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 To investigate whether curcumin alleviates septic lung injury by inhibiting ferroptosis through modulating the TXNIP/TRX-1/GPX4 pathway.Methods Male C57BL/6 mice were randomly divided into Sham group,cecal ligation puncture(CLP)-induced sepsis group,CLP with curcumin treatment(50,100,and 200 mg/kg)groups,and CLP with both curcumin(200 mg/kg)and TRX-1 inhibitor PX-12(25 mg/kg)treatment group.Inflammatory factors,MDA,MPO,and GSH levels in the lung tissue of the mice were detected.Beas-2B cells stimulated with lipopolysaccharide(LPS;1 μg/mL)were treated with 2.5,5,or 10 μmol/L curcumin or with 10 μmol/L curcumin combined with 5 μmol/L PX-12,and the changes in MDA,Fe2+and ROS levels were assessed.Western blotting was performed to detect the protein expressions of TXNIP,TRX-1,GPX4 and X-CT in both the mouse lung tissues and Beas-2B cells.Results The mice with CLP-induced sepsis showed severe lung injury with elevated expressions of IL-6,IL-1β,TNF-α,MDA and MPO and decreased GSH expression.In Beas-2B cells,LPS stimulation significantly increased MDA and Fe2+levels and ROS release,increased TXNIP protein expression,and lowered the protein expression levels of TRX-1,GPX4 and X-CT,and these changes were also observed in the septic mice.Curcumin treatments at different concentrations obviously alleviated lung injury in the septic mice and reduced LPS-induced injury in Beas-2B cells.Curcumin significantly decreased the release of inflammatory factors,MDA and MPO,increased GSH level,lowered Fe2+,MDA and ROS levels,increased TXNIP protein expression,and lowered the protein expressions of TRX-1,GPX4 and X-CT in both septic mouse lung tissues and LPS-stimulated Beas-2B cells.The protective effect of curcumin was effectively blocked by PX-12 treatment.Conclusion Curcumin inhibits ferroptosis and alleviates septic lung injury in mice by elevating TRX-1 and GPX4 and decreasing TXNIP in the lung tissue.

Objective To establish a method of LC-MS/MS for determining cordycepin(Cor)and 3′-deoxyinosine(3′-Deo)concentration in rat plasma,and to study their pharmacokinetics in rats.Methods Protein was precipitated with methanol using 2-chloadenosine(2-Chl)as an internal standard.The chromatography was performed on Kinetex C18(3 mm×100 mm,2.6 μm,Phenomenex,USA)with gradient elution in aqueous(5 mmol·L-1 ammonium acetate)-methanol solution as mobile phase.ESI ion source was used for mass spectrometry,and positive ion multiple reaction monitoring(MRM)was used for scanning detection.The pharmacokinetics of Cor and 3′-Deo after oral administration of Cor(10 mg·kg-1)were studied in rats.Results Cor at 0.5-100 ng·mL-1 and 3′-Deo at 1-200 ng·mL-1 had good linearity,and the lower limits of quantification were 0.5 and 1 ng·mL-1,respectively.After oral administration of Cor in rats,the plasma concentration of Cor was low,which was mainly converted into the metabolite 3′-Deo.The Cmax of Cor and 3′-Deo were(5.4±3.4)and(142.0±50.0)ng·mL-1,and AUC0-360min min were(658.4±459.3)and(18 034.9±4 981.1)ng·min·mL-1,respectively.Conclusion The method is simple,sensi-tive,and accurate,which is suitable for determining Cor and 3′-Deo concentration in plasma and the pharmacokinetic study.

