Protein liquid-liquid phase separation (LLPS), a pivotal phenomenon intricately linked to cellular processes, is regulated by various other proteins. However, there is still a lack of high-throughput methods for screening protein regulators of LLPS in target proteins. Here, we developed a CRISPR/Cas9-based screening method to identify protein phase separation regulators by integrating bimolecular fluorescence complementation (BiFC) and fluorescence-activated cell sorting (FACS). Using this newly developed method, we screened the RNA-binding proteins that regulate PABPN1 phase separation and identified the tumor suppressor QKI as a promoter of PABPN1 phase separation. Furthermore, QKI exhibits decreased expression levels and diminished nuclear localization in colorectal cancer cells, resulting in reduced PABPN1 phase separation, which, in turn, promotes alternative polyadenylation (APA), cell proliferation, and migration in colorectal cancer.
Humans ; Colorectal Neoplasms/genetics* ; RNA-Binding Proteins/genetics* ; Poly(A)-Binding Protein I/genetics* ; CRISPR-Cas Systems ; Flow Cytometry ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement

Objective:Using the established epilepsy patient database to validate the efficacy of the web-based epilepsy diagnosis and anti-seizure medications (ASM) selection tool, EpiPick, for domestic epilepsy patients.Methods:The retrospective collection of clinical data was conducted on patients aged 10 and above who were diagnosed with epilepsy at the Comprehensive Epilepsy Center of the First Affiliated Hospital of Kunming Medical University from January 2017 to December 2020, with regular follow-up and complete information. According to the first ASM recommended by the EpiPick tool and whether they are consistent with the actual ASM used by patients, patients were divided into EpiPick group and clinical group to verify the effectiveness of the EpiPick tool in selecting ASM. The drug retention rate, Engel score, and cumulative probability of no consecutive episodes within 30 months after using the first ASM were compared between the 2 groups, and Kaplan-Meier survival curves were drawn. Finally, the diagnostic results provided by the EpiPick tool were compared with the actual types of epileptic seizures diagnosed clinically, and consistency tests were performed.Results:A total of 364 epilepsy patients were included, including 237 in the EpiPick group and 127 in the clinical group. The ASM retention rates of patients in the EpiPick group and clinical group were 67.9%(161/237) and 56.7%(72/127), respectively, with statistically significant differences (χ2=4.534, P=0.039). Grades Ⅰ, Ⅱ, Ⅲ and Ⅳ according to the Engel scores in the EpiPick group patients who took the first ASM after diagnosis accounted for 47.3%(112/237), 14.8%(35/237), 12.7%(30/237), and 25.3%(60/237), respectively, compared to the clinical group of 32.3%(41/127), 11.8%(15/127), 11.0%(14/127), and 44.9%(57/127), respectively. There was a statistically significant difference in Engel scores between the 2 groups (χ2=14.968, P=0.002). The cumulative seizure-free rates in the EpiPick group at the 1st, 6th, 12th, 30th month and above after starting the first ASM were 73.8%, 61.2%, 53.2%, and 50.6%, respectively, which in the clinical group were 52.0%, 44.1%, 40.2%, and 33.5%, respectively. The logrank test showed a statistically significant difference in the cumulative probability of consecutive seizure freedom between the 2 groups ( HR=0.644 ,95% CI 0.476-0.871 ,P<0.001). After grouping by seizure type [focal seizures (196 cases) and generalized seizures (168 cases)], the cumulative seizure-free rates at the 1st, 6th, 12th, 30th month and above after starting ASM were significantly higher in the EpiPick group than in the clinical group (comparison between the 2 groups in patients with focal seizures: HR=0.654, 95%CI 0.443-0.964, P=0.004; comparison between the 2 groups in patients with generalised seizures: HR=0.586, 95%CI 0.361-0.954, P=0.014). Among 364 patients, 293 cases were clinically diagnosed with seizure classification consistent with the classification results of EpiPick tool. Agreement between the algorithm and the experts in classifying generalized seizures was 83.9%(104/124), which in classifying focal seizures was 78.8%(189/240; Kappa=0.591, P<0.001). Conclusion:Web-based EpiPick tool is suitable to be used to select the first ASM, and is portable for Chinese non-epilepsy specialists to choose ASM for epilepsy patients.

