This study was aimed at creating an engineered strain of Bacillus subtilis for efficient expression of biologically active type 2 toxin B(TcdB2)derived from a highly virulent strain of Clostridium difficile.The TcdB2 gene was cloned from ST1/RT027 strain genome DNA,incorporated into the PHT01 vector,and then transformed into B.subtilis strain WB800N for prokaryotic expression.Cell toxicity assays revealed that the recombinant TcdB2 exhibited cytotoxic effects in various cells.The engineered B.subtilis strain effectively expressed biologically active TcdB2,thus providing a basis for further exploration of the pathogenic mechanisms of highly virulent strains of C.difficile and establishing a foundation for potential vaccine can-didate targets.

Objective:To investigate the relationship between mutated genes and clinical features in patients with essential thrombocythemia(ET).Methods:The clinical data of 69 patients with ET from October 2018 to March 2022 were retrospectively analyzed.According to driver mutation type,patients were divided into JAK2 group,CALR group and triple-negative group.The sex,age,cardiovascular risk factors,thrombosis,splenomegaly,routine blood test and coagulation status of patients in three groups were analyzed.Results:Among 69 ET patients,46 cases were associated with JAK2 mutation,14 cases with CALR mutation,8 cases with triple-negative mutation,and one with MPL gene mutation.There were no significant differences in age and sex among the three groups(P＞0.05).The highest thrombotic rate was 26.09％(12/46)in JAK2 group,then 12.5％(1/8)in triple-negative group,while no thrombotic events occurred in CALR group.The incidence of splenomegaly was the highest in JAK2 group(34.78％),while no splenomegaly occurred in triple-negative group.The white blood cell(WBC)count in JAK2 group was(9.00±4.86)× 109/L,which was significantly higher than(6.03±2.32)× 109/L in CALR group(P＜0.05).The hemoglobin(Hb)and hematocrit(HCT)in JAK2 group were(148.42±18.79)g/L and(0.44±0.06)％,respectively,which were both significantly higher than(131.00±15.17)g/L and(0.39±0.05)％in triple-negative group(P＜0.05).The platelet(PLT)in JAK2 group was(584.17±175.77)× 109/L,which was significantly lower than(703.07±225.60)× 109/L in CALR group(P＜0.05).The fibrinogen(Fg)in JAK2 and triple-negative group were(2.64±0.69)g/L and(3.05±0.77)g/L,respectively,which were both significantly higher than(2.24±0.47)g/L in CALR group(P＜0.05,P＜0.01).The activated partial thromboplastin time(APTT)in triple-negative group was(28.61±1.99)s,which was significantly decreased compared with(31.45±3.35)s in CALR group(P＜0.05).Conclusions:There are differences in blood cell count and coagulation status among ET patients with different driver gene mutations.Among ET patients,JAK2 mutation is most common.Compared with CALR group,the thrombotic rate,WBC and Fg significantly increase in JAK2 group,while PLT decrease.Compared with triple-negative group,the incidence of splenomegaly and HCT significantly increase.Compared with CALR group,Fg significantly increases but APTT decreases in triple-negative group.

Threatened abortion is a common disease of obstetrics and gynecology and one of the diseases responding specifically to traditional Chinese medicine （TCM）. The China Association of Chinese Medicine organized experts in TCM obstetrics and gynecology， Western medicine obstetrics and gynecology， and pharmacology to deeply discuss the advantages of TCM and integrated Chinese and Western medicine treatment as well as the medication plans for threatened abortion. After discussion， the experts concluded that chromosome， endocrine， and immune abnormalities were the key factors for the occurrence of threatened abortion， and the Qi and blood disorders in thoroughfare and conception vessels were the core pathogenesis. In the treatment of threatened abortion， TCM has advantages in preventing miscarriages， alleviating clinical symptoms and TCM syndromes， relieving anxiety， regulating reproductive endocrine and immune abnormalities， personalized and diversified treatment， enhancing efficiency and reducing toxicity， and preventing the disease before occurrence. The difficulty in diagnosis and treatment of threatened abortion with traditional Chinese and Western medicine lies in identifying the predictors of abortion caused by maternal factors and the treatment of thrombophilia. Recurrent abortion is the breakthrough point of treatment with integrated traditional Chinese and Western medicine. It is urgent to carry out high-quality evidence-based medicine research in the future to improve the modern diagnosis and treatment of threatened abortion with TCM.

