Objective To investigate the effects of Caenorhabditis elegans(C.elegans)endogenous U6 promoters on dpy-10 gene editing efficiency.Methods We screened endogenous U6 small nuclear RNA(snRNA)genes of C.elegans from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6r07e5.16 promoter in the pSX524 plasmid(Peft-3::cas9::tbb-2 terminator::U6 r07e5.16::dpy-10 sgRNA)through molecular cloning.Gene editing was performed in wild-type C.elegans using a standardized microinjection protocol.Gene editing efficiency and the high-efficiency gene editing index were quantified based on the screening of dpy-10 mutant phenotypes in the F1 progeny.Results A total of 15 U6 snRNA genes(r07e5.16,f35c11.9,t20d3.13,k09b11.15,k09b11.16,w05b2.8,c28a5.7,f54c8.9,k09b11.11,k09b11.12,k09b11.14,t20d3.12,f54c8.8,f54c8.10,and k09b11.13)were identified from the WormBase database.Based on the editing efficiency and high-efficiency gene editing index,the activity of these promoters was evaluated,and 4 U6 promoters(w05b2.8,c28a5.7,f54c8.9,and k09b11.11)were found to have significantly enhanced gene editing success rates,outperforming other promoters,including U6r07e5.16 and U6k09b11.12,which are commonly used in the C.elegans research community.Notably,the gRNAF+E scaffold did not show superior editing efficiency over the gRNA scaffold when paired with the optimal U6w05b2.8 promoter.Conclusion In this study,U6 promoters that significantly improve gene editing efficiency in C.elegans are identified and the critical role of promoter optimization in CRISPR-Cas9 systems is highlighted.These findings provide a valuable foundation for improving genome editing strategies and offer new ideas for optimizing the CRISPR technology applied in nematode research.

Objective To optimize the real-time quantitative polymerase chain reaction(RT-qPCR)data analysis process through mathematical principles by replacing the biased 2-??CT method with a more rigorous 2-CT method,thereby improving the accuracy of gene expression quantification analysis.Methods Essentially,the CT value serves as the exponent in a base-2 exponential equation within the logic of comparative CT method.In the traditional 2-??CT method,the arithmetic means of raw CT and ΔCT values are directly calculated and the exponential nature of CT data is overlooked,which may introduce systematic bias to the calculation results.We propose a new method,entitled the 2-CT method,in which all calculations are based on the transformation of CT values into 2-CT.This includes computing the relative initial expression levels of target and reference genes within each sample,the relative abundance of the target gene,and its fold change across groups.Statistical comparisons are then performed based on fold change values.By strictly adhering to the exponential nature of of CT values,the biases introduced by arithmetic averaging at the CT or ΔCT level are avoided.We applied this method to multiple RT-qPCR datasets to evaluate the differences between the traditional 2-??CT and the proposed 2-CT methods in gene expression quantification,as well as the effect of the differences.Results In the original dataset from LIVAK and SCHMITTGEN,the two methods produced similar results.However,in the cadmium exposure experiment,findings from the 2-??CT method indicated that 8-hour cadmium exposure caused an increase of irg-6 gene expression in Caenorhabditis elegans from 1.314-fold to 7.125-fold(P=0.000 2).In contrast,findings from the 2-CT method showed a fold change from 1.0 to 4.124(P=0.001 5),a 70%difference between the two methods.Conclusion The2-CT method provides a mathematically more rigorous approach that more accurately reflects gene expression changes,particularly in experiments with high CT variability.It offers a more reliable computational paradigm for quantitative gene expression analysis.

