Objective To construct a novel enzyme-free colorimetric biosensor(T-CHA)based on tri-ple-helix molecular probe(THMP)and catalytic hairpin assembly(CHA)reaction for visual detection and precise quantitative analysis of low-abundance alpha-fetoprotein(AFP).Methods The absorbance produced by AFP induced T-CHA system was recorded by gel electrophoresis and ultraviolet-visible spectrophotometer to evaluate the feasibility of T-CHA strategy.The molar concentration ratio of THMP and CHA,reaction time of CHA,temperature and pH value of buffer solution were optimized respectively.The color reaction of T-CHA was induced by different concentrations of AFP to explore the detection efficiency of T-CHA.Results Under the op-timal experimental conditions,the logarithmic values of AFP at different concentrations showed a linear rela-tionship from 5 μg/mL to 10 ng/mL,and the detection limit was 2.29 μg/mL.The T-CHA system could com-plete accurate quantitative analysis of low abundance AFP and visual free-labeled detection within 105 min,moreover the sensitivity of T-CHA was superior to that of ELISA.Conclusion Utilizing the low background leakage of THMP and the high catalytic efficiency of CHA,the T-CHA system could realize the early accurate diagnosis of hepatocellular carcinoma,moreover which has a certain application prospect in the real time detec-tion.

Objective:To investigate the clinicopathological characteristics, immunophenotypes, molecular features, and differential diagnosis of BAP1 mutated clear cell renal cell carcinoma (CCRCC) for better understanding this entity.Methods:Clinical data, histological morphology, immunophenotypes and molecular characteristics of 18 BAP1 mutated CCRCC cases diagnosed at the Department of Pathology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China from January 2020 to December 2022 were analyzed. The patients were followed up.Results:There were 17 males and 1 female patients, aged from 39 to 72 years, with an average age of 56.3 years. Sixteen patients with primary CCRCC were followed up for an average of 24 months, 7 patients had metastases occurred from 4 to 22 months postoperatively. Thirteen of the 16 patients were alive at the time of the last follow-up while 3 patients died 12, 15, and 20 months after the surgery, respectively. One patient underwent retroperitoneal mass resection, but had lung metastasis 32 months after surgery. One case received cervical tumor resection and died at 22 months after the surgery. Characteristic CCRCC regions were identified in 11 of the 18 cases. The tumor cells were arranged in papillary, alveolar, and large nest patterns. Abundant lymphoid tissue, necrosis, and psammoma bodies were seen. Tumor cells showed abundant eosinophilic cytoplasm, and sometimes exhibited rhabdoid differentiation. Round eosinophilic globules were located in the cytoplasm and extracellular matrix. There were 9 cases with WHO/International Society of Urological Pathology grade 3, and 9 cases with grade 4. PAX8 (18/18), carbonic anhydrase 9 (CA9, 16/18), CD10 (18/18), and vimentin (18/18) were positive in the vast majority of tumors.TFE3 was expressed in 5 cases, with strong expression in only 1 case. Eighteen cases were all positive for P504s. Twelve cases harbored a BAP1 mutation combined with von Hippel-Lindau (VHL) mutation, and 2 cases had mutations in BAP1, VHL and PBRM1 simultaneously. SETD2 mutation was not found in any of the cases.Conclusions:BAP1 mutated CCRCC contained papillary, alveolar, and large nest patterns, eosinophilic cytoplasm, high-grade nucleoli, and collagen globules, with P504s positivity. In practical work, when encountering CCRCC containing these features, pathologists should consider the possibility of BAP1 mutations and conduct related molecular tests.