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannan-binding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP. Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays. Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05). Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Objective:To discuss the biological function of the spacer domain of ADAM metalloproteinase with thrombospondin type 1 motifs 13(ADAMTS13)in the cleaving process of von Willebrand factor(vWF),and to clarify the role of ADAMTS13 in the pathogenesis of thrombotic thrombocytopenic purpura(TTP).Methods:The point mutation method was introduced sequentially into the amino acid residues TEDRLPR of the ADAMTS13 spacer domain(mutants M1-M7)by site-directed mutagenesis.The constructed ADAMTS13 and its mutants plasmids were transfected into the human embryonic kidney HEK293 cells,and the recombinant proteins were purified after stable expression.The cleavage capabilities of both wild type and mutant ADAMTS13 were observed under denaturation conditions,shear stress,and after treatment with ADAMTS13 antibodies.Results:The fluorescence resonance energy transfer(FRET)assay results showed that compared with wild type ADAMTS13,the cleavage abilities of ADAMTS13 mutant M4(R635A)and mutant M7(R638A)on the FRET-vWF73 were decreased(P＜0.05).Under denaturation conditions,the wild-type ADAMTS13 could cleave the vWF multimers;compared with wild-type ADAMTS13,the cleavage activities of ADAMTS13 mutant M4(R635A)and mutant M7(R638A)were significantly decreased(P＜0.01).Under in vitro shear stress,compared with wild type ADAMTS13,the abilities of ADAMTS13 mutant M4(R635A)and mutant M7(R638A)to cleave vWF multimers were significantly decreased(P＜0.01).Compared with wild type ADAMTS13,the binding affinity between vWF and ADAMTS13 mutant M4(R635A)and mutant M7(R638A)had no significant difference(P＞0.05),indicating there were multiple binding sites between C-terminal of ADAMTS13 and vWF.The ADAMTS13 antibodies were able to inhibit the cleavage ability of both wild-type and mutant ADAMTS13 to some extent.Conclusion:The activity of ADAMTS13 after spacer domain mutation is decreased.The ADAMTS13 mutant M4(R635A)and mutant M7(R638A)may be the important action sites for AD AMTS 13 in substrate recognition.

