Heterochromatin formation and remodeling by IRTKS condensates counteract cellular senescence.

Heterochromatin, a key component of the eukaryotic nucleus, is fundamental to the regulation of genome stability, gene expression and cellular functions. However, the factors and mechanisms involved in heterochromatin formation and maintenance still remain largely unknown. Here, we show that insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein, is indispensable for constitutive heterochromatin formation via liquid‒liquid phase separation (LLPS). In particular, IRTKS droplets can infiltrate heterochromatin condensates composed of HP1α and diverse DNA-bound nucleosomes. IRTKS can stabilize HP1α by recruiting the E2 ligase Ubc9 to SUMOylate HP1α, which enables it to form larger phase-separated droplets than unmodified HP1α. Furthermore, IRTKS deficiency leads to loss of heterochromatin, resulting in genome-wide changes in chromatin accessibility and aberrant transcription of repetitive DNA elements. This leads to activation of cGAS-STING pathway and type-I interferon (IFN-I) signaling, as well as to the induction of cellular senescence and senescence-associated secretory phenotype (SASP) responses. Collectively, our findings establish a mechanism by which IRTKS condensates consolidate constitutive heterochromatin, revealing an unexpected role of IRTKS as an epigenetic mediator of cellular senescence.

[Study on mechanism of enhancing antioxidant activity in fermented Ziziphi Spinosae Semen with Poria cocos].

In this study, the antioxidant property changes in fermented Ziziphi Spinosae Semen(FZSS) with Poria cocos were analyzed by DPPH, ABTS and FRAP methods. Then the content determination of active ingredients and ~1H nuclear magnetic resonance(~1H-NMR) spectroscopy were also used to investigate the mechanism of FZSS with P. cocos in enhancing the antioxidant activity. The results showed that the content of active ingredients such as total phenols, total saponins and total polysaccharides were significantly increased during the fermentation time. The results of ~1H-NMR metabonomics showed that the contents of amino acids such as leucine, lysine, valine and alanine, nitrogen compounds such as creatine, creatinine, and betaine, and secondary metabolites, for instance, jujuboside A and spinosin were higher after fermentation, and above components showed positive correlation with antioxidant capacity in Pearson correlation analysis. Therefore, it was inferred that the enhancement of antioxidant activity of FZSS may be the result of the joint action of various chemical components. This study preliminarily clarified the mechanism of FZSS in enhancing the antioxidant activity, and provided new research ideas for the product development and utilization of FZSS.

IRTKS is correlated with progression and survival time of patients with gastric cancer.

BACKGROUND AND OBJECTIVES: IRTKS functions as a novel regulator of tumour suppressor p53; however, the role of IRTKS in pathogenesis of gastric cancer is unclear. DESIGN: We used immunohistochemistry to detect IRTKS levels in 527 human gastric cancer specimens. We generated both IRTKS-deficient and p53-deficient mice to observe survival time of these mice and to isolate mouse embryonic fibroblasts (MEFs) for evaluating in vivo tumorigenicity. Co-immunoprecipitation was used to study the interaction among p53, MDM2 and IRTKS, as well as the ubiquitination of p53. RESULTS: IRTKS was significantly overexpressed in human gastric cancer, which was conversely associated with wild-type p53 expression. Among patients with wild-type p53 (n=206), those with high IRTKS expression (n=141) had a shorter survival time than those with low IRTKS (n=65) (p=0.0153). Heterozygous p53+/- mice with IRTKS deficiency exhibited significantly delayed tumorigenesis and an extended tumour-free survival time. p53+/- MEFs without IRTKS exhibited attenuated in vivo tumorigenicity. IRTKS depletion upregulated p53 and its target genes, such as BAX and p21. Intriguingly, IRTKS overexpression promoted p53 ubiquitination and degradation in MEFs and gastric cancer cells. Under DNA damage conditions, IRTKS was phosphorylated at Ser331 by the activated Chk2 kinase and then dissociated from p53, along with the p53-specific E3 ubiquitin ligase MDM2, resulting in attenuated p53 ubiquitination and degradation. CONCLUSION: IRTKS overexpression is negatively correlated with progression and overall survival time of patients with gastric cancer with wild-type p53 through promotion of p53 degradation via the ubiquitin/proteasome pathway.