AIM:To analyze the expression profiles of long non-coding RNAs(lncRNAs)in the aortas of ath-erosclerotic mice,and explore the role and mechanism of lncRNA AI662270 in regulating macrophage inflammation and lipid phagocytosis.METHODS:Twenty 8-week-old male apolipoprotein E gene knockout(ApoE-/-)mice were randomly divided into experimental and control groups,with 10 mice in each group.The experimental group was fed a high-fat diet for 12 weeks to induce atherosclerosis,while the control group received a normal diet.Body weight and blood lipid levels were measured,and atherosclerotic lesions were detected using Oil Red O staining.High-throughput RNA sequencing(RNA-seq)was used to analyze the lncRNA expression profiles in mouse aortas,and differentially expressed lncRNAs were validated by RT-qPCR.Antisense oligonucleotides(ASO)were used to knock down the expression of lncRNA AI662270 in mouse macrophage RAW264.7 cells.Six treatment groups were established:blank(no treatment)group,ox-idized low-density lipoprotein(oxLDL)group(80 mg/L oxLDL),negative control ASO(ASO-NC)group(transfected with ASO-NC),oxLDL+ASO-NC group(transfected with ASO-NC and treated with 80 mg/L oxLDL),ASO-AI662270 group(transfected with ASO-AI662270),and oxLDL+ASO-AI662270 group(transfected with ASO-AI662270 and treated with 80 mg/L oxLDL).Tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)levels were measured using Enzyme-linked immunosorbent assay(ELISA).NF-κB p65,pNF-κB p65,IκBα and pIκBα levels were detected by Western blot,and NF-κB p65 in the cell nucleus was examined using fluorescent probes.RESULTS:The atherosclerosis mouse model showed significant differences in body weight,serum lipids,and aortic plaque area compared to the control group(P<0.01).A total of 29 differentially expressed lncRNAs were identified involved in metabolic pathways,autophagosome for-mation,and cell adhesion molecule expression(P<0.05).LncRNA AI662270 was significantly upregulated in lesions and oxLDL-stimulated macrophages(P<0.05).Knockdown of AI662270 significantly reduced TNF-α and IL-6 expression in oxLDL-induced RAW264.7 cells(P<0.01),suppressed lipid phagocytosis,and inhibited pNF-κB p65 and pIκBα ex-pression(P<0.05).Additionally,the knockdown of AI662270 reduced the nuclear content of NF-κB p65.CONCLU-SION:The lncRNA expression profiles in the aortas of atherosclerotic mice were significantly altered.LncRNA AI662270 is crucial in modulating inflammation and lipid phagocytosis in mouse macrophages through the NF-κB signaling pathway.

Objective Constructing a risk prediction model of IgA nephropathy proteinuria treated by traditional Chinese medicine based on random survival forest model,Screening prognostic risk factors of IgA nephropathy proteinuria.Methods Collecting retrospectively clinical data of 129 cases diagnosed with IgA nephropathy,randomly divided them into training set(60%)and test set(40%).The risk prediction model of IgA nephropathy proteinuria was constructed in the training set with the random survival forest model,and the prognostic risk factors were screened by VIMP method.The accuracy of risk prediction model was validated in the test set with time-dependent ROC curve(tdROC).Results According to the result of VIMP,the prognostic risk factors for IgA nephropathy proteinuria are in the order of eGFR,hypertension,traditional Chinese medicine,24 hUPRO>1 g,genomo sclerosis ratio,Lee grading,fat,hyperlipidemia,hypertrophymia,hyparmane ledmia,Anemia,age and gender.The eGFR was negatively and non-linearly associated with the risk rate of developing persistent proteinuria.Glomerulosclerosis ratio greater than 0.3 is approximately linearly and positively associated with the risk rate of persistent proteinuria.Conclusion Random survival forest model has good predictive performance in the risk prediction model of IgA nephropathy proteinuria treated by traditional Chinese medicine.This risk model can determine the result of IgA nephropathy treated by traditional Chinese medicine,and which is helpful for clinical follow-up monitoring and formulation of individualized treatment plans.

We conducted a prospective study to assess the non-inferiority of adjuvant chemotherapy alone versus adjuvant concurrent chemoradiotherapy (CCRT) as an alternative strategy for patients with early-stage (FIGO 2009 stage IB-IIA) cervical cancer having risk factors after surgery. The condition was assessed in terms of prognosis, adverse effects, and quality of life. This randomized trial involved nine centers across China. Eligible patients were randomized to receive adjuvant chemotherapy or CCRT after surgery. The primary end-point was progression-free survival (PFS). From December 2012 to December 2014, 337 patients were subjected to randomization. Final analysis included 329 patients, including 165 in the adjuvant chemotherapy group and 164 in the adjuvant CCRT group. The median follow-up was 72.1 months. The three-year PFS rates were both 91.9%, and the five-year OS was 90.6% versus 90.0% in adjuvant chemotherapy and CCRT groups, respectively. No significant differences were observed in the PFS or OS between groups. The adjusted HR for PFS was 0.854 (95% confidence interval 0.415-1.757; P = 0.667) favoring adjuvant chemotherapy, excluding the predefined non-inferiority boundary of 1.9. The chemotherapy group showed a tendency toward good quality of life. In comparison with post-operative adjuvant CCRT, adjuvant chemotherapy treatment showed non-inferior efficacy in patients with early-stage cervical cancer having pathological risk factors. Adjuvant chemotherapy alone is a favorable alternative post-operative treatment.
