Macro-micro fracture mechanism and acoustic emission characteristics of brittle rock induced by loading rate effect.

The deterioration and deformation of brittle rock generally appear in railway tunnels with the operation of large buried deep tunnel. To investigate the macro-micro fracture and acoustic emission evolution characteristics of brittle rock, this paper conducted the uniaxial compression, scanning electron microscope (SEM), and acoustic emission (AE) signal monitoring under different loading rates. The results showed that the loading rate has an obvious enhancement effect on mechanical parameters. The increased loading rate extends the elastic deformation and improves the bearing strength, elastic modulus and deformation modulus. The fracture patterns include shear fracture, composite fracture, and tension fracture. The oblique shear fracture is transformed into composite fracture and tension fracture with the loading rate increasing. The microscopic fracture shows that increasing loading rate inhibits the evolution of oblique shear fractures and promotes the expansion of tensile fractures. The roughness level of tensile fractures is significantly lower than that of oblique shear fracture and composite fracture. The AE parameters and deformation behavior are characterized by simultaneous evolution. The AE amplitude changes from low-level and low-density distribution to high-level and high-density distribution as the loading rate increases. The AE activity intensity of tensile fracture is significantly greater than that of oblique shear fracture and composite fracture. The warning timeliness of cumulative AE events and cumulative AE energy is generally earlier than the AE b-value under the same loading rate, and the early warning timeliness of cumulative AE events is similar to that of cumulative AE energy.

Novel role for Ddx39 in differentiation and telomere length regulation of embryonic stem cells.

Erk signaling is indispensable for the self-renewal and differentiation of mouse embryonic stem cells (ESCs), as well as telomere homeostasis. But how Erk regulates these biological processes remains unclear. We identified 132 Erk2 interacting proteins by co-immunoprecipitation and mass spectrometric analysis, and focused on Ddx39 as a potential Erk2 substrate. We demonstrated that Erk2 phosphorylates Ddx39 on Y132 and Y138. Ddx39 knockout (KO) ESCs are defective in differentiation, due to reduced H3K27ac level upon differentiation. Phosphorylation of Ddx39 promotes the recruitment of Hat1 to acetylate H3K27 and activate differentiation genes. In addition, Ddx39 KO leads to telomere elongation in ESCs. Ddx39 is recruited to telomeres by the telomere-binding protein Trf1, consequently disrupting the DNA loop formed by Trf1 and suppressing the alternative lengthening of telomeres (ALT). Phosphorylation of Ddx39 weakens its interaction with Trf1, releasing it from telomeres. Thus, ALT activity is enhanced, and telomeres are elongated. Altogether, our studies reveal an essential role of Ddx39 in the differentiation and telomere homeostasis of ESCs.

Hypoxic tumor-derived exosomal miR-4488 induces macrophage M2 polarization to promote liver metastasis of pancreatic neuroendocrine neoplasm through RTN3/FABP5 mediated fatty acid oxidation.

Tumor-associated macrophages (TAMs) represent a predominant cellular component within the tumor microenvironment (TME) of pancreatic neuroendocrine neoplasms (pNENs). There is a growing body of evidence highlighting the critical role of exosomes in facilitating communication between tumor cells and TAMs, thereby contributing to the establishment of the premetastatic niche. Nonetheless, the specific mechanisms through which exosomes derived from tumor cells influence macrophage polarization under hypoxic conditions in pNENs, and the manner in which these interactions support cancer metastasis, remain largely unexplored. Recognizing the capacity of exosomes to transfer miRNAs that can modify cellular behaviors, our research identified a significant overexpression of miR-4488 in exosomes derived from hypoxic pNEN cells. Furthermore, we observed that macrophages that absorbed circulating exosomal miR-4488 underwent M2-like polarization. Our investigations revealed that miR-4488 promotes M2-like polarization by directly targeting and suppressing RTN3 in macrophages. This suppression of RTN3 enhances fatty acid oxidation and activates the PI3K/AKT/mTOR signaling pathway through the interaction and downregulation of FABP5. Additionally, M2 polarized macrophages contribute to the formation of the premetastatic niche and advance pNENs metastasis by releasing MMP2, thereby establishing a positive feedback loop involving miR-4488, RTN3, FABP5, and MMP2 in pNEN cells. Together, these findings shed light on the role of exosomal miRNAs from hypoxic pNEN cells in mediating interactions between pNEN cells and intrahepatic macrophages, suggesting that miR-4488 holds potential as a valuable biomarker and therapeutic target for pNENs.

