Spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium

Molecular knowledge of human gastric corpus epithelium remains incomplete. Here, by integrated analyses using single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) techniques, we uncovered the spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium. Specifically, we identified a stem/progenitor cell population in the isthmus of human gastric corpus, where EGF and WNT signaling pathways were activated. Meanwhile, LGR4, but not LGR5, was responsible for the activation of WNT signaling pathway. Importantly, FABP5 and NME1 were identified and validated as crucial for both normal gastric stem/progenitor cells and gastric cancer cells. Finally, we explored the epigenetic regulation of critical genes for gastric corpus epithelium at chromatin state level, and identified several important cell-type-specific transcription factors. In summary, our work provides novel insights to systematically understand the cellular diversity and homeostasis of human gastric corpus epithelium in vivo.

Chromatin Remodeling Factor SMARCA5 is Essential for Hippocampal Memory Maintenance via Metabolic Pathways in Mice / 神经科学通报·英文版

Gene transcription and new protein synthesis regulated by epigenetics play integral roles in the formation of new memories. However, as an important part of epigenetics, the function of chromatin remodeling in learning and memory has been less studied. Here, we showed that SMARCA5 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 5), a critical chromatin remodeler, was responsible for hippocampus-dependent memory maintenance and neurogenesis. Using proteomics analysis, we found protein expression changes in the hippocampal dentate gyrus (DG) after the knockdown of SMARCA5 during contextual fear conditioning (CFC) memory maintenance in mice. Moreover, SMARCA5 was revealed to participate in CFC memory maintenance via modulating the proteins of metabolic pathways such as nucleoside diphosphate kinase-3 (NME3) and aminoacylase 1 (ACY1). This work is the first to describe the role of SMARCA5 in memory maintenance and to demonstrate the involvement of metabolic pathways regulated by SMARCA5 in learning and memory.

Recent advances in prostate cancer: WNT signaling, chromatin regulation, and transcriptional coregulators / 亚洲男科学杂志(英文版)

Prostate cancer is one of the most common diseases in men worldwide. Surgery, radiation therapy, and hormonal therapy are effective treatments for early-stage prostate cancer. However, the development of castration-resistant prostate cancer has increased the mortality rate of prostate cancer. To develop novel drugs for castration-resistant prostate cancer, the molecular mechanisms of prostate cancer progression must be elucidated. Among the signaling pathways regulating prostate cancer development, recent studies have revealed the importance of noncanonical wingless-type MMTV integration site family (WNT) signaling pathways, mainly that involving WNT5A, in prostate cancer progression and metastasis; however, its role remains controversial. Moreover, chromatin remodelers such as the switch/sucrose nonfermentable (SWI/SNF) complex and chromodomain helicase DNA-binding proteins 1 also play important roles in prostate cancer progression through genome-wide gene expression changes. Here, we review the roles of noncanonical WNT signaling pathways, chromatin remodelers, and epigenetic enzymes in the development and progression of prostate cancer.

Effects and mechanism of p53 gene deletion on energy metabolism during the pluripotent transformation of spermatogonial stem cells / 生理学报

Previous studies have shown that long-term spermatogonial stem cells (SSCs) have the potential to spontaneously transform into pluripotent stem cells, which is speculated to be related to the tumorigenesis of testicular germ cells, especially when p53 is deficient in SSCs which shows a significant increase in the spontaneous transformation efficiency. Energy metabolism has been proved to be strongly associated with the maintenance and acquisition of pluripotency. Recently, we compared the difference in chromatin accessibility and gene expression profiles between wild-type (p53+/+) and p53 deficient (p53-/-) mouse SSCs using the Assay for Targeting Accessible-Chromatin with high-throughput sequencing (ATAC-seq) and transcriptome sequencing (RNA-seq) techniques, and revealed that SMAD3 is a key transcription factor in the transformation of SSCs into pluripotent cells. In addition, we also observed significant changes in the expression levels of many genes related to energy metabolism after p53 deletion. To further reveal the role of p53 in the regulation of pluripotency and energy metabolism, this paper explored the effects and mechanism of p53 deletion on energy metabolism during the pluripotent transformation of SSCs. The results of ATAC-seq and RNA-seq from p53+/+ and p53-/- SSCs revealed that gene chromatin accessibility related to positive regulation of glycolysis and electron transfer and ATP synthesis was increased, and the transcription levels of genes encoding key glycolytic enzymes and regulating electron transport-related enzymes were markedly increased. Furthermore, transcription factors SMAD3 and SMAD4 promoted glycolysis and energy homeostasis by binding to the chromatin of the Prkag2 gene which encodes the AMPK subunit. These results suggest that p53 deficiency activates the key enzyme genes of glycolysis in SSCs and enhances the chromatin accessibility of genes associated with glycolysis activation to improve glycolysis activity and promote transformation to pluripotency. Moreover, SMAD3/SMAD4-mediated transcription of the Prkag2 gene ensures the energy demand of cells in the process of pluripotency transformation and maintains cell energy homeostasis by promoting AMPK activity. These results shed light on the importance of the crosstalk between energy metabolism and stem cell pluripotency transformation, which might be helpful for clinical research of gonadal tumors.