Objective To investigate whether curcumin alleviates septic lung injury by inhibiting ferroptosis through modulating the TXNIP/TRX-1/GPX4 pathway.Methods Male C57BL/6 mice were randomly divided into Sham group,cecal ligation puncture(CLP)-induced sepsis group,CLP with curcumin treatment(50,100,and 200 mg/kg)groups,and CLP with both curcumin(200 mg/kg)and TRX-1 inhibitor PX-12(25 mg/kg)treatment group.Inflammatory factors,MDA,MPO,and GSH levels in the lung tissue of the mice were detected.Beas-2B cells stimulated with lipopolysaccharide(LPS;1 μg/mL)were treated with 2.5,5,or 10 μmol/L curcumin or with 10 μmol/L curcumin combined with 5 μmol/L PX-12,and the changes in MDA,Fe2+and ROS levels were assessed.Western blotting was performed to detect the protein expressions of TXNIP,TRX-1,GPX4 and X-CT in both the mouse lung tissues and Beas-2B cells.Results The mice with CLP-induced sepsis showed severe lung injury with elevated expressions of IL-6,IL-1β,TNF-α,MDA and MPO and decreased GSH expression.In Beas-2B cells,LPS stimulation significantly increased MDA and Fe2+levels and ROS release,increased TXNIP protein expression,and lowered the protein expression levels of TRX-1,GPX4 and X-CT,and these changes were also observed in the septic mice.Curcumin treatments at different concentrations obviously alleviated lung injury in the septic mice and reduced LPS-induced injury in Beas-2B cells.Curcumin significantly decreased the release of inflammatory factors,MDA and MPO,increased GSH level,lowered Fe2+,MDA and ROS levels,increased TXNIP protein expression,and lowered the protein expressions of TRX-1,GPX4 and X-CT in both septic mouse lung tissues and LPS-stimulated Beas-2B cells.The protective effect of curcumin was effectively blocked by PX-12 treatment.Conclusion Curcumin inhibits ferroptosis and alleviates septic lung injury in mice by elevating TRX-1 and GPX4 and decreasing TXNIP in the lung tissue.

Objective: To study the structures of ginseng glycopeptides, and to explore the protective effect on the apoptosis of PCI 2 cells treated with amyloid beta25-35 (Aβ25-35), and to lay a theoretical foundation for the development of ginseng anti-Alzheimer' s disease (AD) drugs. Methods: The structures of ginseng glycopeptides were analyzed by reversed-phase high performance liquid chromatography coupled with Q Exactive Orbitrap mass spectrometry. The PCI2 cells were divided into 6 groups, and treated with the medium including different concentrations 0, 6. 25, 12. 50, 25. 00, 50. 00, and 100. 00 mol • L_ 1 ) of Aβ25-35 , and the survival rates of PC12 cells were measured by cell counting kit-8 (CCK-8) method. The PC12 cells were divided into blank control group, model group, and ginseng glycopeptides administration group. The medium including 50. 00 jumol • L_ 1 Aβ25-35, was added in model group, and different concentrations 0. 03, 0.10, 0. 30, and of 1. 00 g • L_ 1 ) ginseng glycopeptides+ 50 jumol • L_ 1 Aβ25-35 were added in administration groups. The survival rates of PC12 cells were measured by CCK-8 method. The apoptotic rates of PCI 2 cells after treated with glycopeptides and Aβ25-35, were detected by Annexin V-FITC/PI method. The PCI 2 cells were divided into blank control group, model group (50.00 jumol • L_ 1 Aβ25-35) and different concentrations (0 . 1 0 and 0.30 g • L_ 1 ) of ginseng glycopeptides administration groups; the percentages of PCI 2 cells in different cell cycles were tested by flow cytometry. Results: More than 20 glycopeptide structures were obtained, such as the peptide chain was NLSHYHSGSS, the glycosyl group was Nl-HexNAc, and the peptide chain was SGSSSSSSSEDDGMGR, the glycosyl group was S6-HexNAc. When the concentration of Aβ25-35 was 50 jumol • L_ 1 , the survival rate of PC12 was (55. 45 + 2. 3 4) % and the survival rate was significantly lower than that in blank control group (P < 0 . 0 1) . Compared with blank control group, the survival rate of the PC12 cells in model group was significantly decreased (P < 0 01); compared with model group, the survival rates of the PCI2 cells in administration groups were significantly increased (P< 0. 05). Compared with blank control group, the apoptotic rate of the PC12 cells in model group were increased (P < 0. 01); compared with model group, the apoptotic rates of the PC12 cells in administration groups were decreased (P < 0 . 05). Compared with blank control group, the percentage of PC12 cells in S phase in model group was increased (P

