn screening data (n=5 907 547) from February 2010 to May 2019 from 31 hospitals in China and verified data (n=3 028) from 34 hospitals of the same period were collected to establish the artificial intelligence model for the prediction of inherited metabolic diseases in neonates. The validity of the artificial intelligence disease risk prediction model was verified by 360 814 newborns' screening data from January 2018 to September 2018 through a single-blind experiment. The effectiveness of the artificial intelligence disease risk prediction model was verified by comparing the detection rate of clinically confirmed cases, the positive rate of initial screening and the positive predictive value between the clinicians and the artificial intelligence prediction model of inherited metabolic diseases. Results: A total of 3 665 697 newborns' screening data were collected including 3 019 cases' positive data to establish the 16 artificial intelligence models for 32 inherited metabolic diseases. The single-blind experiment (n=360 814) showed that 45 clinically diagnosed infants were detected by both artificial intelligence model and clinicians. A total of 2 684 cases were positive in tandem mass spectrometry screening and 1 694 cases were with high risk in artificial intelligence prediction model of inherited metabolic diseases, with the positive rates of tandem 0.74% (2 684/360 814)and 0.46% (1 694/360 814), respectively. Compared to clinicians, the positive rate of newborns was reduced by 36.89% (990/2 684) after the application of the artificial intelligence model, and the positive predictive values of clinicians and artificial intelligence prediction model of inherited metabolic diseases were 1.68% (45/2 684) and 2.66% (45/1 694) respectively. Conclusion: An accurate, fast, and the lower false positive rate auxiliary diagnosis system for neonatal inherited metabolic diseases by artificial intelligence technology has been established, which may have an important clinical value.

Comparison of RNA m6A and DNA methylation profiles between mouse female germline stem cells and STO cells.

N6-methyladenosine (m6A) methylation modification is the most prevalent and abundant internal modification of eukaryotic mRNAs. Increasing evidence has shown that mRNA m6A plays important roles in the development of stem cells. However, to the best of our knowledge, no reports about the roles of mRNA m6A in mouse female germline stem cells (mFGSCs) have been published. In this study, we compared the genome-wide profiles of mRNA m6A methylation and DNA methylation between FGSCs and sandosinbred mice (SIM) embryo-derived thioguanine and ouabain-resistant (STO) cells. qRT-PCR revealed that the expression levels of mRNA m6A-related genes (Mettl3, Alkbh5, Ythdf1, Ythdf2, Ythdc1, and Ythdc2) in FGSCs were significantly higher than those in STO cells. m6A RNA immunoprecipitation sequencing (MeRIP-seq) data further showed that the unique m6A-methylated mRNAs in FGSCs and STO cells were related to cell population proliferation and somatic development, respectively. Additionally, knockdown of Ythdf1 inhibited FGSC self-renewal. Comparison of methylated DNA immunoprecipitation sequencing (MeDIP-seq) results between FGSCs and STO cells identified that DNA methylation contributed to FGSC proliferation by suppressing the somatic program. These results suggested that m6A regulated FGSC self-renewal possibly through m6A binding protein YTHDF1, and DNA methylation repressed somatic programs in FGSCs to maintain FGSC characteristics.

C28 induced autophagy of female germline stem cells in vitro with changes of H3K27 acetylation and transcriptomics.

There are a few studies indicating that small molecular compounds affect the proliferation, differentiation, apoptosis, and autophagy of female germline stem cells (FGSCs). However, whether small molecular compound 28 (C28) affect development of FGSCs remains unknown. In this study, we found that C28 reduced the viability and proliferation of FGSCs, respectively. Additionally, western blotting showed that the expression of autophagy marker light chain 3 beta II (LC3B-II) was significantly increased and expression of sequestosome-1 (SQSTM1) was significantly reduced in C28-treated groups. Immunofluorescence showed that, in C28-treated groups, the number of LC3B-II-positive puncta was increased significantly. These results indicated that C28 induced autophagy of FGSCs in vitro. Furthermore, data from Chromatin Immunoprecipitation Sequencing for H3K27ac showed that autophagy-related biological processes such as regulation of mitochondrial membrane potential, Golgi vesicle transport, and cellular response to reactive oxygen species were different after C28-treated. In addition, RNA-Seq showed that the expression of genes (Trib3, DDIT3, and ATF4) related to endoplasmic reticulum (ER) stress was enhanced by C28. These results suggest that the changes of H3K27ac and ER stress might be associated with C28-induced FGSC autophagy.