Curcumin alleviates the manganese-induced neurotoxicity by promoting autophagy in rat models of manganism / 北京大学学报(医学版)

OBJECTIVE@#To investigate the protective effects of curcumin(CUR) and its mechanism on a rat model of neurotoxicity induced by manganese chloride (MnCl2), which mimics mangnism.@*METHODS@#Sixty male SD rats were randomly divided into 5 groups, with 12 rats in each group. Control group received 0.9% saline solution intraperitoneally (ip) plus double distilled water (dd) H2O intragastrically (ig), MnCl2 group received 15 mg/kg MnCl2(Mn2+ 6.48 mg/kg) intraperitoneally plus dd H2O intragastrically, CUR group received 0.9% saline solution intraperitoneally plus 300 mg/kg CUR intragastrically, MnCl2+ CUR1 group received 15 mg/kg MnCl2 intraperitoneally plus 100 mg/kg curcumin intragastrically, MnCl2+ CUR2 group received 15 mg/kg MnCl2 intraperitoneally plus 300 mg/kg CUR intragastrically, 5 days/week, 4 weeks. Open-field and rotarod tests were used to detect animals' exploratory behavior, anxiety, depression, movement and balance ability. Morris water maze (MWM) experiment was used to detect animals' learning and memory ability. ICP-MS was used to investigate the Mn contents in striata. The rats per group were perfused in situ, their brains striata were removed by brains model and fixed for transmission electron microscope (TEM), histopathological and immunohistochemistry (ICH) analyses. The other 6 rats per group were sacrificed. Their brains striata were removed and protein expression levels of transcription factor EB (TFEB), mammalian target of rapamycin (mTOR), p-mTOR, Beclin, P62, microtubule-associated protein light chain-3 (LC3) were detected by Western blotting. Terminal deoxynucleotidyl transterase-mediated dUTP nick end labeling (TUNEL) staining was used to determine neurocyte apoptosis of rat striatum.@*RESULTS@#After exposure to MnCl2 for four weeks, MnCl2-treated rats showed depressive-like behavior in open-field test, the impairments of movement coordination and balance in rotarod test and the diminishment of spatial learning and memory in MWM (P < 0.05). The striatal TH+ neurocyte significantly decreased, eosinophilic cells, aggregative α-Syn level and TUNEL-positive neurocyte significantly increased in the striatum of MnCl2 group compared with control group (P < 0.05). Chromatin condensation, mitochondria tumefaction and autophagosomes were observed in rat striatal neurocytes of MnCl2 group by TEM. TFEB nuclear translocation and autophagy occurred in the striatum of MnCl2 group. Further, the depressive behavior, movement and balance ability, spatial learning and memory ability of MnCl2+ CUR2 group were significantly improved compared with MnCl2 group (P < 0.05). TH+ neurocyte significantly increased, the eosinophilic cells, aggregative α-Syn level significantly decreased in the striatum of MnCl2+ CUR2 group compared with MnCl2 group. Further, compared with MnCl2 group, chromatin condensation, mitochondria tumefaction was alleviated and autophagosomes increased, TFEB-nuclear translocation, autophagy was enhanced and TUNEL-positive neurocyte reduced significantly in the striatum of MnCl2+ CUR2 group (P < 0.05).@*CONCLUSION@#Curcumin alleviated the MnCl2-induced neurotoxicity and α-Syn aggregation probably by promoting TFEB nuclear translocation and enhancing autophagy.