Objective: To investigate the expression of ribosomal protein L26 ( RPL26) in gastric cancer cells (GC) and its effect on cell apoptosis and proliferation . Methods: The expression of RPL26 in GES-1 and GC cell lines was detected by Western blot. GC cell line HGC-27 was used to construct RPL26 overexpression cell line , and GC cell lines HGC-27 and AGS cells were used to construct RPL26 knockdown cell line . The overexpression and knockdown efficiency of RPL26 were detected by Western blot. Cell counting kit-8 (CCK-8) , colony formation assay and Transwell assay were used to detect the effects of the overexpression and knockdown of RPL26 on the pro- liferation and migration of GC cells . Western blot was used to detect the expression of Phosphatidylinositol-3-kinase (PI3K) / protein kinase B (AKT) signaling pathway related factors PI3K , AKT , phosphorylated phosphatidylinosi- tol-3-kinase (p-PI3K) , phosphorylated protein kinase B ( p-AKT) and downstream factors B-Cell lymphoma-2 (Bcl-2) , Bcl-2 associated X protein (Bax) and Cyclin A , G1 /S-specific Cyclin D1(Cyclin D1) , Cyclin-depend- ent kinases (CDK)4 and CDK2 in overexpression and knockdown of RPL26 stably transfected cell lines . Results: Compared with GES-1 , RPL26 was highly expressed in HGC-27 cells ( tHGC-27 = 4. 97 ; P < 0. 01) and elevated in AGS , but the difference was not statistically significant. In HGC-27 and AGS cells , CCK-8 and colony formation assays showed that the proliferation ability of cells decreased after the knockdown of RPL26. Transwell assay showed that the migration ability of cells decreased after the knockdown of RPL26. Western blot showed that Bcl-2 expression was decreased in HGC-27 , AGS cells after the knockdown of RPL26 ( tHGC-27 = 11 . 50 , tAGS = 4. 77 ; P < 0. 001 , P < 0. 01) , and Bax expression increased ( tHGC-27 = 9. 63 , tAGS = 4. 05 ; P < 0. 001 , P < 0. 05) . In HGC-27 cells , the ratios of p-PI3K/PI3K and p-AKT/AKT significantly decreased after the knockdown of RPL26 ( tp-PI3K/PI3K = 3 . 86 , tp-AKT/AKT = 8. 29 ; P < 0. 05 , P < 0. 01) . Cyclin A , Cyclin D1 , CDK4 , CDK2 protein expressions de- creased ( t = 9. 61 , 5 . 10 , 11 . 64 , 7. 81 ; P < 0. 01 or P < 0. 001) , while the overexpression of RPL26 in HGC-27 cells showed the opposite trend . Conclusion The knockdown of RPL26 may arrest the cell cycle in G1 /S phase by inhibiting the PI3K/AKT signaling pathway , thereby inhibiting cell proliferation and promoting apoptosis .

Objective:To analyze the efficacy of anterior cervical Hybrid surgery and posterior cervical expansive open-door laminoplasty(EODL)in the treatment of multilevel cervical spondylotic myelopathy,and to discuss the selection of surgical methods for the patients with multilevel cervical spondylotic myelopathy.Methods:The retrospective analysis was conducted of 70 patients with multilevel cervical spondylotic myelopathy who underwent surgery at Affilated Beijing Traditional Chinese Medicine Hospital of Capital Medical University from July 2017 to July 2020.Based on the different surgical methods,the patients were divided into anterior group(n=35)and posterior group(n=35).The patients in anterior group underwent Hybrid surgery[anterior cervical discectomy and fusion(ACDF)combined with artificial cervical disc replacement(ACDR)],and the patients in posterior group underwent EODL.The hospitalization time,operation time,intraoperative blood loss,and postoperative drainage volume of the patients in two groups were recorded;the efficacy was evaluated by Japanese orthopaedic association(JOA)score,JOA improvement rate,neck disability index(NDI),visual analogue scale(VAS)for pain,and postoperative satisfaction score;the complications of the patients in two groups after surgery were recorded.Results:Compared with posterior group,the intraoperative blood loss,postoperative drainage volume,hospitalization time,and operation time of the patients in anterior group were significantly decreased(P<0.01),and the preoperative score had no significant difference(P>0.05).At the final follow-up after surgery,compared with posterior group,the JOA score and JOA improvement rate of the patients in anterior group were significantly increased(P<0.01),and the NDI score and VAS score were significantly decreased(P<0.01).Compared with before surgery,the JOA scores of the patients in two groups at the final follow-up after surgery were increased(P<0.01),and the NDI and VAS scores were significant decreased(P<0.01).The postoperative satisfaction of the patients in two groups was high based on the postoperative satisfaction score.There was no significant difference in the incidence of postoperative complication of the patients between two groups(P>0.05).Conclusion:Both the anterior cervical Hybrid surgery and EODL achieve the satisfactory results in the treatment of multilevel cervical spondylotic myelopathy.Hybrid surgery has the advantages of less bleeding and shorter surgery time,and the most suitable surgical method should be chosen clinically based on the actual situation of the patients.