Aim To explore the role of endoplasmic re-ticulum stress( ERS) in Astragaloside Ⅳ-induced car-dioprotection against ischemia/reperfusion injury in rats. Methods A model of myocardial ischemia 30 min followed by 120 min reperfusion was made by liga-ting coronary artery in male Wistar rats. Rats were di-vided randomly into 4 groups: sham group, ischemia/reperfusion group, ERS inhibitor TUDCA group, As-tragaloside Ⅳgroup. Myocardial samples were collect-ed from the risk zones during ischemia and reperfu-sion, ERS was determined by measuring levels of glu-cose regulated protein 78 ( GRP78 ) , an established marker of ERS with Western blot. Immunofluorescence study was used to test GRP78 intensity with laser scan-ning confocal microscopy, TTC method was used to measure the infarct size,hematoxylin-eosin staining was used to observe the changes of morphological changes of myocardium. Results There was no statistical difference in GRP78 expression during ischemia com-pared to the sham group, but was markedly increased upon reperfusion. Astragaloside Ⅳ could mimic TUD-CA and significantly decreased the GRP78 expression, reduced infarct size and improved the morphology of myocardial tissue with a significant statistical difference compared with the control group ( P<0. 05 ) . Conclu-sions ERS is induced upon reperfusion but not during ischemia in isolated rat hearts. Astragaloside Ⅳ pre-vents myocardial reperfusion injury presumably by the inhibition of ERS.

Objective To investigate the role of transforming growth factor-β activated kinase-1 (TAK1) signaling pathway in the activation of bone marrow derived macrophages (BMDM) induced by high glucose.Methods Purity of mouse BMDM was detected by flow cytometry.The mice macrophages cultured in vitro were stimulated by high glucose and treated with TAK1 specific inhibitor 5Z-7-oxozeaenol.Cells were divided into normal control group (RPMI 1640),osmolality control group (25 mmol/L mannitol),high glucose group (33 mmol/L D-glucose) and inhibitor group (33 mmol/L D-glucose+300 nmol/L 5Z-7-oxozeaenol).Immunocytochemistry and flow cytometry were used to detect macrophage subtype.The expression of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis Factor-α (TNF-α) mRNA were determined by real time PCR.Expressions of p-TAK1,TAK1 binding protein (TAB1),p-JNK,p-p38 MAPK and NF-κB p65 proteins were analyzed by Western blotting.Results The purity of BMDM was about 99.36％.Compared with normal control group,high glucose group had increased percentage of M1 macrophages,increased expression of MCP-1 and TNF-α mRNA (all P ＜ 0.05).Moreover,p-TAK1,TAB1,p-JNK,p-p38 MAPK and NF-κB p65 proteins expression also increased significantly in high glucose group (all P ＜ 0.05).After treatment with inhibitor 5Z-7-oxozeaenol,the effects induced by high glucose were inhibited (P ＜ 0.05).Conclusions High glucose can induce M1 macrophage activation and expression of inflammatory cytokine of BMDM,which can be inhibited 5Z-7-oxozeaenol through inhibiting TAK1/MAPK and TAK1/NF-κB pathway.

