A histidine cluster determines YY1-compartmentalized coactivators and chromatin elements in phase-separated enhancer clusters.

As an oncogenic transcription factor, Yin Yang 1 (YY1) regulates enhancer and promoter connection. However, gaps still exist in understanding how YY1 coordinates coactivators and chromatin enhancer elements to assemble enhancers and super-enhancers. Here, we demonstrate that a histidine cluster in YY1's transactivation domain is essential for its formation of phase separation condensates, which can be extended to additional proteins. The histidine cluster is also required for YY1-promoted cell proliferation, migration, clonogenicity and tumor growth. YY1-rich nuclear puncta contain coactivators EP300, BRD4, MED1 and active RNA polymerase II, and colocalize with histone markers of gene activation, but not that of repression. Furthermore, YY1 binds to the consensus motifs in the FOXM1 promoter to activate its expression. Wild-type YY1, but not its phase separation defective mutant, connects multiple enhancer elements and the FOXM1 promoter to form an enhancer cluster. Consistently, fluorescent in situ hybridization (FISH) assays reveal the colocalization of YY1 puncta with both the FOXM1 gene locus and its nascent RNA transcript. Overall, this study demonstrates that YY1 activates target gene expression through forming liquid-liquid phase separation condensates to compartmentalize both coactivators and enhancer elements, and the histidine cluster of YY1 plays a determinant role in this regulatory mechanism.

Study on Lgr5 expression in pancreas tissues and organoids by lineage tracing.

Leucine rich repeat containing G protein-coupled receptor 5(Lgr5) is widely expressed in multiple tissues and can be used as a stem cell marker in a variety of epithelial organs (including the small intestine, colon, stomach and hair follicles). In this study, we used Lgr5-CreERT2+/- and Rosa26-mTmG hybridized transgenic mice to investigate the expression of Lgr5 in both ductal epithelial cells during pancreas development and in vitro cultured pancreatic duct organoids. After induction with Tamoxifen, the Lgr5 expression was analyzed by detecting the enhanced green fluorescence protein in the pancreatic tissue sections in adult animals and embryos at different developmental stages. The results showed that Lgr5 expression was detected neither in adult pancreatic duct epithelia nor in the embryonic pancreatic tissues at day 15.5 or in newborn mice. However, when 4-hydroxy-Tamoxifen was supplemented to the culture medium, EGFP could be detected in the primary pancreatic duct organoids from Lgr5-Cre ERT2+/-; Rosa26-mTmG mice. These results suggested that Lgr5 was not expressed in adult and embryonic pancreatic tissues; but could be expressed in the cultured pancreas ductal organoids. The research lays the foundation for exploring specific gene expression patterns in stem/progenitor cells during pancreatic development.

Copper promotes sheep pancreatic duct organoid growth by activation of an antioxidant protein 1-dependent MEK-ERK pathway.

Proper amounts of copper supplemented in livestock feed improve the physical growth and traits of farm animals. The pancreas is an important organ with both exocrine and endocrine portions. To investigate the role and mechanism of copper in the sheep pancreas, we first established sheep pancreatic duct organoids (sPDOs). We found that an appropriate amount of copper benefited the formation and growth of sPDOs, whereas excess or deficient copper damaged sPDOs. We found that the proliferation-stimulating effect of copper was related to the copper chaperone antioxidant protein 1 (ATOX1)-dependent activation of MEK-ERK1/2 signaling. Atox1 knockdown suppressed the cell proliferation of sPDOs, even in the presence of the MEK activator. These results indicate that moderate concentrations of copper promote sPDO growth through ATOX1-regulated cell proliferation by activation of MEK-ERK. Moreover, our study indicates that organoids may be a useful model to study organ growth mechanisms in livestock.

Molecular chaperone HspB2 inhibited pancreatic cancer cell proliferation via activating p53 downstream gene RPRM, BAI1, and TSAP6.

Heat shock proteins (HSPs) were known as the molecular chaperones, which play a pivotal role in the protein quality control system, ensuring correct folding of proteins, and facilitating the correct refolding of damaged proteins via the transient interaction with their substrate proteins. They also practice in the regulation of cell cycles and are involved in apoptosis. We found that HspB2 was almost completely silent in pancreatic cancer and few studies investigated the role of HspB2 in cancer cells, particularly in pancreatic cancer. Here, we reported that HspB2 effectively inhibited cell proliferation in Panc-1 cells. Specifically, we demonstrated that HspB2 could combine mut-p53 and change the DNA binding site of mutant p53, subsequently upregulated the expression of RPRM, BAI-1, and TSAP6 which were the downstream genes of wt-p53, participate in mediating downstream responses to p53, including inhibiting cell proliferation and angiogenesis. The main aim of this study is to investigate the relationship between HspB2 and p53, and provide a novel treatment strategy for pancreatic cancer.