Characterization of chromatin accessibility in psoriasis / 医学前沿

The pathological hallmarks of psoriasis involve alterations in T cell genes associated with transcriptional levels, which are determined by chromatin accessibility. However, to what extent these alterations in T cell transcriptional levels recapitulate the epigenetic features of psoriasis remains unknown. Here, we systematically profiled chromatin accessibility on Th1, Th2, Th1-17, Th17, and Treg cells and found that chromatin remodeling contributes significantly to the pathogenesis of the disease. The chromatin remodeling tendency of different subtypes of Th cells were relatively consistent. Next, we profiled chromatin accessibility and transcriptional dynamics on memory Th/Treg cells. In the memory Th cells, 803 increased and 545 decreased chromatin-accessible regions were identified. In the memory Treg cells, 713 increased and 1206 decreased chromatin-accessible regions were identified. A total of 54 and 53 genes were differentially expressed in the peaks associated with the memory Th and Treg cells. FOSL1, SPI1, ATF3, NFKB1, RUNX, ETV4, ERG, FLI1, and ETC1 were identified as regulators in the development of psoriasis. The transcriptional regulatory network showed that NFKB1 and RELA were highly connected and central to the network. NFKB1 regulated the genes of CCL3, CXCL2, and IL1RN. Our results provided candidate transcription factors and a foundational framework of the regulomes of the disease.

Transcriptome Analysis of Schwann Cells at Various Stages of Myelination Implicates Chromatin Regulator Sin3A in Control of Myelination Identity / 神经科学通报·英文版

Enhancing remyelination after injury is of utmost importance for optimizing the recovery of nerve function. While the formation of myelin by Schwann cells (SCs) is critical for the function of the peripheral nervous system, the temporal dynamics and regulatory mechanisms that control the progress of the SC lineage through myelination require further elucidation. Here, using in vitro co-culture models, gene expression profiling of laser capture-microdissected SCs at various stages of myelination, and multilevel bioinformatic analysis, we demonstrated that SCs exhibit three distinct transcriptional characteristics during myelination: the immature, promyelinating, and myelinating states. We showed that suppressor interacting 3a (Sin3A) and 16 other transcription factors and chromatin regulators play important roles in the progress of myelination. Sin3A knockdown in the sciatic nerve or specifically in SCs reduced or delayed the myelination of regenerating axons in a rat crushed sciatic nerve model, while overexpression of Sin3A greatly promoted the remyelination of axons. Further, in vitro experiments revealed that Sin3A silencing inhibited SC migration and differentiation at the promyelination stage and promoted SC proliferation at the immature stage. In addition, SC differentiation and maturation may be regulated by the Sin3A/histone deacetylase2 (HDAC2) complex functionally cooperating with Sox10, as demonstrated by rescue assays. Together, these results complement the recent genome and proteome analyses of SCs during peripheral nerve myelin formation. The results also reveal a key role of Sin3A-dependent chromatin organization in promoting myelinogenic programs and SC differentiation to control peripheral myelination and repair. These findings may inform new treatments for enhancing remyelination and nerve regeneration.

Effect of Buyi Pishen acupuncture on inflammatory factor and cartilage matrix in adjuvant arthritis rats / 中国针灸