Objective:To study the diagnostic value of artificial intelligence B-ultrasound image computer-aided diagnosis system (hereinafter referred to as intelligent ultrasound system) in adult goiter.Methods:In June 2022 and March 2023, two phases of thyroid disease survey were carried out in 4 cities in Anhui Province. One village was selected in each city, and 250 adults were selected as survey subjects in each village. Adult bilateral thyroid area was scanned by both intelligent ultrasound system and conventional ultrasound scanning equipment, and the effectiveness of intelligent ultrasound system in the diagnosis of goiter was analyzed based on the results of conventional ultrasound examination. Receiver operating characteristic (ROC) curve was drawn, and Kappa test was used to analyze the consistency between intelligent ultrasound system and conventional ultrasound examination in the diagnosis of goiter. At the same time, Spearman correlation analysis and Bland-Altman method were used to evaluate the consistency of the two methods in measuring thyroid volume.Results:After screening and removing outliers and missing values, a total of 910 adults were included, including 253 males (27.80%) and 657 females (72.20%). The age was (45.92 ± 10.20) years old, ranging from 18 to 60 years old. The sensitivity, specificity, and accuracy of the intelligent ultrasound system for diagnosing adult goiter were 80.00%, 99.67%, and 99.56%, respectively. The area under the ROC curve (AUC) was 0.996, which was consistent with the results of conventional ultrasound examination for diagnosing goiter ( κ = 0.67, P < 0.001). After controlling for variables such as gender, thyroid function, and thyroid nodules, the intelligent ultrasound system showed good consistency with conventional ultrasound examination in the diagnosis of goiter in females, adults with thyroid dysfunction, and adults without thyroid nodules ( κ = 0.66, 0.80, 0.80, P < 0.001). The consistency in the diagnosis of goiter in adults with thyroid nodules was moderate ( κ = 0.56, P < 0.001). Spearman correlation analysis showed a highly positive correlation between the measurement results of adult thyroid volume by intelligent ultrasound system and conventional ultrasound examination ( r = 0.88, P < 0.001). The Bland-Altman method results showed that only 4.62% (42/910) of points in adults were outside the 95% consistency limit, indicating good consistency between intelligent ultrasound system and conventional ultrasound examination in measuring thyroid volume (< 5%). The proportion of points outside the 95% consistency limit in males, adults with thyroid dysfunction, and adults with thyroid nodules was 6.72% (17/253), 5.83% (12/206), and 6.45% (12/186), respectively. Conclusions:The intelligent ultrasound system has certain diagnostic value for adult goiter and has good consistency with conventional ultrasound examination for thyroid volume measurement. However, the accuracy of diagnosis for males and adults with thyroid nodules still needs to be improved.

Objective To investigate the clinical application value of MRI Radiomics score(Radscore)combined with clinicopatho-logical features in predicting postoperative progression-free survival(PFS)of patients with endometrial cancer(EC).Methods A total of 127 patients with EC were selected.The radiomic features of the lesions were extracted from T2 WI,diffusion weighted imaging(DWI)and apparent diffusion coefficient(ADC)images.The features were screened by random forest model and Radscore was calcu-lated.Simultaneously,clinical and pathological characteristics of patients were collected and incorporated,and multivariate Cox regression analysis was used to screen the risk factors related to PFS.The MRI Radscore and clinicopathological features were mapped to the nomogram,and the performance of nomogram was evaluated by receiver operating characteristic(ROC)curve and calibration curve.Results Multivariate Cox regression analysis showed that progesterone receptor(PR),human epididymis protein 4(HE4)and MRI Radscore were independent risk factors for predicting PFS in patients with EC(P＜0.05).The area under the curve(AUC)of the predicted PFS at 1,3 and 5 years after surgery were 0.91,0.804 and 0.776,respectively.Calibration curves showed that nomogram had a good fit in predicting PFS in patients with EC 1,3 and 5 years after surgery.Conclusion The nomogram con-structed based on multi-sequence MRI Radscore and clinicopathological features has favorable accuracy and stability in predicting postoperative PFS in individuals diagnosed with EC.