Btbd8 deficiency reduces susceptibility to colitis by enhancing intestinal barrier function and suppressing inflammation.

Introduction: BTBD8 has been identified as a susceptible gene for inflammatory bowel diseases (IBD). However, the function of BTBD8 in normal development and IBD pathogenesis remains unknown. Methods: We administered drinking water with 3% dextran sodium sulfate (DSS) to wild-type (WT) and Btbd8 knockout (KO) mice for seven consecutive days to induce IBD. Subsequently, we further examined whether Btbd8 KO affects intestinal barrier and inflammation. Results: We demonstrated that Btbd8 deficiency partially protects mice from DSS-induced IBD, even though no obvious phenotypes were observed in Btbd8 KO mice. Btbd8 deletion leads to strengthened tight junctions between intestinal epithelial cells, elevated intestinal stem cell activity, and enhanced mucus layer. All these three mechanisms work together to improve the intestinal barrier integrity in Btbd8 KO mice. In addition, Btbd8 deficiency mitigates inflammation by reducing the expression of IL-1ß and IL-6 by macrophages. Discussion: Our studies validate the crucial role of Btbd8 in IBD pathogenesis, and reveal that Btbd8 deficiency may ameliorate DSS-induced IBD through improving the intestinal barrier integrity, as well as suppressing inflammatory response mediated by macrophages. These findings suggest that Btbd8 could be a promising therapeutic target for the treatment of IBD.

Glycolysis-Stimulated Esrrb Lactylation Promotes the Self-Renewal and Extraembryonic Endoderm Stem Cell Differentiation of Embryonic Stem Cells.

Embryonic stem cells (ESCs) favor glycolysis over oxidative phosphorylation for energy production, and glycolytic metabolism is critical for pluripotency establishment, maintenance, and exit. However, an understanding of how glycolysis regulates the self-renewal and differentiation of ESCs remains elusive. Here, we demonstrated that protein lactylation, regulated by intracellular lactate, contributes to the self-renewal of ESCs. We further showed that Esrrb, an orphan nuclear receptor involved in pluripotency maintenance and extraembryonic endoderm stem cell (XEN) differentiation, is lactylated on K228 and K232. The lactylation of Esrrb enhances its activity in promoting ESC self-renewal in the absence of the LIF and XEN differentiation of ESCs by increasing its binding at target genes. Our studies reveal the importance of protein lactylation in the self-renewal and XEN differentiation of ESCs, and the underlying mechanism of glycolytic metabolism regulating cell fate choice.

The NR2B-targeted intervention alleviates the neuronal injuries at the sub-acute stage of cerebral ischemia: an exploration of stage-dependent strategy against ischemic insults.

Stroke is reported to be the second leading cause of death worldwide, among which ischemic stroke has fourfold greater incidence than intracerebral hemorrhage. Excitotoxicity induced by NMDAR plays a central role in ischemic stroke-induced neuronal death. However, intervention targeted NMDARs against ischemic stroke has failed, which may result from the complex composition of NMDARs and the dynamic changes of their subunits. In this current study, the levels of NR1, NR2A and NR2B subunits of NMDARs were observed upon different time points during the reperfusion after 1 h ischemia with the western blot assay. It was found that the changes of NR1 subunit were only detected after ischemia 1 h/reperfusion 1 day (1 d). While, the changes of NR2A and NR2B subunits may last to ischemia 1 h/reperfusion 7 day(7 d), indicating that NR2subunits may be a potential target for ischemia-reperfusion injuries at the sub-acute stage of ischemic stroke. Simultaneously, mitochondrial injuries in neurons were investigated with transmission electron microscopy (TEM), and mitochondrial dysfunction was evaluated with mitochondrial membrane proteins oxidative respiratory chain complex and OCR. When the antagonist of NMDARs was used before ischemic exposure, the neuronal mitochondrial dysfunction was alleviated, suggesting that these aberrant deviations of NMDARs from basal levels led to mitochondrial dysfunction. Furthermore, when the antagonist of NR2B was administrated intracerebroventricularly at the sub-acute cerebral ischemia, the volume of cerebral infarct region was decreased and the neural functions were improved. To sum up, the ratio of NR2B-containing NMDARs is vital for mitochondrial homeostasis and then neuronal survival. NR2B-targeted intervention should be chosen at the sub-acute stage of cerebral ischemia.