Guanine-rich RNA binding protein GRSF1 inhibits myoblast differentiation through repressing mitochondrial ROS production.

Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the RNA-binding protein (RBP) family. GRSF1 regulates RNA metabolism through RNA processing, transport and translation in the cytoplasm and mitochondria. However, its role in myogenesis has not been investigated. Here, we demonstrated that the expression of mitochondrial GRSF1 was negatively related to the differentiation of mouse skeletal myoblasts. Interference with GRSF1 promotes myogenesis both in vitro and in vivo without affecting MyoD expression or cell proliferation. Further studies illustrated that GRSF1 regulated myogenic differentiation through direct targeting of mitochondrial GPX4, a key regulator of the cellular redox status, leading to the modulation of ROS levels, which is important for myogenesis. Our findings underscore a critical function for GRSF1 during skeletal myogenesis linked to its regulation of muscle redox homeostasis.

MiR-17 and miR-19 cooperatively promote skeletal muscle cell differentiation.

Skeletal myogenesis is a highly coordinated process that involves cell proliferation, differentiation and fusion controlled by a complex gene regulatory network. The microRNA gene cluster miR-17-92 has been shown to be related to this process; however, the exact role of each cluster member remains unclear. Here, we show that miR-17 and miR-20a could effectively promote the differentiation of both C2C12 myoblasts and primary bovine satellite cells. In contrast, miR-18a might play a negative role in C2C12 cell differentiation, while miR-19 and miR-92a had little influence. Transcriptome and target analyses revealed that miR-17 could act on Ccnd2, Jak1 and Rhoc genes that are critical for cell proliferation and/or fusion. Notably, the addition of miR-19 could reverse the lethal effect of miR-17 and could thus facilitate the maturation of myotubes. Furthermore, by co-injecting the lentiviral shRNAs of miR-17 and miR-19 into mouse tibialis anterior muscles, we demonstrated the wound healing abilities of the two miRNAs. Our findings indicate that in combination with miR-19, miR-17 is a potent inducer of skeletal muscle differentiation.

Zinc chelator TPEN induces pancreatic cancer cell death through causing oxidative stress and inhibiting cell autophagy.

The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems including immunodeficiency and sensory dysfunctions. Previous studies had shown that Zn availability was also important for tumor growth and progression. The aim of the present study was to investigate the potential mechanisms of N,N,N,N-Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (a membrane permeable zinc chelator) induced pancreatic cancer cell death. The text of inductively coupled plasma-mass spectrometry (ICP-MS) showed in human pancreatic cancer samples that the zinc content in cancer was higher than that in adjacent tissues. The pancreatic cancer cell lines Panc-1, 8988T, BxPc-3, and L3.6 were used in this study. Our results indicated that TPEN markedly induced cell death, via increasing reactive oxygen species (ROS) and restraining autophagy. Our data also indicated that TPEN-stimulated mitochondrial metabolism produced much ROS. Meanwhile, TPEN reduced the levels of glutathione (GSH) and triggered ROS outbreak, which were the main causes of cell death. In addition, cell autophagy was significantly depressed in Panc-1 cells treated by TPEN, which was due to the ability of disrupting lysosomal by TPEN. Thus, we thought zinc depletion by TPEN was a potential therapeutic strategy for pancreatic cancer.

Mutual inhibitions between epidermal growth factor receptor signaling and miR-124a control pancreatic progenitor proliferation.

Pancreatic stem/progenitor cells convert from a proliferative to a differentiated fate passing through proliferation cease to a resting state. However, the molecular mechanisms of cell cycle arrest are poorly understood. In this study, we demonstrated that the microRNA-124a (miR-124a) inhibited the proliferation of pancreatic progenitor cells both in vitro and ex vivo and promoted a quiescent state. The miR-124a directly targeted SOS Ras/Rac guanine nucleotide exchange factor 1 (SOS1), IQ motif-containing GTPase-activating protein 1 (IQGAP1), signal transducer and activator of transcription 3 (STAT3), and cyclin D2 (CCND2), thereby inactivating epidermal growth factor receptor (EGFR) downstream signaling pathways including mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK), phosphatidylinositol 3-kinase-protein kinase B (PI3K/AKT) and Janus kinase (JAK)/STAT3. miR-124a blocked cell proliferation mainly through targeting STAT3 to inhibit PI3K/AKT and JAK/STAT3 signaling. Moreover, miR-124a expression was negatively regulated by EGFR downstream PI3K/AKT signaling. These results indicated that miR-124a and EGFR signaling mutually interact to form a regulating circuit that determines the proliferation of pancreatic progenitor cells.