Female ; Humans ; Uterine Cervical Neoplasms/drug therapy* ; Prospective Studies ; Quality of Life ; Neoplasm Staging ; Chemoradiotherapy ; Chemotherapy, Adjuvant/adverse effects* ; Adjuvants, Immunologic ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use* ; Retrospective Studies

OBJECTIVES: The International Federation of Gynecology and Obstetrics (FIGO) 2000 scoring system classifies gestational trophoblastic neoplasia (GTN) patients into low- and high-risk groups, so that single- or multi-agent chemotherapy can be administered accordingly. However, a number of FIGO-defined low-risk patients still exhibit resistance to single-agent regimens, and the risk factors currently adopted in the FIGO scoring system possess inequable values for predicting single-agent chemoresistance. The purpose of this study is therefore to evaluate the efficacy of risk factors in predicting single-agent chemoresistance and explore the feasibility of simplifying the FIGO 2000 scoring system for GTN. METHODS: The clinical data of 578 GTN patients who received chemotherapy between January 2000 and December 2018 were retrospectively reviewed. Univariate and multivariate logistic regression analyses were carried out to identify risk factors associated with single-agent chemoresistance in low-risk GTN patients. Then, simplified models were built and compared with the original FIGO 2000 scoring system. RESULTS: Among the eight FIGO risk factors, the univariate and multivariate analyses identified that pretreatment serum human chorionic gonadotropin (hCG) level and interval from antecedent pregnancy were consistently independent predictors for both first-line and subsequent single-agent chemoresistance. The simplified model with two independent factors showed a better performance in predicting single-agent chemoresistance than the model with the other four non-independent factors. However, the addition of other co-factors did improve the efficiency. Overall, simplified models can achieve favorable performance, but the original FIGO 2000 prognostic system still features the highest discrimination. CONCLUSIONS Pretreatment serum hCG level and interval from antecedent pregnancy were independent predictors for both first-line and subsequent single-agent chemoresistance, and they had greater weight than other non-independent factors in predicting single-agent chemoresistance. The simplified model composed of certain selected factors is a promising alternative to the original FIGO 2000 prognostic system, and it shows comparable performance.
Female ; Gestational Trophoblastic Disease/drug therapy* ; Humans ; Multivariate Analysis ; Pregnancy ; Retrospective Studies ; Risk Factors

We aimed to evaluate the effectiveness and safety of single-course initial regimens in patients with low-risk gestational trophoblastic neoplasia (GTN). In this trial (NCT01823315), 276 patients were analyzed. Patients were allocated to three initiated regimens: single-course methotrexate (MTX), single-course MTX + dactinomycin (ACTD), and multi-course MTX (control arm). The primary endpoint was the complete remission (CR) rate by initial drug(s). The primary CR rate was 64.4% with multi-course MTX in the control arm. For the single-course MTX arm, the CR rate was 35.8% by one course; it increased to 59.3% after subsequent multi-course MTX, with non-inferiority to the control (difference -5.1%,95% confidence interval (CI) -19.4% to 9.2%, P = 0.014). After further treatment with multi-course ACTD, the CR rate (93.3%) was similar to that of the control (95.2%, P = 0.577). For the single-course MTX + ACTD arm, the CR rate was 46.7% by one course, which increased to 89.1% after subsequent multi-course, with non-inferiority (difference 24.7%, 95% CI 12.8%-36.6%, P < 0.001) to the control. It was similar to the CR rate by MTX and further ACTD in the control arm (89.1% vs. 95.2%, P =0.135). Four patients experienced recurrence, with no death, during the 2-year follow-up. We demonstrated that chemotherapy initiation with single-course MTX may be an alternative regimen for patients with low-risk GTN.
