反向虚拟筛选平台及应用

靶标确证是老药新用、药物毒副作用研究的关键。基于分子对接方法 Auto Dock Vina和内部构建的疾病靶标数据库,采用分布式架构,构建了反向虚拟筛选平台。应用该平台对药物吡斯的明进行靶标确证,最终成功找到其靶标乙酰胆碱酯酶,验证了平台的实用性和准确性。     

HMGB3 promotes PARP inhibitor resistance through interacting with PARP1 in ovarian cancer

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) resistance remains a therapeutic challenge in ovarian cancer. High-mobility group box 3 (HMGB3) plays significant roles in the development of drug resistance of many cancers. However, the function of HMGB3 in PARPi resistance is poorly understood. In the current study, we clarified that HMGB3 was aberrantly overexpressed in high-grade serous ovarian carcinoma (HGSOC) tissues, and high HMGB3 levels indicated shorter overall survival and drug resistance in HGSOC. The overexpression of HMGB3 increased the insensitivity of ovarian cancer to PARPi, whereas HMGB3 knockdown reduced PARPi resistance. Mechanistically, PARP1 was identified as a novel interaction partner of HMGB3, which could be blocked using olaparib and was enhanced upon DNA damage conditions. We further showed that loss of HMGB3 induced PARP1 trapping at DNA lesions and inhibited the PARylation activity of PARP1, resulting in an increased DNA damage response and cell apoptosis. The PARPi-resistant role of HMGB3 was also verified in a xenograft mouse model. In conclusion, HMGB3 promoted PARPi resistance via interacting with PARP1, and the targeted inhibition of HMGB3 might overcome PARPi resistance in ovarian cancer therapy.Subject terms: Chemotherapy, Ovarian cancer, Ovarian cancer, Cancer therapeutic resistance     

鼠类对栓皮栎和锥栗种子的贮藏:验证高单宁假说

种子中的单宁影响鼠类的取食和贮藏策略.高单宁假说认为鼠类倾向于优先贮藏单宁含量高的种子、取食单宁含量低的种子.同域分布的鼠类对种子的取食和贮藏选择是否符合高单宁假说的预测尚缺乏足够的实验证据.本研究通过围栏实验研究了四川都江堰地区亚热带常绿阔叶林中的小泡巨鼠(Leopoldamyys edwardsi)、针毛鼠(Niv...     

Impact of nonrandom selection mechanisms on the causal effect estimation for two-sample Mendelian randomization methods

Nonrandom selection in one-sample Mendelian Randomization (MR) results in biased estimates and inflated type I error rates only when the selection effects are sufficiently large. In two-sample MR, the different selection mechanisms in two samples may more seriously affect the causal effect estimation. Firstly, we propose sufficient conditions for causal effect invariance under different selection mechanisms using two-sample MR methods. In the simulation study, we consider 49 possible selection mechanisms in two-sample MR, which depend on genetic variants (G), exposures (X), outcomes (Y) and their combination. We further compare eight pleiotropy-robust methods under different selection mechanisms. Results of simulation reveal that nonrandom selection in sample II has a larger influence on biases and type I error rates than those in sample I. Furthermore, selections depending on X+Y, G+Y, or G+X+Y in sample II lead to larger biases than other selection mechanisms. Notably, when selection depends on Y, bias of causal estimation for non-zero causal effect is larger than that for null causal effect. Especially, the mode based estimate has the largest standard errors among the eight methods. In the absence of pleiotropy, selections depending on Y or G in sample II show nearly unbiased causal effect estimations when the casual effect is null. In the scenarios of balanced pleiotropy, all eight MR methods, especially MR-Egger, demonstrate large biases because the nonrandom selections result in the violation of the Instrument Strength Independent of Direct Effect (InSIDE) assumption. When directional pleiotropy exists, nonrandom selections have a severe impact on the eight MR methods. Application demonstrates that the nonrandom selection in sample II (coronary heart disease patients) can magnify the causal effect estimation of obesity on HbA1c levels. In conclusion, nonrandom selection in two-sample MR exacerbates the bias of causal effect estimation for pleiotropy-robust MR methods.     

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca²⁺-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T₀ regenerants and sprdr6 null T₁ progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

A DNA-free CRISPR-Cas9 genome editing tool based on an optimized protoplast regeneration protocol of wild tomato creates stable and inheritable diploid and tetraploid regenerants.     