Identifying cortical specific long noncoding RNAs modified by m6A RNA methylation in mouse brains.

Proper development of the mammalian cerebral cortex relies on precise gene expression regulation. Increasing evidence shows that cortical development is regulated by both mRNAs and long noncoding RNAs (lncRNAs), which also are modified by N6-methyladenosine (m6A). Patterns of m6A-methylation in lncRNAs in the developing cortex have not been uncovered. Here we reveal differentially expressed lncRNAs and report stage-specific m6A-methylation patterns in lncRNAs expressed in mouse embryonic (E) and postnatal (P) cortices using RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation (MeRIP) sequencing. Many lncRNAs show temporal differential expression, and display genic distribution in the genome. Interestingly, we detect temporal-specific m6A-methylation with consensus m6A motif GGACU in the last exon in most lncRNAs. And m6A methylation levels of lncRNAs are positively correlated with the transcript abundance of lncRNAs that have no significantly differential expression in E- and P-stages. Furthermore, the transcript abundance has a positive correlation between the m6A genic lncRNAs and their nearest m6A methylated mRNAs. Our work reveals a fundamental expression reference of lncRNAs and their nearest mRNAs, and highlights an importance of m6A-mediated epitranscriptomic modifications in lncRNAs that are temporally expressed in the developing cortex.

Fe(II) interaction with cement phases: Method development, wet chemical studies and X-ray absorption spectroscopy.

Fe(II) interaction with cement phases was studied by means of co-precipitation and sorption experiments in combination with X-ray absorption fine structure (XAFS) spectroscopy. Oxidation of Fe(II) was fast in alkaline conditions and therefore, a methodology was developed which allowed Fe(II) to be stabilised in the sorption experiments and to prepare samples for spectroscopy. X-ray diffraction (XRD) of the co-precipitation samples showed uptake of a small portion of Fe(II) by calcium-silicate-hydrates (C-S-H) in the interlayer indicated by an increase in the interlayer spacing. Fe(II) incorporation by AFm phases was not indicated. Wet chemical experiments using 55Fe radiotracer revealed linear sorption of Fe(II) irrespective of the Ca/Si ratio of C-S-H and equilibrium pH. The Kd values for Fe(II) sorption on C-S-H are more than three orders of magnitude lower as compared to Fe(III), while they are comparable to those of other bivalent metal cations. XAFS spectroscopy showed Fe(II) binding by C-S-H in an octahedral coordination environment. The large number of neighbouring atoms rules out the formation of a single surface-bound Fe(II) species. Instead the data suggest presence of Fe(II) in a structurally bound entity. The data from XRD and XAFS spectroscopy suggests the presence of both surface- and interlayer-bound Fe(II) species.

Integrated Glycosylation Patterns of Glycoproteins and DNA Methylation Landscapes in Mammalian Oogenesis and Preimplantation Embryo Development.

Glycosylation is one of the most fundamental post-translational modifications. However, the glycosylation patterns of glycoproteins have not been analyzed in mammalian preimplantation embryos, because of technical difficulties and scarcity of the required materials. Using high-throughput lectin microarrays of low-input cells and electrochemical techniques, an integration analysis of the DNA methylation and glycosylation landscapes of mammal oogenesis and preimplantation embryo development was performed. Highly noticeable changes occurred in the level of protein glycosylation during these events. Further analysis identified several stage-specific lectins including LEL, MNA-M, and MAL I. It was later confirmed that LEL was involved in mammalian oogenesis and preimplantation embryogenesis, and might be a marker of FGSC differentiation. Modified nanocomposite polyaniline/AuNPs were characterized by electron microscopy and modification on bare gold electrodes using layer-by-layer assembly technology. These nanoparticles were further subjected to accuracy measurements by analyzing the protein level of ten-eleven translocation protein (TET), which is an important enzyme in DNA demethylation that is regulated by O-glycosylation. Subsequent results showed that the variations in the glycosylation patterns of glycoproteins were opposite to those of the TET levels. Moreover, analysis of correlation between the changes in glyco-gene expression and female germline stem cell glycosylation profiles indicated that glycosylation was related to DNA methylation. Subsequent integration analysis showed that the trend in the variations of glycosylation patterns of glycoproteins was similar to that of DNA methylation and opposite to that of the TET protein levels during female germ cell and preimplantation embryo development. Our findings provide insight into the complex molecular mechanisms that regulate human embryo development, and a foundation for further elucidation of early embryonic development and informed reproductive medicine.