OBJECTIVE@#To observe the effect of Buyi Pishen acupuncture (acupuncture for invigorating spleen and kidney) on inflammatory factor and synovial cartilage matrix in adjuvant arthritis (AA) rats, and to explore the mechanism of acupuncture for rheumatoid arthritis (RA).@*METHODS@#A total of 60 clean male Wistar rats were randomized into a normal group, a model group, a tripterygium wilfordii polyglycoside tablet (TWP) group and an acupuncture group, 15 rats in each group. Rats in the model group, the TWP group and the acupuncture group received intradermal injection of Freund's complete adjuvant (FCA) at right hind foot pad to induce the AA model. TWP suspension of 8 mg/kg was given by gavage in the TWP group. Acupuncture was applied at "Shenshu" (BL 23), "Pishu" (BL 20) and right "Housanli" (ST 36), "Sanyinjiao" (SP 6), "Yanglingquan" (GB 34) in the acupuncture group, 15 min a time, once a day. The intervention was given 15 days in both TWP group and acupuncture group. The foot-pad swelling degree before modeling, before and after intervention and the arthritis index (AI) score before and after intervention were calculated; the serum levels of interleukin (IL)-1β, IL-4, IL-10 and tumor necrosis factor-α (TNF-α) were detected by ELISA method; the ultrastructure and histomorphological changes of synovium issue were observed by transmission electron microscope and HE staining; the positive expression of matrix metalloproteinase (MMP)-3 and MMP-9 in synovium issue was detected by immunohistochemistry method.@*RESULTS@#Before intervention, foot-pad swelling degree of the model group, the TWP group and the acupuncture group was increased compared with the normal group (P<0.01). After intervention, foot-pad swelling degree and AI score were increased compared with the normal group (P<0.01), foot-pad swelling degree and AI scores in the TWP group and the acupuncture group were lower than the model group (P<0.05), and those in the acupuncture group were decreased compared with the TWP group (P<0.05). The model group exhibited unclear nuclear membrane of synovial cells, chromatin pyknosis, massive inflammatory cell infiltration and hyperplasia in synovial tissue; the TWP group and the acupuncture group exhibited clear and smooth nuclear membrane of synovial cells, inapparent chromatin pyknosis, less inflammatory cell infiltration and hyperplasia in synovial tissue, the acupuncture group exhibited less matrix destruction as well. Compared with the normal group, serum levels of IL-1β and TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were increased (P<0.01), while serum levels of IL-4 and IL-10 were decreased (P<0.01) in the model group. Compared with the model group, serum levels of IL-1β and TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were decreased (P<0.05, P<0.01), while serum levels of IL-4 and IL-10 were increased (P<0.05) in the TWP group and the acupuncture group; compared with the TWP group, serum level of TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were decreased (P<0.05), while serum levels of IL-4 and IL-10 were increased (P<0.05) in the acupuncture group.@*CONCLUSION@#Buyi Pishen acupuncture can effectively improve the injury of articular cartilage in AA rats, its mechanism maybe related to reducing the inflammatory reaction in synovium and inhibiting the degradation of articular cartilage matrix.

Evaluation of the sperm DNA fragmentation index in infertile Japanese men by in-house flow cytometric analysis / 亚洲男科学杂志(英文版)

Semen analysis has long been used to evaluate male fertility. Recently, several sperm function tests have been developed. Of those, the sperm DNA fragmentation index (DFI), which describes the status of the sperm DNA, is thought to be a suitable parameter for evaluating male fertility. However, there have been no large-scale studies on the sperm DFI of Japanese men. Therefore, we investigated the feasibility of using an in-house flow cytometry-based sperm DFI analysis based on the sperm DNA fragmentation test of sperm chromatin structure assay (SCSA) to assess male fertility in Japan. This study enrolled 743 infertile and 20 fertile Japanese men. To evaluate reproducibility, inter- and intraobserver precision was analyzed. A receiver operating characteristic curve analysis was used to set a cutoff value for the sperm DFI to identify men who could father children by timed intercourse or intrauterine insemination. The variability of the sperm DFI among fertile volunteers was determined. The relationship between semen parameters and the sperm DFI was assessed by Spearman's rho test. A precision analysis revealed good reproducibility of the sperm DFI. The cutoff value of sperm DNA fragmentation in infertile men was 24.0%. Semen volume had no relationship with the sperm DFI. Sperm concentration, sperm motility, total motile sperm count, and percentage of normal-shaped sperm were significantly and negatively correlated with the sperm DFI. The median sperm DFI was smaller in fertile volunteers (7.7%) than that in infertile men (19.4%). Sperm DNA fragmentation analysis can be used to assess sperm functions that cannot be evaluated by ordinary semen analysis.

Nuclear peripheral chromatin-lamin B1 interaction is required for global integrity of chromatin architecture and dynamics in human cells

The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.

Non-coding RNAs and chromatin: key epigenetic factors from spermatogenesis to transgenerational inheritance

Cellular fate and gene expression patterns are modulated by different epigenetic factors including non-coding RNAs (ncRNAs) and chromatin organization. Both factors are dynamic throughout male germ cell differentiation on the seminiferous tubule, despite the transcriptional inactivation in the last stages of spermatogenesis. Sperm maturation during the caput-to-cauda transit on the epididymis involves changes in chromatin organization and the soma-to-germ line transference of ncRNAs that are essential to obtain a functional sperm for fertilization and embryo development. Here, the male environment (diseases, drugs, mental stress) is crucial to modulate these epigenetic factors throughout sperm maturation, affecting the corresponding offspring. Paternal transgenerational inheritance has been directly related to sperm epigenetic changes, most of them associated with variations in the ncRNA content and chromatin marks. Our aim is to give an overview about how epigenetics, focused on ncRNAs and chromatin, is pivotal to understand spermatogenesis and sperm maturation, and how the male environment impacts the sperm epigenome modulating the offspring gene expression pattern.