RBM47/SNHG5/FOXO3 axis activates autophagy and inhibits cell proliferation in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is the main type of thyroid carcinoma. Despite the good prognosis, some PTC patients may deteriorate into more aggressive diseases, leading to poor survival. Molecular technology has been increasingly used in the diagnosis and treatment of thyroid carcinoma. In this study, we identified that RNA Binding Motif Protein 47 (RBM47) was downregulated in PTC tissues and cells, and overexpression of RBM47 could activate autophagy and inhibit proliferation in PTC cells. RBM47 promotes but can not bind directly to Forkhead Box O3 (FOXO3). FOXO3 activates Autophagy Related Gene 3 (ATG3), ATG5, and RBM47 to form a loop and promote autophagy. RBM47 can bind directly to and stabilized lncRNA Small Nucleolar RNA Host Gene 5 (SNHG5) to inhibit PTC cells proliferation and activate autophagy in vitro and in vivo. SNHG5 inhibits ubiquitination and degradation of FOXO3 by recruiting Ubiquitin Specific Peptidase 21 (USP21), then promotes the translocation of FOXO3 from cytoplasm to nucleus. Our study revealed the regulatory mechanism of RBM47/SNHG5/FOXO3 axis on cell proliferation and autophagy in PTC, which may provide valuable insight for the treatment of PTC.Subject terms: Oncogenes, Head and neck cancer     

TGF-β induces GBM mesenchymal transition through upregulation of CLDN4 and nuclear translocation to activate TNF-α/NF-κB signal pathway

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor. The unregulated expression of Claudin-4 (CLDN4) plays an important role in tumor progression. However, the biological role of CLDN4 in GBM is still unknown. This study aimed to determine whether CLDN4 mediates glioma malignant progression, if so, it would further explore the molecular mechanisms of carcinogenesis. Our results revealed that CLDN4 was significantly upregulated in glioma specimens and cells. The inhibition of CLND4 expression could inhibit mesenchymal transformation, cell invasion, cell migration and tumor growth in vitro and in vivo. Moreover, combined with in vitro analysis, we found that CLDN4 can modulate tumor necrosis factor-α (TNF-α) signal pathway. Meanwhile, we also validated that the transforming growth factor-β (TGF-β) signal pathway can upregulate the expression of CLDN4, and promote the invasion ability of GBM cells. Conversely, TGF-β signal pathway inhibitor ITD-1 can downregulate the expression of CLDN4, and inhibit the invasion ability of GBM cells. Furthermore, we found that TGF-β can promote the nuclear translocation of CLDN4. In summary, our findings indicated that the TGF-β/CLDN4/TNF-α/NF-κB signal axis plays a key role in the biological progression of glioma. Disrupting the function of this signal axis may represent a new treatment strategy for patients with GBM.Subject terms: CNS cancer, Epithelial-mesenchymal transition     

PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence

Host defense systems employ posttranslational modifications to protect against invading pathogens. Here, we found that protein inhibitor of activated STAT 1 (PIAS1) interacts with the nucleoprotein (NP), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) of influenza A virus (IAV). Lentiviral-mediated stable overexpression of PIAS1 dramatically suppressed the replication of IAV, whereas siRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression significantly increased virus growth. The expression of PIAS1 was significantly induced upon IAV infection in both cell culture and mice, and PIAS1 was involved in the overall increase in cellular SUMOylation induced by IAV infection. We found that PIAS1 inhibited the activity of the viral RNP complex, whereas the C351S or W372A mutant of PIAS1, which lacks the SUMO E3 ligase activity, lost the ability to suppress the activity of the viral RNP complex. Notably, the SUMO E3 ligase activity of PIAS1 catalyzed robust SUMOylation of PB2, but had no role in PB1 SUMOylation and a minimal role in NP SUMOylation. Moreover, PIAS1-mediated SUMOylation remarkably reduced the stability of IAV PB2. When tested in vivo, we found that the downregulation of Pias1 expression in mice enhanced the growth and virulence of IAV. Together, our findings define PIAS1 as a restriction factor for the replication and pathogenesis of IAV.     

ERK-mediated Cytoplasmic Retention of USP11 Contributes to Breast Cancer Cell Proliferation by Stabilizing Cytoplasmic p21

Breast cancer ranks as the most frequently diagnosed cancer among women worldwide. Elevated cytoplasmic p21 levels are often found in breast cancer tissues and related to a poor prognosis. However, the underlying mechanisms that lead to the stabilization of cytoplasmic p21 protein, which normally has a very short half-life, remain obscure. In this study, we found that there was a strong correlation between p21 and USP11 in the cytoplasm of breast cancer tissues and cells. Furthermore, we revealed that ERK1/2 phosphorylated USP11 at the Ser905 site, which promoted the cytoplasmic localization of USP11. In the cytoplasm, USP11 colocalized and interacted with p21. As a result, USP11 catalyzed the removal of polyubiquitin chains bound to cytoplasmic p21 and resulted in its stabilization. Functionally, USP11-mediated stabilization of cytoplasmic p21 induced breast cancer cell proliferation in vitro and in vivo. Our findings provide the first evidence that ubiquitinated p21 in the cytoplasm can be recycled through USP11-mediated deubiquitination, and we identified the USP11-p21 axis in the cytoplasm as a potential therapeutic target for breast cancer control.