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 overcome the limitations of existing human respiratory syncytial virus(hRSV)animal models,such as semi-permissiveness and short duration of infection,this study established an IL2rg gene knockout(IL2rg-/-)rat model using TALEN gene editing technology.Methods The animal model was infected with hRSV intranasally.Clinical characteristics,body weight,and temperature changes were observed over the infection period(0～35 days).The total viral loads in respiratory organs,such as the nasal tissue,trachea,and lungs,were measured at various time points(4,11,20,and 35 days post-infection).Pathological analysis was conducted on target organs at the endpoint of observation(35 days post-infection).Changes in peripheral blood T,B,NK,and NKT cells and various cytokines were assessed at various time points(4,20,and 35 days post-infection).Results(1)IL2rg/-knockout rats sustained high viral loads in the nasal cavity upon intranasal inoculation with hRSV.The average peak titer rapidly reached 1 × 1010 copies/g in nasal tissue and 1 × 107 copies/g up to 5 weeks post-infection.(2)However,no significant pathological changes were noted in nasal,tracheal,or lung tissues.(3)An increase was observed in the content of peripheral blood B cells in hRSV-infected IL2rg--rats.(4)IL-6 and MCP-1 were increased in the early stage of infection and then decreased at the end of the observation period.Conclusions This study established a new IL2rg-/-rat model using TALEN technology and found that this model effectively supported high-level replication and long-term infection of hRSV,providing a good basis for antiviral drug screening and in vivo efficacy evaluation of anti-hRSV antibodies.

ObjectiveThrough improving the potential of vascular endothelial growth factor receptor (VEGFR)-humanized mouse model (hKDR^+/+) with C57BL/6N background to allow the growth of different mouse tumor cell lines, to establish novel tumor-bearing mouse models which can support in vivo tumorigenesis of different mouse tumor cell lines and be used to evaluate drugs targeting VEGFR.MethodsFirstly, a method to evaluate the in vivo activity of antibody targeting VEGFR based on the hKDR^+/+ humanized mouse model was established. Recombinant activating gene 1 (Rag1) knockout mice (Rag1^-/-) were established using CRISPR/Cas9 technology. Then these Rag1^-/- mice were crossed with hKDR^+/+ mice to get a double gene modified homozygous hKDR^+/+/Rag1^-/- mouse model by screening. Finally, tumor cell lines derived from different mouse strains were tested on the double gene-modified mouse model to compare the differences in tumor growth. ResultsAntibodies designed for VEGFR showed significant anti-tumor activity in hKDR^+/+ mice, which significantly reduced tumor volume and weight compared with the PBS group (P<0.01, P<0.05). The number of B cells and T cells in the peripheral blood of Rag1^-/- mice and hKDR^+/+/Rag1^-/- mice decreased (P<0.05, P<0.001). Tumors were observed in hKDR^+/+/Rag1^-/-, Rag1^-/-, wild-type, and hKDR^+/+ mice after 7 d of inoculation of MC38 cells derived from C57BL/6 mice. Tumors were only observed in groups of hKDR^+/+/Rag1^-/- and Rag1^-/- mice, but not in the wild-type and hKDR^+/+ mice after 10 d of inoculation with CT26 cells derived from BALB/c mice. After 3 weeks of inoculation, the tumor volume of hKDR^+/+/Rag1^-/- mice was significantly larger than that of Rag1^-/- mice (P<0.01). ConclusionRag1 knockout mice were obtained and a novel hKDR^+/+/Rag1^-/- double genes modified mouse model was further screened. The tumor cell lines from different mouse strain origins were more prone to growth in mice with Rag1 gene deficiency. The results suggest that the reduced immune response of hKDR^+/+ humanized mice will improve the capacity of supporting the growth of mouse tumor lines in the model. As a result, more tumor-bearing mouse models may be constructed for the evaluation of drugs targeting VEGFR in this way.