Aim We used bone marrow-derived macro-phages ( BMMs ) , to explore the mechanism of macro-phage activation and the effect of TGF-β activated ki-nase-1 ( TAK1 ) inhibitor 5 Z-7-oxozeaenol on it under AGEs conditions. Methods The BMMs were obtained from C57 mice, and purity of BMMs was detected by flow cytometry. Cell viability was tested after treatment with different concentrations of TAK1 inhibitors. Laser confocal microscopy was used to detect macrophage M1 subtype . Flow cytometry was used to analyse the macro-phage activated by AGEs. TNF-α and MCP-1 mRNA levels were evaluated by qRT-PCR. Western blot was used to detect the expression levels of TAK1 signal pathway protein. Results AGEs stimulation could in-crese the activity of M1 macrophages,and 5Z-7-oxoze-aenol could inhibit the differentiation of BMMs. Com-pared with control group, AGEs increased the expres-sion of MCP-1 and TNF-α mRNA(P<0. 01). p-TAK1, TAB1,p-JNK,p-p38MAPK and NF-κBp65 proteins ex-pression also increased significantly ( P <0. 05 ) . After treatment with inhibitor, transcription levels of MCP-1 and TNF-α decreased significantly ( P < 0. 05 , P <0. 01 ) . 5 Z-7-oxozeaenol treatment downregulated the expression of p-TAK1,TAB1,p-JNK,p-p38MAPK and
NF-κBp65 proteins ( P <0. 05 ) . Conclusions AGEs can induce BMMs to M1 phenotypic polarization. 5Z-7-oxozeaenol reduces the expression of inflammatory cyto-kine via inhibiting TAK1/MAPKs, MAPKs/NF-κB pathways.

Objective To investigate the renoprotective effect of transforming growth factor beta activator kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (OZ) in diabetic db/db mice and the mechanism.Methods Twenty-four male db/db mice were randomly divided into two groups:db/db mice (db/db,n=12) and db/db mice with 5Z-7-oxozeaenol treatment (db/db+OZ,n=12).Another group of wild type mice (n=12) was held as the control group.OZ 2 mg/kg was administrated by intraperitoneal injection every other day.At week 8 and 12 after 5Z-7-oxozeaenol treatment,blood glucose (BG),body weight (BW),kidney weight (KW) and urinary albumin excretion rate (UAER) were evaluated.Kidney pathological lesions were detected by light and electron microscopy.NF-κB p65,monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-ot (TNF-o) were detected by immunohistochemistry.Western blotting was used to detect p-TAK1,TAB1,p-p38MAPK and IL-1β expression,while ICAM-1 and MCP-1 mRNA levels were evaluated by real-time PCR.Results Compared with control group,the levels of BG,BW,KW and UAER were higher (P ＜ 0.01) in db/db mice group,while BW,KW and UAER levels were significantly decreased in db/db + OZ group compared with that in db/db mice group (P ＜ 0.05).In week 8 and 12 db/db mice,glomerular volume and extracellular matrix were increased,while pathological lesions in kidney tissue were positively improved by TAK1 inhibitor.Immunohistochemistry showed that NF-κB p65,MCP-1 and TNF-α expression levels were apparently increased in db/db mice group compared with that in control group (P ＜ 0.05) and were significantly inhibited by TAK1 inhibitor (P ＜ 0.05).Western blotting showed that p-TAK1,TAB1,p-p38MAPK and IL-1β expression levels were higher in db/db mice group than that in control group (P ＜ 0.05) and lower in db/db+ OZ group than that in db/db mice group (P ＜ 0.05).Moreover,real-time PCR showed that the expressions of ICAM-1 and MCP-1 mRNA were higher in db/db mice group than that in control group and lower in db/db+OZ group than that in db/db mice group (P ＜0.05).Conclusions TAK1 Inhibitor can down-regulate MAPK and NF-κB pathway to restrain the reaction of inflammation and alleviate kidney injury in diabetic db/db mice.