Effect of interactions of chromatin regulatory elements with different promoters on the regulation of gene expression / 生物工程学报

The effect of altering the promoter region of ubiquitous chromatin-opening element (UCOE) and matrix attachment region (MAR) on stable and efficient expression of genes was investigated. Four different promoters were tested, namely, oct4 containing an enhancer region, sox2 having a CpG island, nanog having no regulatory elements, and CMV containing a CpG island and an enhancer region. Eight reporter plasmids were constructed: pOCT4-UCOE, pOCT4-MAR, pSOX2-UCOE, pSOX2-MAR, pNANOG-UCOE, pNANOG-MAR, pCMV-UCOE, and pCMV-MAR. Stable and efficient expression was observed when UCOE combined with the oct4 promoter, whereas the sox2 was the best promoter suited for MAR. Comparison of the stable clones of oct4-UCOE and sox2-MAR showed that UCOE-regulated expression is more stable and efficient than MAR-regulated expression. When CpG island-containing promoter is linked with UCOE, stable and efficient expression could be observed. These data suggest that an enhancer region in the promoter leads to high, yet unstable expression when combined with UCOE, whereas CpG islands stabilize expression. In conclusion, UCOE and MAR interact with regulatory elements on the promoter by altering the chromatin open state and chromatin loop to regulate gene expression.

Advances of CRISPR/dCas9 system in live cell imaging / 生物工程学报

The study of distinct genes, chromosomes and the spatio-temporal relationships between them is of great significance in genetics, developmental biology and biomedicine. CRISPR/Cas9 has become the most widely used gene editing tool due to its excellent targeting ability. Recently, researchers have developed a series of advanced live cell imaging techniques based on the nuclease-inactivated mutant of Cas9 (dCas9), providing rapid and convenient tools for high-resolution imaging of specific sites in the chromatin and genome. This review summarizes the advances of CRISPR/dCas9 system in live cell imaging from three aspects, including the strategies of cell delivery, optimization of the fluorescence signals, as well as orthogonal and multicolor imaging. Furthermore, we shed light on the development trends and prospects of this field.

A review on the genetic mechanism of chromatin remodeling in children with neurodevelopmental disorders / 中国当代儿科杂志

Neural development is regulated by both external environment and internal signals, and in addition to transcription factors, epigenetic modifications also play an important role. By focusing on the genetic mechanism of ATP-dependent chromatin remodeling in children with neurodevelopmental disorders, this article elaborates on the effect of four chromatin remodeling complexes on neurogenesis and the development and maturation of neurons and neuroglial cells and introduces the clinical research advances in neurodevelopmental disorders.

Follicular Lesions with Papillary Nuclear Characteristics: Differences in Chromatin Detected by Computerized Image Analysis

ABSTRACT Objective: Follicular lesions of the thyroid with papillary carcinoma nuclear characteristics are classified as infiltrative follicular variant of papillary thyroid carcinoma-FVPTC (IFVPTC), encapsulated/well demarcated FVPTC with tumour capsular invasion (IEFVPTC), and the newly described category "non-invasive follicular thyroid neoplasm with papillary-like nuclear features" (NIFTP) formerly known as non-invasive encapsulated FVPTC. This study evaluated whether computerized image analysis can detect nuclear differences between these three tumour subtypes. Materials and methods: Slides with histological material from 15 cases of NIFTP and 33 cases of FVPTC subtypes (22 IEFVPTC, and 11 IFVPTC) were analyzed using the Image J image processing program. Tumour cells were compared for both nuclear morphometry and chromatin textural characteristics. Results: Nuclei from NIFTP and IFVPTC tumours differed in terms of chromatin textural features (grey intensity): mean (92.37 ± 21.01 vs 72.99 ± 14.73, p = 0.02), median (84.93 ± 21.17 vs 65.18 ± 17.08, p = 0.02), standard deviation (47.77 ± 9.55 vs 39.39 ± 7.18; p = 0.02), and coefficient of variation of standard deviation (19.96 ± 4.01 vs 24.75 ± 3.31; p = 0.003). No differences were found in relation to IEFVPTC. Conclusion: Computerized image analysis revealed differences in nuclear texture between NIFTP and IFVPTC, but not for IEFVPTC.