Classical swine fever virus replicated poorly in cells from MxA transgenic pigs.

BACKGROUND: In addition to their value as livestock, pigs are susceptible to classical swine fever virus (CSFV) and can serve as reservoirs for CSFV, allowing it to develop into an epizootic. CSFV, a pestivirus of the Flaviviridae family, has a single-stranded RNA genome. Recent research has indicated that the human MxA protein inhibits the life cycles of certain RNA viruses, such as members of the Bunyaviridae family, the Flaviviridae family and others. RESULTS: To produce pigs with antiviral protection against CSFV, transgenic pigs expressing human MxA were generated by nuclear transplantation. Cells from three MxA transgenic piglets were used to investigate in vitro antiviral activity of MxA aganist CSFV, and the results of in vitro indirect immunofluorescence assays, virus titration and real-time PCR indicated that the MxA transgenic pig has an antiviral capacity against CSFV. CONCLUSIONS: Transgene with human MxA on pigs is feasible. High levels of MxA expression do inhibit CSFV in vitro at early time points post-infection at 60-96dpi.

Inhibiting cyprinid herpesvirus-3 replication with CRISPR/Cas9.

OBJECTIVES: The potential of CRISPR/Cas9 gene editing to repress CyHV-3 was tested in vitro. RESULTS: By targeting two basic target genes necessary for the early transcription of CyHV-3, we show that virus transcription and particle release were significantly decreased by CRISPR/Cas9, as measured by quantitative real-time PCR and virus titration experiments, respectively. CONCLUSIONS: (A) The effectiveness is confirmed of the CRISPR/Cas9 system at repressing exogenous genes, including large viral genomic DNA, by introducing site-specific mutations in vitro. (B) The CyHV-3 virus replicates poorly in Cas9-positive cells. (C) The inhibition of thymidine kinase alone cannot block viral particle release.

Divergence and codon usage bias of Betanodavirus, a neurotropic pathogen in fish.

Betanodavirus is a small bipartite RNA virus of global economical significance that can cause severe neurological disorders to an increasing number of marine fish species. Herein, to further the understanding of the evolution of betanodavirus, Bayesian coalescent analyses were conducted to the time-stamped entire coding sequences of their RNA polymerase and coat protein genes. Similar moderate nucleotide substitution rates were then estimated for the two genes. According to age calculations, the divergence of the two genes into the four genotypes initiated nearly simultaneously at ∼700 years ago, despite the different scenarios, whereas the seven analyzed chimeric isolates might be the outcomes of a single genetic reassortment event taking place in the early 1980s in Southern Europe. Furthermore, codon usage bias analyses indicated that each gene had influences in addition to mutational bias and codon choice of betanodavirus was not completely complied with that of fish host.

Notch Signaling in Pancreatic Development.

The Notch signaling pathway plays a significant role in embryonic cell fate determination and adult tissue homeostasis. Various studies have demonstrated the deep involvement of Notch signaling in the development of the pancreas and the lateral inhibition of Notch signaling in pancreatic progenitor differentiation and maintenance. The targeted inactivation of the Notch pathway components promotes premature differentiation of the endocrine pancreas. However, there is still the contrary opinion that Notch signaling specifies the endocrine lineage. Here, we review the current knowledge of the Notch signaling pathway in pancreatic development and its crosstalk with the Wingless and INT-1 (Wnt) and fibroblast growth factor (FGF) pathways.

Evolution of mammalian and avian bornaviruses.

Recently, Avian Bornavirus (ABV) was identified to be a new member of the Bornaviridae family consisting solely of the mammal-infecting Borna disease virus (BDV). Here, to gain more insights into the evolution of these bornaviruses, the time-stamped N gene sequences of BDV genotype 1 (BDV1) and ABV were subjected to Bayesian coalescent analyses. The nucleotide substitution rates and the divergence times were estimated. Age calculations suggested that the first diversification event of the analyzed BDV1 isolates might have taken place about 300years ago, and revealed that ABV was an old virus newly recognized. Great differences were observed in the rate of nucleotide substitution and the pattern of codon usage bias between BDV1 and ABV. Moreover, the analyzed bornaviruses might be descended from an AT-rich ancestor.