Antineoplastic Combined Chemotherapy Protocols/adverse effects* ; Dactinomycin/adverse effects* ; Female ; Gestational Trophoblastic Disease/drug therapy* ; Humans ; Methotrexate/therapeutic use* ; Pregnancy ; Retrospective Studies

ObjectiveTo explore the mechanism of Yishen Huoxue prescription in renal interstitial fibrosis （RIF） from the perspective of endothelial cell and cell energy metabolism. MethodThe model was successfully established by unilateral ureteral obstruction （UUO）. Seventy-five SPF C57BL/6 mice were randomly divided into a model group， a resveratrol group （50 mg·kg^-1·d^-1）， three Yishen Huoxue prescription low， medium， and high-dose groups （7.1， 14.2， 28.4 g·kg^-1·d^-1）， with 15 mice in each group. In addition， another 15 mice were used to prepare sham operation model. Mice in the sham operation group and the model group were gavaged with equal volume of normal saline. All mice were sacrificed on 7， 14， and 21 d after modeling. The protein expression of platelet endothelial cell adhesion molecule 31 （CD31） was detected by immunohistochemical S-P method. The expression of α-smooth muscle actin （α-SMA）， collagen Ⅳ （Col-Ⅳ）， angiopoietin 1（Ang-1） and tyrosine kinase receptors 2 （Tie-2）， vascular endothelial growth factor （VEGF）， vascular endothelial cadherin （VE-cadherin）， and occludin in renal tissues was detected by Western blotting. The mRNA expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin in renal tissues were detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the levels of reactive oxygen species （ROS） in mice were detected by enzyme linked immunosorbent assay （ELISA）. ResultAs compared with the sham operation group， the expression of CD31 in renal tissues of the model group was significantly decreased and worsened with the extension of modeling time （P<0.05）， α-SAM and Col-Ⅳ protein expression levels were significantly increased （P<0.01）， but the expression of CD31 was stable in 14-21 d. ROS levels were significantly increased （P<0.01）， and the protein and mRNA expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin were significantly down-regulated （P<0.01）. As compared with the model group， the expression of CD31 was increased （P<0.05）， and α-SAM and Col-Ⅳ in the resveratrol group and the medium and high-dose Yishen Huoxue prescription groups were significantly decreased （P<0.01）. The ROS content was significantly decreased （P<0.01）， and the protein and mRNA expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin were up-regulated （P<0.01）， As compared with the resveratrol group， the protein expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin in the medium and low-dose Yishen Huoxue prescription groups were significantly different （P<0.01）. There was no significant difference in the mRNA expressions of CD31 and Ang-1/Tie-2 in the high-dose Yishen Huoxue prescription group， and no significant difference in the ROS level in the medium-dose Yishen Huoxue prescription group. ConclusionThe anti-RIF effect of Yishen Huoxue prescription may be related to promoting vascular endothelial repair， regulating mitochondrial ROS to reduce oxidative stress， protecting the integrity of renal endothelial structure， delaying cell apoptosis， and maintaining cell energy metabolism.

Objective:To assess the efficacy and safety of 100 units of botulinum toxin A (BTX-A) intradetrusor injection in patients with overactive bladder.Methods:From April 2016 to December 2018, 17 tertiary hospitals were selected to participate in this prospective, multicenter, randomized, double-blind, placebo-controlled study. Two phases of study were conducted: the primary phase and the extended phase. This study enrolled patients aged 18 to 75 years who had been inadequately managed by anticholinergic therapy (insufficient efficacy or intolerable side effects) and had spontaneous voiding with overactive bladder. Exclusion criteria included patients with severe cardiac, renal and hepatic disorders, patients with previous botulinum toxin treatment for 6 months or allergic to BTX-A, patients with urinary tract infections, patients with urinary stones, urinary tract tumors, diabetes mellitus, and bleeding tendency. Eligible patients were randomly assigned to BTX-A group and placebo control group in a ratio of 2∶1. Two groups of patients received 20 intradetrusor injections of BTX-A 100U or placebo at the depth of the submucosal muscle layer respectively under cystoscope, including 5 injections at the base of the bladder, 3 injections to the bladder triangle, 5 injections each to the left and right walls and 2 injections to the top, sparing the bladder neck. As a placebo control group, patients received same volume of placebo containing no BTX-A and only adjuvant freeze-dried preparations for injection with the same method. A combination of gelatin, sucrose, and dextran served as adjuvants. Average micturition times per 24 hours, urinary incontinence (UI) episodes per day, average micturition volume per day, OAB symptom score(OABSS), and quality of life (QOL) score were recorded at baseline