Single-cell RNA-Seq analysis reveals dynamic trajectories during mouse liver development.

BACKGROUND: The differentiation and maturation trajectories of fetal liver stem/progenitor cells (LSPCs) are not fully understood at single-cell resolution, and a priori knowledge of limited biomarkers could restrict trajectory tracking. RESULTS: We employed marker-free single-cell RNA-Seq to characterize comprehensive transcriptional profiles of 507 cells randomly selected from seven stages between embryonic day 11.5 and postnatal day 2.5 during mouse liver development, and also 52 Epcam-positive cholangiocytes from postnatal day 3.25 mouse livers. LSPCs in developing mouse livers were identified via marker-free transcriptomic profiling. Single-cell resolution dynamic developmental trajectories of LSPCs exhibited contiguous but discrete genetic control through transcription factors and signaling pathways. The gene expression profiles of cholangiocytes were more close to that of embryonic day 11.5 rather than other later staged LSPCs, cuing the fate decision stage of LSPCs. Our marker-free approach also allows systematic assessment and prediction of isolation biomarkers for LSPCs. CONCLUSIONS: Our data provide not only a valuable resource but also novel insights into the fate decision and transcriptional control of self-renewal, differentiation and maturation of LSPCs.

Cinobufagin Is a Selective Anti-Cancer Agent against Tumors with EGFR Amplification and PTEN Deletion.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor, and almost half of the patients carrying EGFR-driven tumor with PTEN deficiency are resistant to EGFR-targeted therapy. EGFR amplification and/or mutation is reported in various epithelial tumors. This series of studies aimed to identify a potent compound against EGFR-driven tumor. We screened a chemical library containing over 600 individual compounds purified from Traditional Chinese Medicine against GBM cells with EGFR amplification and found that cinobufagin, the major active ingredient of Chansu, inhibited the proliferation of EGFR amplified GBM cells and PTEN deficiency enhanced its anti-proliferation effects. Cinobufagin also strongly inhibited the proliferation of carcinoma cell lines with wild-type or mutant EGFR expression. In contrast, the compound only weakly inhibited the proliferation of cancer cells with low or without EGFR expression. Cinobufagin blocked EGFR phosphorylation and its downstream signaling, which additionally induced apoptosis and cytotoxicity in EGFR amplified cancer cells. In vivo, cinobufagin blocked EGFR signaling, inhibited cell proliferation, and elicited apoptosis, thereby suppressing tumor growth in both subcutaneous and intracranial U87MG-EGFR xenograft mouse models and increasing the median survival of nude mice bearing intracranial U87MG-EGFR tumors. Cinobufagin is a potential therapeutic agent for treating malignant glioma and other human cancers expressing EGFR.

Mutagenesis of PhaR, a Regulator Gene of Polyhydroxyalkanoate Biosynthesis of Xanthomonas oryzae pv. oryzae Caused Pleiotropic Phenotype Changes.

Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials produced in various microorganisms under nutrient-limited conditions. PhaR is a regulatory protein involved in PHA synthesis. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important bacterial pathogens in rice and has PHA biosynthesis genes in its genome, but the biological function of phaR in Xoo is unknown. In this study, we investigated the effects of the mutagenesis of phaR gene in Xoo strain PXO99A. Compared to the wildtype, the PhaR gene knock-out mutant PXO99ΔphaR was hypermotile and showed decreased growth rates in both rich and limited nutrient media. PXO99ΔphaR also showed almost 75% decrease in extracellular polysaccharide (EPS) production. When inoculated in rice leaves by leaf-clipping method, PXO99ΔphaR displayed reduced virulence in terms of lesion length and bacterial multiplication compared with the wildtype strain. PXO99ΔphaR also showed enhanced hypersensitive response (HR) induction in the leaves of non-host Nicotiana benthamiana with elevated hpa1 gene expression. Introduction of a cosmid containing the phaR coding sequence restored the phenotypes of the mutant to those of the wildtype strain. These results suggest that PhaR gene is an important gene that affects multiple bacterial characteristics, including EPS production, growth rate, defense response induced harpin production and motility, related to its virulence in plant.