Homozygous Loss of Septin12, but not its Haploinsufficiency, Leads to Male Infertility and Fertilization Failure.

The SEPTIN12 gene has been associated with male infertility. Male Septin12 +/- chimera mice were infertile, supporting the prevailing view that SEPTIN12 haploinsufficiency causes male infertility. In this study, we identified a heterozygous mutation on SEPTIN12, c.72C>A (p.Cys24Ter) in the male partner of a patient couple, who had a previous fertilization failure (FF) after intracytoplasmic sperm injection (ICSI) and became pregnant after ICSI together with artificial oocyte activation (AOA). To investigate the role of SEPTIN12 in FF and oocyte activation, we constructed Septin12 knockout mice. Surprisingly, Septin12 -/- male mice, but not Septin12 +/- male mice, are infertile, and have reduced sperm counts and abnormal sperm morphology. Importantly, AOA treatment enhances the 2-cell embryo rate of ICSI embryos injected with Septin12 -/- sperm, indicating that FF caused by male Septin12 deficiency is overcome by AOA. Mechanistically, loss of PLCζ around the acrosome might be the reason for FF of Septin12 -/- sperm. Taken together, our data indicated that homozygous knockout of Septin12, but not Septin12 haploinsufficiency, leads to male infertility and FF.

Hbxip is essential for murine embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1.

HBXIP, also named LAMTOR5, has been well characterized as a transcriptional co-activator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5, and that depletion of Hbxip compromises the self-renewal of embryonic stem cells (ESCs), with reduced expression of pluripotency genes, reduced cell proliferation and decreased colony-forming capacity. In addition, both Hbxip-/- ESCs and E7.5 embryos displayed defects in ectodermal and mesodermal differentiation. Mechanistically, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, displayed differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/- and p18-/- mice, lacking subunits of the Ragulator complex, also shared similar phenotypes, embryonic lethality and retarded growth around E7-E8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation.

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live mouse cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female mouse embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain plays critical roles in XCI. In Ssb knockdown cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.

lncRNA 5430416N02Rik Promotes the Proliferation of Mouse Embryonic Stem Cells by Activating Mid1 Expression through 3D Chromatin Architecture.

Both 3D chromatin architecture and long non-coding RNAs (lncRNAs) play essential roles in pluripotency maintenance. However, whether lncRNAs are involved in organizing 3D chromatin structure remains largely unexplored. We identified 39 lncRNAs bound by Klf4, among which we further revealed the 5430416N02Rik promoter is a chromatin interaction hub. Knockout of the 5430416N02Rik locus reduces the proliferation rate of embryonic stem cells (ESCs). Moreover, deleting both the promoter and the gene body of 5430416N02Rik causes a more severe proliferation defect and has a more profound impact on the transcriptome than deleting the gene body alone. The reduced proliferation of the 5430416N02Rik locus knockout ESCs is mainly due to the downregulation of Mid1, the expression of which requires the inter-chromosomal interaction between Mid1 and 5430416N02Rik loci. In summary, our data demonstrated that the lncRNA 5430416N02Rik gene locus maintains the fast proliferation of ESCs by activating the expression of Mid1 through chromatin interaction.

Cops5 safeguards genomic stability of embryonic stem cells through regulating cellular metabolism and DNA repair.