Evolution of the viral hemorrhagic septicemia virus: divergence, selection and origin.

Viral hemorrhagic septicemia virus (VHSV) is an economically significant rhabdovirus that affects an increasing number of freshwater and marine fish species. Extensive studies have been conducted on the molecular epizootiology, genetic diversity, and phylogeny of VHSV. However, there are discrepancies between the reported estimates of the nucleotide substitution rate for the G gene and the divergence times for the genotypes. Herein, Bayesian coalescent analyses were conducted to the time-stamped entire coding sequences of the six VHSV genes. Rate estimates based on the G gene indicated that the marine genotypes/subtypes might not all evolve slower than their major European freshwater counterpart. Age calculations on the six genes revealed that the first bifurcation event of the analyzed isolates might have taken place within the last 300 years, which was much younger than previously thought. Selection analyses suggested that two codons of the G gene might be positively selected. Surveys of codon usage bias showed that the P, M and NV genes exhibited genotype-specific variations. Furthermore, we proposed that VHSV originated from the Pacific Northwest of North America.

Detoxification of Aflatoxin B1 by Phytochemicals in Agriculture and Food Science.

Aflatoxin B1 (AFB1), the most toxic and harmful mycotoxin, has a high likelihood of occurring in animal feed and human food, which seriously affects agriculture and food safety and endangers animal and human health. Recently, natural plant products have attracted widespread attention due to their low toxicity, high biocompatibility, and simple composition, indicating significant potential for resisting AFB1. The mechanisms by which these phytochemicals resist toxins mainly involve antioxidative, anti-inflammatory, and antiapoptotic pathways. Moreover, these substances also inhibit the genotoxicity of AFB1 by directly influencing its metabolism in vivo, which contributes to its elimination. Here, we review various phytochemicals that resist AFB1 and their anti-AFB1 mechanisms in different animals, as well as the common characteristics of phytochemicals with anti-AFB1 function. Additionally, the shortcomings of current research and future research directions will be discussed. Overall, this comprehensive summary contributes to the better application of phytochemicals in agriculture and food safety.

miR-375 inhibits proliferation of mouse pancreatic progenitor cells by targeting YAP1.

BACKGROUND/AIMS: The Hippo signaling pathway regulates expansion and differentiation of stem cells and tissue progenitor cells during organ development and tissue regeneration. Previous studies have shown that YAP1, a potent effector of the Hippo signaling pathway, plays a crucial role in pancreas development, but the function of YAP1 in pancreatic progenitor cells is less known. METHODS: The spatio-temporal expression pattern of YAP1 in mouse developing pancreata was detected by in situ hybridization. The effect of silencing YAP1 on the proliferation of pancreatic progenitor cells was analyzed by CCK-8 assay and Ki67 immunostaining. The regulation of miR-375 on YAP1 expression was determined by dual luciferase reporter assay, QRT-PCR and western blot. Finally, the influence of miR-375 on proliferation of pancreatic progenitor cells was analyzed by CCK-8 assay and Ki67 immunostaining. RESULTS: We found that YAP1 was highly expressed in embryonic and adult pancreatic progenitor cells. Knocking down YAP1 by siRNA inhibited the proliferation of pancreatic progenitor cells. The mouse YAP1 was a target gene of miR-375, and miR-375 could target the 3' UTR of YAP1 mRNA to decrease its protein and mRNA levels. Similar to silencing YAP1 by siRNA, the proliferation of pancreatic progenitor cells was inhibited significantly by miR-375. CONCLUSION: Our results indicate that YAP1 is necessary for the proliferation of pancreatic progenitor cells and miR-375 participates in regulating YAP1 expression during pancreatic progenitor cells differentiation.

[Regulation of differentiation of mesenchymal stem cells by the Hippo pathway effectors TAZ/YAP].