Breast cancer has become the most common malignant tumor in the world. Heat shock protein 90 (HSP90) is a kind of molecular chaperone which can promote protein folding and maintain protein stability. HSP90 includes HSP90α, HSP90β, GRP94 and TRAP1 subtypes. Previous studies have found that the level of HSP90 is significantly increased in malignant tumors such as breast cancer, and is closely related to the occurrence and development of tumors. Meanwhile, the research on inhibitors targeting HSP90 has also attracted much attention. In this paper, we reviewed the expression of four HSP90 subtypes in breast cancer and their relationship with the clinicopathologic feature and prognosis of patients, discussed the research progress of specific inhibitors of HSP90 subtypes in breast cancer, and analyzed the application prospect of HSP90 as biomarkers for breast cancer prognosis monitoring and therapeutic targets.

Leukemia is a type of common hematopoietic system tumor with fever, leukocytosis, anemia and hemorrhage as the main clinical symptoms. Leukemia can be divided into acute leukemia and chronic leukemia. TCM believes that leukaemia is closely related to latent pathogen and fire toxin. Based on the theory of "excessive qi is fire", this article believed that the pathogenesis of leukemia hemorrhage is the transformation of bone marrow pathological qi into fire, clarified the relationship between leukemia cells and "fire", and proposed that the basic principle of treatment should be strengthening the body and reduce fire, supplementing with methods such as clearing heat and detoxifying, tonifying qi and nourishing yin, so as to provide references for clinical treatment.

Objective:To analyze the expression of human leukocyte antigen G (HLA-G) in human T-cell leukemia virus type 1 (HTLV-1)-positive T cells, and to investigate its role in the occurrence and development of HTLV-1 infection.Methods:The expression of HLA-G in HTLV-1-positive T cell lines (MT2 and MT4) was detected by Western blot and real-time PCR. HLA-G gene in MT2 and MT4 cells was knocked down by siRNA, and the effects of HLA-G on the expression of HTLV-1 Tax and P19 at mRNA and protein levels were detected by Western blot and real-time PCR. Moreover, the changes in cytokine expression in MT2 and MT4 cells were monitored at RNA level after HLA-G gene silencing. The proliferation ability of MT2 and MT4 cells was analyzed by CCK8. Signal transducer and activator of transcription 3 (STAT3) pathway-related proteins were detected by Western blot.Results:Compared with HTLV-1-negative T cells (Jurkat and MOLT4), the expression of HLA-G increased significantly in MT2 and MT4 cells. After knocking down the HLA-G gene with siRNA in MT2 and MT4 cells, the expression of HTLV-1 Tax and P19 at mRNA and protein levels was decreased, and the expression of antiviral cytokines IFN-γ and TNF-α was increased. The proliferation of MT2 and MT4 cells and STAT3 phosphorylation in these cells were decreased.Conclusions:HTLV-1 could induce T cells to overexpress the immune tolerance molecule HLA-G. Silencing HLA-G gene in HTLV-1-positive T cells could promote the production of antiviral cytokines and reduce IL-6 expression and STAT3 phosphorylation, thereby effectively inhibiting the replication of HTLV-1.

Objective:To investigate the relationship between single nucleotide polymorphism (SNPs) of Zeste homolog enhancer 2 (EZH2) gene and the risk of breast cancer.Methods:Recruiting 1 039 breast cancer patients and 1 040 controls at 22 referral hospitals nationwide in China, the genotype distribution of 3 SNPs loci of EZH2 genes was observed to detect the correlation between different genotypes and the risk of breast cancer genotypes EZH2 expression in breast cancer tissues and its correlation with patient prognosis were analyzed using breast cancer data from the database.Results:EZH2 rs6464926 CC genotype was compared with TT genotype (TT vs. CC: OR=1.362, 95% CI: 1.063-1.746, P=0.015) and dominant model (TC+TT vs .CC: OR=1.22, 95% CI: 1.004-1.483, P=0.045) .In women with BMI ≥24 kg/m 2, the TC genotype ( P=0.050), TT genotype ( P=0.025) and dominant model (TC+TT, P=0.021) of rs6464926 locus were significantly different from CC genotype in cancer risk. rs6464926 was correlated with EZH2 gene expression ( P=6.89E-47). EZH2 gene is highly expressed in breast cancer tissues, and patients with high expression were associated with shorter OS ( HR=1.27, P=0.013), DMFS ( HR=1.37, P<0.01), and RFS ( HR=1.44, P<0.01). Conclusions:The polymorphism rs6464926 of EZH2 gene is associated with breast cancer susceptibility in Chinese women. rs6464926 might regulate breast cancer risk and prognosis by changing EZH2 expression.