Objective To investigate the association of PR gene exon 5 region H770H (rs1042839) single nucleotide polymorphism (SNP) with the genetic susceptibility to endometriosis (EM) in southern Han Chinese women. Methods Totally 431 EM patients and 499 non-EM women were collected and separated into EM group and control group, that all cases were confirmed by operation and pathology. A case-control study was performed in EM and control groups to evaluate the association of these SNP with the susceptibility to EM by using a fluorescent quantitative PCR-based high resolution melting (HRM) method. Results The C and T of PR H770H allele frequencies among the EM and control groups were 97.9%(844/862), 2.1% (18/862) and 99.4% (992/998), 0.6% (6/998), respectively. The CC, CT and TT of PR H770H genotype frequencies among the EM and control groups were 95.8%(413/431), 4.2%(18/431), 0 and 98.8%(493/499), 1.2%(6/499), 0, respectively. There were statistical significances in the PR H770H alleles and genotypes distributions between the two groups (χ2=7.386, P=0.007;χ2=8.135, P=0.004). Carrying allele C reduced the risk of EM (OR=0.986, 95%CI: 0.976-0.996), while carrying allele T enhanced the risk of EM (OR=3.319, 95%CI: 1.323-8.325); carrying genotype CC reduced the risk of EM 0.970 time (OR=0.970, 95%CI: 0.949-0.991), whereas carrying genotype CT enhanced the risk of EM 3.473 times (OR=3.473, 95%CI:1.391-8.671). Conclusion There is significant association between the polymorphism of PR H770H and genetic susceptibility to EM in southern Han Chinese women.

OBJECTIVETo assess the association of a single nucleotide polymorphism(SNP) in tumor suppressor gene P53 with the risk of endometriosis (EM) in Han Chinese women.

METHODSFor 460 EM patients, 113 patients with endometrial carcinoma and 530 matched unrelated controls, a rs1042522(C/G) SNP of the P53 gene was genotyped by polymerase chain reaction-single strand polymorphism (PCR-SSP) and DNA sequencing.

RESULTSA significant difference has been detected in the distribution of rs1042522 alleles and genotypes between the EM patients and controls (P< 0.01). Allele G has increased the risk of EM by 1.209 times, while allele C has reduced this risk by 0.837 times. Compared with GG genotype, GC and CC genotypes have both increased the risk for EM (OR=2.073, 95%CI: 1.521-2.820, and OR=1.930, 95%CI: 1.363-2.733, respectively). Significant differences were also detected in the distribution of rs1042522 alleles and genotypes between endometrial carcinoma patients and controls (P< 0.01). Allele G has increased the risk to endometrial carcinoma by 1.311 times, while allele C has reduced this risk by 0.757 times. Compared with GG genotypes, individuals with GC and CC genotypes are more likely to be affected with endometrial carcinoma (OR=2.778, 95%CI: 1.585-4.870, and OR=2.864, 95%CI: 1.557-5.263, respectively).

CONCLUSIONOur study has suggested a significant association between the rs1042522(G/C) polymorphism and susceptibility to EM in Han Chinese women. The mechanism of EM is similar to carcinoma from genetics point of view.

Alleles ; Asian Continental Ancestry Group ; Base Sequence ; China ; Endometrial Neoplasms ; genetics ; Endometriosis ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Odds Ratio ; Polymorphism, Single Nucleotide ; Tumor Suppressor Protein p53 ; genetics