Objective: To explore the characteristics and correlations of vaginal flora in women with cervical lesions. Methods: A total of 132 women, including 41 women diagnosed with normal cervical (NC), 39 patients with low-grade cervical intraepithelial neoplasia (CIN 1), 37 patients with high-grade cervical intraepithelial neoplasia (CIN 2/3) and 15 patients with cervical squamous cell carcinoma (SCC), who came from the gynecological clinic of Second Hospital of Shanxi Medical University during January 2018 to June 2018, were enrolled in this study according to the inclusive and exclusive criteria strictly. The vaginal flora was detected by 16S rDNA sequencing technology. Co-occurrence network analysis was used to investigate the Spearman correlations between different genera of bacteria. Results: The dominant bacteria in NC, CIN 1 and CIN 2/3 groups were Lactobacillus [constituent ratios 79.4% (1 869 598/2 354 098), 63.6% (1 536 466/2 415 100) and 58.3% (1 342 896/2 301 536), respectively], while Peptophilus [20.4% (246 072/1 205 154) ] was the dominant bacteria in SCC group. With the aggravation of cervical lesions, the diversity of vaginal flora gradually increased (Shannon index: F=6.39, P=0.001; Simpson index: F=3.95, P=0.012). During the cervical lesion progress, the ratio of Lactobacillus gradually decreased, the ratio of other anaerobes such as Peptophilus, Sneathia, Prevotella and etc. gradually increased, and the differential bacteria (LDA score >3.5) gradually evolved from Lactobacillus to other anaerobes. The top 10 relative abundance bacteria, spearman correlation coefficient>0.4 and P<0.05 were selected. Co-occurrence network analysis showed that Prevotella, Peptophilus, Porphyrinomonas, Anaerococcus, Sneathia, Atopobium, Gardnerella and Streptococcus were positively correlated in different stages of cervical lesions, while Lactobacillus was negatively correlated with the above anaerobes. It was found that the relationship between vaginal floras in CIN 1 group was the most complex and only Peptophilus was significantly negatively correlated with Lactobacillus in SCC group. Conclusions: The increased diversity and changed correlations between vaginal floras are closely related to cervical lesions. Peptophilus is of great significance in the diagnosis, prediction and early warning of cervical carcinogenesis.
Female ; Humans ; Vagina/microbiology* ; Uterine Cervical Neoplasms/genetics* ; Uterine Cervical Dysplasia ; Cervix Uteri ; Lactobacillus/genetics* ; Papillomavirus Infections

OBJECTIVE: To investigate the effects of miR-144-3p on cell proliferation, cell cycle and apoptosis of blast phase chronic myelogenous leukemia (CML) K562 cells. METHODS: K562 cells were cultured in vitro and mimics negative control, hsa-miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor were respectively transfected into K562 cells with transfection reagents. The cells were divided into five groups including blank control, mimics negative control, miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor. After transfection, the cell proliferation activity was detected by CCK-8 assay. The cell cycle distribution and apoptosis were detected by flow cytometry. RESULTS: Compared with the blank control and mimics negative control groups, the proliferation rate of miR-144-3p mimics group was significantly decreased (P<0.05), the proportion of S phase cells was markedly increased (P<0.05), while the proportion of G₁ phase cells was obviously decreased (P<0.05), and the apoptosis rate was significantly increased (P<0.05). Compared with the blank control and inhibitor negative control groups, the proliferation rate of miR-144-3p inhibitor group was obviously increased (P<0.05), the proportion of S phase cells was markedly decreased (P<0.05), while the proportion of G₁ phase cells was obviously increased (P<0.05), and the apoptosis rate was significantly decreased (P<0.05). CONCLUSION miR-144-3p can inhibit the proliferation and promote apoptosis of K562 cells, affect the cell cycle, and block K562 cells in S phase, which indicates that miR-144-3p is involved in the cell cycle activity of CML during blastic phase.
Humans ; Apoptosis/genetics* ; Cell Cycle/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; K562 Cells ; MicroRNAs/metabolism*

Neoadjuvant therapy has been widely applied in the treatment of rectal cancer, which can shrink tumor size, lower tumor staging and improve the prognosis. It has been the standard preoperative treatment for patients with locally advanced rectal cancer. The efficacy of neoadjuvant therapy for rectal cancer patients varies between individuals, and the results of tumor regression are obviously different. Some patients with good tumor regression even achieve pathological complete response (pCR). Tumor regression is of great significance for the selection of surgical regimes and the determination of distal resection margin. However, few studies focus on tumor regression patterns. Controversies on the safe distance of distal resection margin after neoadjuvant treatment still exist. Therefore, based on the current research progress, this review summarized the main tumor regression patterns after neoadjuvant therapy for rectal cancer, and classified them into three types: tumor shrinkage, tumor fragmentation, and mucin pool formation. And macroscopic regression and microscopic regression of tumors were compared to describe the phenomenon of non-synchronous regression. Then, the safety of non-surgical treatment for patients with clinical complete response (cCR) was analyzed to elaborate the necessity of surgical treatment. Finally, the review studied the safe surgical resection range to explore the safe distance of distal resection margin.
Humans ; Neoadjuvant Therapy/methods* ; Margins of Excision ; Treatment Outcome ; Rectal Neoplasms/pathology* ; Rectum/pathology* ; Neoplasm Staging ; Retrospective Studies