Although there is insufficient evidence supporting the link between septate uterus and infertility, there are many studies demonstrated the effect of spetal incision on pregnancy in women diagnosed with septate uterus associated with infertility. Hysteroscopic metroplasty can significantly improve the reproductive performance of those with septate uterus. Some Müllerian malformations can be healed by surgery. The accurate diagnosis and appropriate therapeutic approch are fundamental for successful treatment. Any attempt at surgical correction of uterine abnormalities must be aimed at preserving or improving reproductive function. Among congenital uterine anomalies, septate uterus is the most amenable to simple hysteroscopic treatment. The resection of the septum is performed as standard treatment worldwide.
Female ; Humans ; Septate Uterus

Objective To discuss the application of combined probiotics pretreatment and late triple therapy with helicobacter pylori infection in children.Methods 300 children with helicobacter pylori infection were randomly divided into three groups according to the way of random table.Group A was treated with standard triple therapy, omeprazole,amoxicillin and clarithromycin treatment,and 10 d was 1 treatment course.B group was given probiotic pretreatment,prior to the triple therapy using compound lactobacillus acidophilus piece 1 piece /time,cold water to take after meals,taking 2 weeks,follow -up treatment with triple therapy for 10d.In group C,triple therapy before using compound lactobacillus acidophilus piece 1 piece /time,cold water to take after meals,taking 2 weeks,follow -up treatment with triple therapy for 10d.To take one week after the completion of compound lactobacillus acidophilus piece 1 piece /time,cold water after meals.The clinical therapeutic effects were recorded.Results The helicobacter pylori clearance rate of group A was 55.00%,that of group B was 86.00%,that in group C was 89.00%,the helico-bacter pylori clearance rate of group B and group C was significantly higher than that in group A,the difference was statistically significant (χ2 =23.103 7,28.670 6,all P <0.05).The treatment effect between group B and group C had no statistically significant difference (χ2 =0.411 4,P >0.05 ).In A group,nausea and vomiting occurred in 5 cases,2 cases of diarrhea,abdominal distension abdominal pain in 4 cases,skin rash in 5 cases.In group B,nausea and vomiting occurred in 2 cases,0 cases of diarrhea,abdominal distension abdominal pain in 1 case,skin rash in 1 case.In group C,nausea and vomiting occurred in 1 case,0 cases of diarrhea,abdominal distension abdominal pain 0 cases,skin rashes in 1 case.The incidence rate of adverse reactions in group B and group C was lower than that in group A,the difference was statistically significant (χ2 =11.965 8,8.000 0,all P <0.05).The incidence rate of ad-verse reactions between group B and group C had no statistically significant difference (χ2 =0.687 3,P >0.05). Conclusion Joint probiotics pretreatment and late triple therapy application in helicobacter pylori in children can promote helicobacter pylori clearance,reduce the triple therapy drug adverse reactions,it is worthy of popularization and application in clinic.

Objective To investigate the clinical significance of detecting cardiac troponin Ⅰ (cTnⅠ) and CK -MB in children with hand-foot-and-mouth disease and myocardial injury.Methods 90 children with hand-foot-and-mouth disease (observation group) were detected the level of serum cTnⅠ and CK-MB.At the same time,40 healthy children were chosen as control group.Results Compared with the control group,the levels of CK-MB and cTnl in observation group were significantly higher than those of the control group (t =8.92,5.46,all P ＜ 0.01),which indicated that children with hand,foot and mouth disease was easier to merge myocardial injury.CK-MB and cTnl levels in high-risk group were significantly higher than those in normal children group (t =9.17,6.13,all P ＜ 0.01),and the levels of CK-MB and cTnl were positively correlated with severe degree (r =0.767,0.683,all P ＜ 0.01).For children with hand,foot and mouth disease merged myocarditis,cTnl diagnostic sensitivity (53.8％) was lower than that of CK-MB diagnostic sensitivity (71.8％),but the specificity was better than that of CK-MB,and the diagnosis of cTnl detection window 2 weeks longer than CK-MB,but after 2 weeks both lose their clinical diagnostic significance.Conclusion Children with hand,foot and mouth disease easily merge myocardial damage,dynamic measuring CK-MB and cTnl levels could help early diagnosis of children with hand,foot and mouth disease whether merged myocardial damage,both applications can also complement each other,more timely and accurate reflection of disease progression and recovery,it is worth promoting.