The highly conserved COP9 signalosome (CSN), composed of 8 subunits (Cops1 to Cops8), has been implicated in pluripotency maintenance of human embryonic stem cells (ESCs). Yet, the mechanism for the CSN to regulate pluripotency remains elusive. We previously showed that Cops2, independent of the CSN, is essential for the pluripotency maintenance of mouse ESCs. In this study, we set out to investigate how Cops5 and Cops8 regulate ESC differentiation and tried to establish Cops5 and Cops8 knockout (KO) ESC lines by CRISPR/Cas9. To our surprise, no Cops5 KO ESC clones were identified out of 127 clones, while three Cops8 KO ESC lines were established out of 70 clones. We then constructed an inducible Cops5 KO ESC line. Cops5 KO leads to decreased expression of the pluripotency marker Nanog, proliferation defect, G2/M cell-cycle arrest, and apoptosis of ESCs. Further analysis revealed dual roles of Cops5 in maintaining genomic stability of ESCs. On one hand, Cops5 suppresses the autophagic degradation of Mtch2 to direct cellular metabolism toward glycolysis and minimize reactive oxygen species (ROS) production, thereby reducing endogenous DNA damage. On the other hand, Cops5 is required for high DNA damage repair (DDR) activities in ESCs. Without Cops5, elevated ROS and reduced DDR activities lead to DNA damage accumulation in ESCs. Subsequently, p53 is activated to trigger G2/M arrest and apoptosis. Altogether, our studies reveal an essential role of Cops5 in maintaining genome integrity and self-renewal of ESCs by regulating cellular metabolism and DDR pathways.

Antitumor effects of anlotinib in thyroid cancer.

There is no effective treatment for patients with poorly differentiated papillary thyroid cancer or anaplastic thyroid cancer (ATC). Anlotinib, a multi-kinase inhibitor, has already shown antitumor effects in various types of carcinoma in a phase I clinical trial. In this study, we aimed to better understand the effect and efficacy of anlotinib against thyroid carcinoma cells in vitro and in vivo. We found that anlotinib inhibits the cell viability of papillary thyroid cancer and ATC cell lines, likely due to abnormal spindle assembly, G2/M arrest, and activation of TP53 upon anlotinib treatment. Moreover, anlotinib suppresses the migration of thyroid cancer cells in vitro and the growth of xenograft thyroid tumors in mice. Our data demonstrate that anlotinib has significant anticancer activity in thyroid cancer, and potentially offers an effective therapeutic strategy for patients of advanced thyroid cancer type.

Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation.

We double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain of Xist) to generate "ΔA mutant" and to tether effector proteins for dissecting Xist functionality. Based on the observation in live cells that the induced XCI in undifferentiated embryonic stem (ES) cells is counteracted by the intrinsic X chromosome reactivation (XCR), we identified Kat8 and Msl2, homologs of Drosophila dosage compensation proteins, as players involved in mammalian XCR. Furthermore, live-cell imaging revealed the obviously undersized ΔA Xist cloud signals, clarifying an issue regarding the previous RNA fluorescence in situ hybridization results. Tethering candidate proteins onto the ΔA mutant reveals the significant roles of Ythdc1, Ezh2, and SPOC (Spen) in Xist-mediated gene silencing and the significant role of Ezh2 in Xist RNA spreading.

COPS2 Antagonizes OCT4 to Accelerate the G2/M Transition of Mouse Embryonic Stem Cells.

Proper regulation of the cell cycle is essential to safeguard the genomic integrity of embryonic stem cells (ESCs) while maintaining the fast proliferation rate. The pluripotency factor OCT4 has been shown to inhibit CDK1 activation, thus preventing mitotic entry and facilitating the maintenance of genomic integrity. Yet, how ESCs enter mitosis in the presence of OCT4 remains unclear. We previously reported that COPS2 promotes the progression through the G2/M phase of mouse ESCs. In this study, through co-immunoprecipitation and mass spectrometric analysis, we found that COPS2 interacts with OCT4 and CDK1. We further demonstrated that COPS2 stimulates the activity of CDK1/CYCLIN B only when OCT4 is present. Consistently, COPS2 promotes the G2/M transition only in the presence of OCT4 in HeLa cells. Mechanistically, COPS2 attenuates the interaction between OCT4 and CDK1 by sequestering OCT4 and forming a COPS2/CDK1 complex, thus blocking the inhibitory effect of OCT4 on CDK1 activation.