Mesenchymal stem cells (MSCs) are pluripotent cells which can differentiate into several distinct lineages, such as chondrocytes, adipocytes and myofibers. It has been reported that the lineage-specific transcriptional factors including Runt related transcription factor 2 (RUNX2), Peroxisome proliferator-activator receptor gamma (PPARgamma) and Myogenic differentiation 1 (MyoD) may play key regulatory roles among the differentiation of MSCs. Recently, researches have confirmed that the Hippo pathway impacts the differentiation fates of MSCs through regulating the activity of line- age-specific transcription factors by the Hippo pathway effectors Tafazzin (TAZ) and/or Yes-associated protein (YAP). The interaction between TAZ and RUNX2 boosts the osteogenic processes and promotes MSCs differentiating into osteoblast lineage. However, PPARgamma binding to TAZ may inhibit the adipocytes differentiation, and thus overexpression of TAZ in mesenchymal stem cell-like cells increases the expression of myogenic genes and hastens myofiber formation through a MyoD-dependent manner. Moreover, other signaling pathways (such as BMP-2, TNF-alpha, Eph-Ephrin, etc.), small molecules (KR62980, TM-25659, etc.), and mechanistic stimuli can also affect the fate by regulating the activity of TAZ/YAP. In this review, we summarized the signaling pattern of Hippo pathway and the function mechanism of TAZ and/or YAP by enumerating their interaction to several lineage-specific transcriptional factors and relationship with other signal pathways during MSCs differentiation.

Copper chaperone antioxidant 1: multiple roles and a potential therapeutic target.

Copper (Cu) was recently demonstrated to play a critical role in cellular physiological and biochemical processes, including energy production and maintenance, antioxidation and enzymatic activity, and signal transduction. Antioxidant 1 (ATOX1), a chaperone of Cu previously named human ATX1 homologue (HAH1), has been found to play an indispensable role in maintaining cellular Cu homeostasis, antioxidative stress, and transcriptional regulation. In the past decade, it has also been found to be involved in a variety of diseases, including numerous neurodegenerative diseases, cancers, and metabolic diseases. Recently, increasing evidence has revealed that ATOX1 is involved in the regulation of cell migration, proliferation, autophagy, DNA damage repair (DDR), and death, as well as in organism development and reproduction. This review summarizes recent advances in the research on the diverse physiological and cytological functions of ATOX1 and the underlying mechanisms of its action in human health and diseases. The potential of ATOX1 as a therapeutic target is also discussed. This review aims to pose unanswered questions related to ATOX1 biology and explore the potential use of ATOX1 as a therapeutic target.

[Practices of integrating the undergraduate and graduate teaching of Biochemistry and Molecular Biology].

Biochemistry and Molecular Biology are the cornerstone courses of talent training in the field of life science. Taking these course as an example, this study explored reconstructing the knowledge framework, developing teaching cases, sharing teaching resources, innovating teaching means and establishing ideological education patterns. Supported by the scientific research achievements with discipline characteristics and online teaching platform, this research explored and practiced an integrated curriculum reform mode. This mode is guided by scientific research and education, based on the course development, and driven by communication and cooperation. A shared space of "exchange, practice, openness and informatization" was developed to achieve free and independent integration of undergraduate and graduate teaching motivated by learning knowledge, resulting in an effective student training.

A ribosomal gene panel predicting a novel synthetic lethality in non-BRCAness tumors.

Poly (ADP-ribose) polymerase (PARP) inhibitors are one of the most exciting classes of targeted therapy agents for cancers with homologous recombination (HR) deficiency. However, many patients without apparent HR defects also respond well to PARP inhibitors/cisplatin. The biomarker responsible for this mechanism remains unclear. Here, we identified a set of ribosomal genes that predict response to PARP inhibitors/cisplatin in HR-proficient patients. PARP inhibitor/cisplatin selectively eliminates cells with high expression of the eight genes in the identified panel via DNA damage (ATM) signaling-induced pro-apoptotic ribosomal stress, which along with ATM signaling-induced pro-survival HR repair constitutes a new model to balance the cell fate in response to DNA damage. Therefore, the combined examination of the gene panel along with HR status would allow for more precise predictions of clinical response to PARP inhibitor/cisplatin. The gene panel as an independent biomarker was validated by multiple published clinical datasets, as well as by an ovarian cancer organoids library we established. More importantly, its predictive value was further verified in a cohort of PARP inhibitor-treated ovarian cancer patients with both RNA-seq and WGS data. Furthermore, we identified several marketed drugs capable of upregulating the expression of the genes in the panel without causing HR deficiency in PARP inhibitor/cisplatin-resistant cell lines. These drugs enhance PARP inhibitor/cisplatin sensitivity in both intrinsically resistant organoids and cell lines with acquired resistance. Together, our study identifies a marker gene panel for HR-proficient patients and reveals a broader application of PARP inhibitor/cisplatin in cancer therapy.