Proteomic Signatures in Spermatozoa Reveal the Role of Paternal Factors in Recurrent Pregnancy Loss

Advances in three-dimensional genomics / 生物工程学报

Three-dimensional (3D) genomics is an emerging discipline that studies the 3D spatial structure and function of genomes, focusing on the 3D spatial conformation of genome sequences in the nucleus and its biological effects on biological processes such as DNA replication, DNA recombination and gene expression regulation. The invention of chromosome conformation capture (3C) technology speeds up the research on 3D genomics and its related fields. Furthermore, the development of 3C-based technologies, such as the genome-wide chromosome conformation capture (Hi-C) and chromatin interaction analysis using paired-end tag sequencing (ChIA-PET), help scientists get insight into the 3D genomes of various species. Aims of 3D genomics are to reveal the spatial genome organization, chromosomal interaction patterns, mechanisms underlying the transcriptional regulation and formation of biological traits of microorganism, plant, animal. Additionally, the identification of key genes and signaling pathways associated with biological processes and disease via chromosome 3C technology boosts the rapid development of agricultural science, life science and medical science. This paper reviews the research progress of 3D genomics, mainly in the concept of 3D genomics, the development of chromosome 3C technologies and their applications in agricultural science, life science and medical science, specifically in the field of tumor.

Three-dimensional chromosome conformation capture and its derived technologies / 生物工程学报

Linear chromatin is compacted into eukaryotic nucleus through a complex and multi-layered architecture. Consequently, chromatin conformation in a local or long-distance manner is strongly correlated with gene expression. Chromosome conformation capture (3C) technology, together with its variants like 4C/5C/Hi-C, has been well developed to study chromatin looping and whole genome structure. In this review, we introduce new technologies including chromosome capture combined with immunoprecipitation, nuclei acid-based hybridization, single cell and genome sequencing, as well as their application.

Research progress on methylation of histones H3K9 and H3K27 in early-stage embryos / 中华医学遗传学杂志

With the in-depth exploration of all stages in early-stage embryos, in particular zygotic genome activation and first cell lineage differentiation, researchers have found that early embryonic epigenetics follows a strict pattern of temporal and spatial modification. Previous studies have determined the inhibitory effect of H3K9me3 and H3K27me3 on genomic expression, and found that they are involved in many core biological events in the genome such as chromatin reprogramming, genomic imprinting, maintenance of embryonic stem cell pluripotency and somatic cell nuclear transfer, though the detailed molecular mechanism has remained elusive. From the point of developmental biology and epigenetics, this article has expounded the research progress on the methylation of H3K9 and H3K27 histones in early-stage embryos, which may provide a clue for the complex mechanism of embryonic development and improvement of culture method for embryos in vitro.

Fluoxetine Induces Apoptotic and Oxidative Neuronal Death Associated with The Influx of Copper Ions in Cultured Neuronal Cells / 전남의대학술지

We examined the effect of fluoxetine, a selective serotonin reuptake inhibitor antidepressant, on neuronal viability in mouse cortical near-pure neuronal cultures. Addition of fluoxetine to the media for 24 hours induced neuronal death in a concentration-dependent manner. To delineate the mechanisms of fluoxetine-induced neuronal death, we investigated the effects of trolox, cycloheximide (CHX), BDNF, z-VAD-FMK, and various metal-chelators on fluoxetine-induced neuronal death. Neuronal death was assessed by MTT assay. The addition of 20 µM fluoxetine to the media for 24 hours induced 60–70% neuronal death, which was associated with the hallmarks of apoptosis, chromatin condensation and DNA laddering. Fluoxetine-induced death was significantly attenuated by CHX, BDNF, or z-VAD-FMK. Treatment with antioxidants, trolox and ascorbate, also markedly attenuated fluoxetine-induced death. Interestingly, some divalent cation chelators (EGTA, Ca-EDTA, and Zn-EDTA) also markedly attenuated the neurotoxicity. Fluoxetine-induced reactive oxygen species (ROS) generation was measured using the fluorescent dye 2′,7′-dichlorofluorescin diacetate. Trolox and bathocuproine disulfonic acid (BCPS), a cell membrane impermeable copper ion chelator, markedly attenuated the ROS production and neuronal death. However, deferoxamine, an iron chelator, did not affect ROS generation or neurotoxicity. We examined the changes in intracellular copper concentration using a copper-selective fluorescent dye, Phen Green FL, which is quenched by free copper ions. Fluoxetine quenched the fluorescence in neuronal cells, and the quenching effect of fluoxetine was reversed by co-treatment with BCPS, however, not by deferoxamine. These findings demonstrate that fluoxetine could induce apoptotic and oxidative neuronal death associated with an influx of copper ions.