Objective:To verify the effect and safety of low-intensity extracorporeal shockwave therapy(Li-ESWT)in improving the symptoms of ED,and provide some reference for further related large-scale clinical trials.Methods:Twenty-six patients diag-nosed with ED received Li-ESWT with an energy of 0.09 mJ/mm2 for 20 minutes once a week for 6 four-week courses.Before and at 3,6,9,and 12 months after treatment,we obtained the IIEF-5 and Erectile Hardness Scale(EHS)scores of the patients using question-naires,recorded the incidence of treatment-related adverse reactions,compared the erectile function of the patients before and after treatment,and evaluated the effect and safety of Li-ESWT in improving ED-related symptoms.Results:Compared with the baseline,the IIEF-5 scores of the patients were significantly increased(P＜0.01)while the EHS scores slightly increased at 3 months after Li-ESWT treatment(P＞0.05),both IIEF-5 and EHS scores were dramatically increased at 6 months(P＜0.01),and both signifi-cantly higher than at 3 months.At 9 months,EHS scores remained remarkably higher than the baseline(P＜0.01)although IIEF-5 scores slightly lower than at 6 months.At 12 months,however,IIEF-5 scores decreased,though still significantly higher than the base-line(P＜0.01),and EHS scores became lower than at 6 and 9 months(P＜0.05)but still markedly higher than before treatment(P＜0.05).Adverse reactions observed during the intervention mainly included pruritus(4.35％),pain(2.90％),paresthesia(2.17％),and petechiae/ecchymosis(2.90％).Conclusion:Li-ESWT can increase the IIEF-5 and EHS scores and improve the clinical symptoms of ED patients,with a low incidence of adverse reactions during the treatment.

Objective:To evaluate the consistency of the Gleason scores of PCa patients based on preoperative biopsy with those from postoperative pathology,identify the possible factors influencing results of scoring,and construct a risk scoring model.Meth-ods:We collected the demographic and clinical data on the patients with PCa confirmed by preoperative prostate biopsy or postoperative pathology and treated by radical prostatectomy within 6 months after diagnosis.Using paired sample t-test,we identified the difference between the Gleason scores based on preoperative biopsy and those from postoperative pathology,analyzed the demographic and clinical data on the patients for relevant factors affecting the consistency of the Gleason scores,and calculated and visualized the relative risk values of the factors through Poisson regression.From the continuous variables with statistical significance,we screened independent risk factors for the difference in the Gleason scores by Lasso regression analysis,established a risk scoring model,generated risk coeffi-cients,and evaluated the predictive ability of the model using the ROC curve.Based on the results of imaging examination with statisti-cally significant differences,we constructed a column chart by logistic regression and evaluated the predictive validity of the chart using calibration curves,decision curves and ROC curves.Results:The results of paired sample t-test for 210 PCa patients showed statisti-cally significant differences between the Gleason scores from preoperative biopsy and those from postoperative pathology(P＜0.001).There were significant differences in the body weight,BMI and PSA level as well as in all other factors but prostate calcification be-tween the patients with consistent and those with inconsistent Gleason scores(all P＜0.05).An 8-factor prediction model was suc-cessfully constructed,which could predict the consistency of Gleason scores,with a better predicting performance than the single indi-cator within the model.The nomogram exhibited a C-index value of 0.85,with the calibration curve similar to the standard one,the threshold of the decision curve 0.10-0.92,and the area under the ROC curve higher than other predictive indicators.Conclusion:Based on the demographic and clinical data on PCa patients,a risk prediction model and a column chart were successfully constructed,which could effectively predict the difference between the Gleason scores from preoperative prostate biopsy and those from postoperative pathology.