Objective To obtain the basic growth parameters of a self-established F1 hybrid CB6F1 C57-ras transgenic mouse model, and to provide basic information for commercialization of this mouse model. Methods F1 hybrid mice (CB6F1) were produced by crossing C57-ras heterozygous transgenic (c-Ha-ras+/-) male mice and wild-type BALB/cJ female mice.The average litter size, weaning rate, sex ratio, growth performance and C57-ras transgenic positive rate were recorded and analyzed.Results The average litter size was eight, weaning rate was 90%, and sex ratio was approximately in accordance with prediction.The average body weight of newborn mice was 1.73 ±0.05 g.The average body weight of 10-week old c-Ha-ras transgenic female and male mice in CB6F1 background was 24.38 ±1.74 g and 29.42 ±1.72g, respectively, which had a significant difference (P<0.01).The c-Ha-ras transgenic positive rate was 46.9%. which was in accordance with genetic rules.Conclusions The F1 hybrid mice (CB6F1) produced by crossing C57-ras heterozygous transgenic ( c-Ha-ras +/-) male mice and wild-type BALB/cJ female mice show normal growth performance and development characteristics, and it can be used for large-scale commercial supply.

The effects of quercetin-3-O-β-D-glucuronide (Q3GA)on the triglyceride metabolism and oxidative stress in steatotic HepG2 cells and the underlying mechanism were investigated in this study.Significant fat accu-mulation was documented by Oil Red O staining;intracellular triglyceride levels were detected by triglyceride(TG)enzymatic assay.DCFH-DA staining assay was performed to observe reactive oxygen species (ROS)pro-duction of HepG2 cells.The level of malondialdehyde(MDA)and superoxide dismutase(SOD)were assayed by thibabituric acid method and xanthine oxidase method.Changes in the mRNA expression of peroxisome prolifera-tor-activated receptorα(PPARα);carnitine palmitoyltransferase 1 A(CPT1 A);medium chain acyl-CoA dehydro-genase (MCAD);cytochrome P450 4A11(CYP4A11)and acyl-CoA oxidase(ACO);which are related with fatty acid oxidation were assessed by RT-PCR.Our results showed that Q3GA obviously reduced fat deposition and TG content.At the same time;Q3 GA decreased MDA content and significantly increased the SOD activity with reduced ROS production.Moreover;the PPARα;CPT1A;MCAD expression-related fatty acid βoxidation was upregulated with the treament of Q3GA;while without any change of the expression of CYP4A11;ACO.In conclu-sion;Q3GA prevents FFA-induced HepG2 cell steatosis;and enhances mitochondrial fatty acidβoxidation;which may partly be related to its anti-oxidation ability.

Objective To study the value of three-dimensional (3D) visualization and virtual surgery system in piggyback liver transplantation. Methods Two patients who suffered from choledocholithiasis were scanned by 64-slice spiral CT and the data were collected. The segmentation of the hepatic CT images was carried out. The 3D model of the liver and the intrahepatic vessels was reconstructed, and was imported to the FreeForm Modeling System in STL format for smoothing and modifying. Piggyback liver transplantation was simulated with the force-feedback equipment (PHANToM). Results The reconstructed 3D model of the liver was vivid, and the process of the virtual piggyback liver transplantation was verisimilar. Conclusions The 3D model of the liver enables the simulation of piggyback liver transplantation. It can reduce the risk and complications of the surgery, and enhance the communication between doctor and patient through designing surgical plan and demonstrating visualized operation before surgery. Virtual liver transplantation is also helpful during the training of medical workers.