Genome-Wide RNAi Screen Identify Melanoma-Associated Antigen Mageb3 Involved in X Chromosome Inactivation.

Xist (inactivated X chromosome specific transcript) is a prototype long noncoding RNA in charge of epigenetic silencing of one X chromosome in each female cell in mammals. In a genetic screen, we identify Mageb3 and its homologs Mageb1 and Mageb2 as genes functionally required for Xist-mediated gene silencing. Mageb1-3 are previously uncharacterized genes belonging to the MAGE (melanoma-associated antigen) gene family. Mageb1-3 are expressed in undifferentiated ES cells and early stages of in vitro differentiation, a critical time window of X chromosome inactivation. Mageb3 showed both cytoplasmic and nuclear localization without enrichment on the inactive X (Xi). Mageb3 interacted with Polycomb group ring finger 3 (Pcgf3), a RING finger protein involved in recruiting Polycomb activities onto Xi. Mageb3 overexpression stabilized Pcgf3 protein. Mageb1-3 gene knockout affected H3K27me3 enrichment and the spreading of gene silencing along Xi. These data suggested that Mageb3 might regulate the recruitment of the Polycomb complex onto Xi and subsequent H3K27me3 modification through Pcgf3. Moreover, the nucleolar enrichment of Mageb3 was diminished when nuclear matrix factor hnRNP U is overexpressed, implying the interaction between Mageb3 and nuclear matrix, which is another possible mechanism for Mageb3 to regulate X chromosome inactivation.

RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells.

Ten-eleven translocation (TET) proteins play key roles in the regulation of DNA-methylation status by oxidizing 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), which can both serve as a stable epigenetic mark and participate in active demethylation. Unlike the other members of the TET family, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We found that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation.

Pold3 is required for genomic stability and telomere integrity in embryonic stem cells and meiosis.

Embryonic stem cells (ESCs) and meiosis are featured by relatively higher frequent homologous recombination associated with DNA double strand breaks (DSB) repair. Here, we show that Pold3 plays important roles in DSB repair, telomere maintenance and genomic stability of both ESCs and spermatocytes in mice. By attempting to generate Pold3 deficient mice using CRISPR/Cas9 or transcription activator-like effector nucleases, we show that complete loss of Pold3 (Pold3-/-) resulted in early embryonic lethality at E6.5. Rapid DNA damage response and massive apoptosis occurred in both outgrowths of Pold3-null (Pold3-/-) blastocysts and Pold3 inducible knockout (iKO) ESCs. While Pold3-/- ESCs were not achievable, Pold3 iKO led to increased DNA damage response, telomere loss and chromosome breaks accompanied by extended S phase. Meanwhile, loss of Pold3 resulted in replicative stress, micronucleation and aneuploidy. Also, DNA repair was impaired in Pold3+/- or Pold3 knockdown ESCs. Moreover, Pold3 mediates DNA replication and repair by regulating 53BP1, RIF1, ATR and ATM pathways. Furthermore, spermatocytes of Pold3 haploinsufficient (Pold3+/-) mice with increasing age displayed impaired DSB repair, telomere shortening and loss, and chromosome breaks, like Pold3 iKO ESCs. These data suggest that Pold3 maintains telomere integrity and genomic stability of both ESCs and meiosis by suppressing replicative stress.

Simple Meets Single: The Application of CRISPR/Cas9 in Haploid Embryonic Stem Cells.

The CRISPR/Cas9 system provides a powerful method for the genetic manipulation of the mammalian genome, allowing knockout of individual genes as well as the generation of genome-wide knockout cell libraries for genetic screening. However, the diploid status of most mammalian cells restricts the application of CRISPR/Cas9 in genetic screening. Mammalian haploid embryonic stem cells (haESCs) have only one set of chromosomes per cell, avoiding the issue of heterozygous recessive mutations in diploid cells. Thus, the combination of haESCs and CRISPR/Cas9 facilitates the generation of genome-wide knockout cell libraries for genetic screening. Here, we review recent progress in CRISPR/Cas9 and haPSCs and discuss their applications in genetic screening.