Objective To investigate the association of tumor necrosis factor-alpha (TNF-α) gene promoter region - 1031T/C and its combination with interleukin-6 (IL-6 ) gene promoter region -634C/G single nucleotide polymorphisms (SNP) with the genetic susceptibility to endometriosis.Methods Total of 432 endometriosis patients and 499 non-endometriosis women who had received an operation due to tubal ligation,tubal recanalization,laparoscopic hydrotubation,ovarian simple cyst and teratoma were collected and separated into endometriosis group and control group,that all cases were confirmed by operation and pathology.A case-control study was performed in endometriosis and control group to evaluate the association of these SNP with the susceptibility to endometriosis by using a fluorescent quantitative PCR-based high resolution melting ( HRM ) method.Results ( 1 ) TNF-α - 1031T/C genotype:the T and C of TNF-α - 1031T/C allele frequencies in the endometriosis group and control group were 79.2％ (684/864),20.8％ (180/864) and 81.8％ (816/998),18.2％ (182/998),respectively.The TT,TC and CC of TNF-α - 1031T/C genotype frequencies in the two groups were 63.7％ (275/432),31.0％ ( 134/432 ),5.3％ (23/432) and 66.5％ (332/499),30.5％ (152/499),3.0％ ( 15/499),respectively.There were no statistical significances in the TNF-α - 1031T/C alleles and genotypes distributions between the two groups ( P =0.158,P =0.186 ).( 2 ) TNF-α - 1031T/C and IL-6 - 634C/G conjoint genotypes:to research on the TNF-α - 1031T/C and IL-6 -634C/G genotypes for conjoint analysis,the TT + CC,TC + CC,CC +CC,TT + CG,TC + CG,CC + CG,TT + GG,TC + GG and CC + GG combination genotype frequencies in the two groups were 39.4％ ( 170/432 ),19.4％ ( 84/432 ),4.6％ ( 20/432 ),20.6％ ( 89/432 ),8.8％ (38/432),0.9％ (4/432),3.5％ (15/432),2.3％ (10/432),0.5％ (2/432) and 36.7％ ( 183/499),17.4％(87/499),1.4％ (7/499),26.1％ (130/499),10.4％ (52/499),1.2％ (6/499),3.8％ (19/499),2.6％ ( 13/499),0.4％ (2/499),respectively.There were no statistical significances in the combination genotypes distributions between the two groups ( P =0.107 ).As compared with carriers of TT + CC combination genotype,the endometriosis risk of carriers of CC + CC combination genotype enhanced 3.076 times ( 95％ CI:1.268 - 7.457,P =0.009 ),and the endometriosis risk of carriers of other combination genotypes were no statistical significances (all P ＞ 0.05 ).ConclusionsThe study demonstrates that there are no significant association between the SNP of TNF-α - 1031T/C and genetic susceptibility to endometriosis.However the results indicate that there are significant association betweengenetic susceptibility to endometriosis and the combination polymorphisms of TNF-α -1031T/C and IL-6- 634C/G.

Objective To investigate the association of polymorphisms of arylhydrocarbon receptor (AhR) - 1661G/A with glutathione S-transferase pi ( GSTP1 ) - 313A/G and the susceptibility to endometriosis in southern Han Chinese.Methods Total of 432 endometriosis patients undergoing laparoscopic or laparotomy surgery matched with 493 patients with fallopian tube ligation,tubal recanalization,laparoscopic hydrotubation,benign ovarian tumor and teratoma surgeries without endometriosis as control group were enrolled in this study.The single nucleotide polymorphism (SNP) of AhR -1661G/A and GSTP1 -313A/G were detected by using a fluorescent quantitative PCR-based high resolution melting (HRM).Results The numbers of combined genotypes AhR - 1661G/A and GSTP1 -313A/G were 120 patients with AG + AA,64 patients with AG + AG,8 patients with AG + GG,109 patients with GG +AA,84 patients with GG + AG,4 patients with GG + GG,31 patients with AA + AA,10 patients with AA + AG,1 patient with AA + GG at endometriosis group and 131 patients with AG + AA,68 patients with AG + AG,6 patients with AG + GG,157 patients with GG + AA,66 patients with GG + AG,4 patients with GG + GG,35 patients with AA + AA,20 patients with AA + AG,3 patients with AA + GG at endometriosis group.There was no statistically different frequencies of genotypes between endometriosis group and control group (x2 = 12.558,P = 0.128 ).Compared with genotype GG + AA,the risk of endometriosis with genotype GG + AG was increased 1.833 time (95％CI:1.233-2.274).Conclusion The combined genotype GG + AG [ from AhR - 1661G/A (GG) and GSTP1 - 313A/G (AG) ] might be related with susceptibility to endometriosis.