Objective To investigate the effects of S-allylcysteine ( SAC ) , on nitric oxide ( NO ) production and antioxidant enzyme activities in hyperlipidemic rats. Methods Male Wistar rats were randomly divided into seven groups. Five groups including normal control group ( normal diet) , model control group ( high-fat diet, HFD) and SAC low,medium,high treated group (high-fat diet +25,50,100 mg·kg-1 SAC) were sacrificed after 4 weeks dosing,while the other two groups including L-arginine group (normal diet+ 20 mg·kg-1 L-arginine) and SAC+L-arginine group (50 mg·kg-1 SAC+20 mg·kg-1 L-arginine) were sacrificed at 4 h after dosing. The serum, livers and kidneys were collected. The levels of NO, the activities of nitric oxide synthase ( NOS ) , antioxidant enzymes in vivo and L-arginine contents in serum were determined. Results Comparing with model control group, the activities of total NOS in serum and liver were significantly reduced in SAC-treated groups (P<0. 05). The level of L-arginine in SAC-treated groups was (8. 25 ± 1. 15), (7. 76 ± 1. 24) and (7. 22 ± 1. 64)μg·mL-1 , respectively. Compared with model control group, the level of L-arginine were significantly reduced in SAC-treated groups (P<0. 05). Comparing with L-arginine group, the activities of total NOS (T-NOS) and iNOS were reduced in SAC+L-arginine group. SAC treatment (100 mg·kg-1) significantly increased the activities of superoxide dismutase (SOD) (P<0. 01) and the level of glutathione (GSH) (P<0.01), and decreased the level of malondialdehyde (MDA) in serum and liver of hyperlipidemic rats. Conclusion These data suggest that SAC inhibits the NO production by reducing iNOS activity, arginine concentration and exhibited antioxidant activity, which may play a pharmacologically important role in protection from oxidative injury and pathogenesis of atherosclerosis.

Objective To investigate the relationship between the expression of β-catenin and drug-resistance mechanism of choriocarcinoma according to the expression of β-catenin in JEG-3 cells (human choriocarcinoma cell line) and drug resistant JEG-3/VP16 cells.Methods The mRNA and protein expressions of β-catenin were analyzed with reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting.Flow cytometry was used to determine the percentages of β-catenin-positive cells in the two choriocarcinoma cell lines.Results Both drug resistant choriocarcinoma cells and drag sensitive cells were found to express β-catenin; but the expression of β-catenin mRNA (1.43 ±0.24) and protein(1.49 ±0.17)in drug resistant choriocarcinoma cells was found much higher than that in drug sensitive cells(0.65 ±0.14,0.66 ±0.16,P ＜0.01).And according to detect by flow cytometry,we found the number of β-catenin-positive cells in JEG-3/VP16 cells [(40.13 ±5.17) ％] was much more than that in JEG-3 cells [(13.15 ± 1.48) ％,P ＜ 0.01].Conclusions β-catenin was highly expressed in the drug resistant choriocarcinoma cell line (JEG-3/VP16).It indicates β-catenin might be involved in the drug resistance mechanism of choriocareinoma.

Objective To evaluate the clinical efficacy and adverse effects of brucea javanica oil emulsion combined with DP chemotherapy in treating advanced non-small-cell lung cancer.Methods Totally 48 patients with advanced non-small-cell lung cancer were divided into two groups randomly by mechanical sampling method.Twenty-four cases in treatment group were treated by brucea javanica oil emulsion combined with DP chemotherapy, while 24 cases in control group were treated by DP chemotherapy only.The clinical effects were evaluated after treatment of two cycles.Results The short-term effective rate was 54.2% (13/24) in treatment group and 45.8% (11/24) in control group, and there was no significant difference between two groups ( χ2 = 0.333, P = 0.564).The rate of increased and stable life quality was 87.5%(21/24) in treatment group and 58.3%(14/24) in control group,and there was significant difference between two groups (χ2 = 5.169,P = 0.023).The rate of increased and stable weight was 79.2% (19/24) in treatment group and 45.8%( 11/24) in control group, and there was significant difference between two groups (χ2 = 5.689,P = 0.017).The incidence of nausea or vomiting was 45.8% (11/24) in treatment group and 41.7%( 10/24 ) in control group, and there was no significant difference between two groups (χ2 = 0.085, P = 0.771 ).Compared with those in control group, patients in treatment group had less adverse effects in decreasing of peripheral blood leucocytes and showed better immune function.Conclusion Brucea javanica oil emulsion combined with DP chemotherapy in treating advanced non-small-cell lung cancer has good clinical effect, especially enhances the quality of life, improves immune function and decreases the adverse effects of chemotherapy.