and the 2nd, 6th and 12th week after treatment. The primary efficacy endpoint was the change from baseline in the average micturition times per 24 hours at the 6th week after treatment. The secondary efficacy endpoints included the change from baseline in the average micturition times per 24 hours at 2nd and 12th week, as well as the change from baseline in the OABSS, QOL score, average frequency of urgency and UI episodes per day, urgency score, average micturition volume per day at 2nd, 6th and 12th week after treatment. Patients were followed for 12 weeks to assess adverse events (AEs). After assessed at week 12, if the micturition times has decreased less than 50% compared to baseline and the patient is willing to receive retreatment, then patients could enter the extended trial phase. In that phase, patients in both groups were injected with 100 units BTX-A from 12th week onwards and then followed up the same indicators for 12 weeks.Results:216 patients were enrolled in this trial (144 cases in the BTX-A group and 72 cases in the placebo control group). Baseline characteristics such as age (47.75±14.20 in the BTX-A group and 46.39±15.55 in the control group), sex (25 male/117 female in the BTX-A group and 10/61 in the control group), and disease duration (0.51 years in the BTX-A group and 0.60 years in the control group) were balanced between the two groups( P>0.05). A marked reduction from baseline in average micturition times per 24 hours was observed in all treatment groups at the 6th week and the reduction of the two groups was statistically different ( P<0.001 and P=0.008 respectively). Compared with the baseline, the average micturition times per 24 hours at the 6th week decreased from baseline by 2.40(0.70, 4.60)times for the BTX-A group and 0.70(-1.00, 3.30) times for the placebo control group respectively, and the difference between the two groups was considered to be statistically significant ( P=0.003). The change rates of average micturition times per 24 hours from baseline at the 6th week of the two groups were (16±22)% and (8±25)% respectively, and the difference between the two groups was statistically significant ( P=0.014). Compared with the baseline, the average micturition times per 24 hours at 2nd and 12th week decreased by 2.00(0.00, 4.00)and 3.30(0.60, 5.03)for the BTX-A group, 1.00(-1.00, 3.00)and 1.70(-1.45, 3.85)for the placebo control group respectively. The difference between two groups was considered to be statistically significant ( P=0.038 and P=0.012); the changes of average urgency times per day for the BTX-A group and the control group at the 2nd, 6th and 12th week were 2.00(0.00, 4.30)and 2.40(0.30, 5.00), 3.00(0.30, 5.70)and 0.70(-1.30, 2.70), 0.70(-1.30, 3.00) and 1.35(-1.15, 3.50), respectively. There were significant differences between two groups at the 2nd, 6th and 12th week, ( P=0.010, P=0.003 and P=0.025, respectively). The OABSS of the BTX-A group and the control group at the 6th week decreased by 1.00(0.00, 4.00)and 0.50(-1.00, 2.00) compared with the baseline, and the difference between the two groups was statistically significant ( P=0.003). 47 cases of BTX-A group and 34 cases of placebo control group entered the extended trial phase, and 40 and 28 cases completed the extended trial phase, respectively. The average micturition volume per 24 hours changed by -16.60(-41.60, -0.60)ml and -6.40(-22.40, 13.30)ml, (-35.67±54.41)ml and(-1.76±48.69)ml, (-36.14±41.51)ml and (-9.28±44.59)ml, (-35.85±43.35)ml and(-10.41±40.29)ml for two groups at the 12th, 14th, 18th and 24th week, and the difference between two groups was statistically significant at each follow-up time ( P=0.01, 0.006, 0.012 and 0.016, respectively). There was no significant difference in other parameters( P>0.05). However, adverse reactions after intradetrusor injection included increased residual urine volume (27 in the BTX-A group and 3 in the control group), dysuria (21 in the BTX-A group and 6 in the control group), urinary infection (19 in the BTX-A group and 6 in the control group), bladder neck obstruction (3 in the BTX-A group and 0 in the control group), hematuria (3 in the BTX-A group and 1 in the control group), elevated alanine aminotransferase (3 in the BTX-A group and 0 in the control group), etc. During the follow-up period, there was no significant difference in the other adverse events between two groups except the increase of residual urine volume( P<0.05). In the primary trial phase, among the 27 cases with increased residual urine volume in BTA group, only 1 case (3.70%) with PVR more than 300 ml; the PVR of 3 patients in the placebo group was less than 100 ml. The increase of residual urine volume caused by the injection could be improved or disappeared with the passage of time. Conclusions:Intradetrusor injection of Chinese BTX-A improved the average micturition times per 24 hours, the average daily urgent micturition times, OABSS, and average micturition volume per time, and reduced the adverse effects in patients with overactive bladder.Chinese BTX-A at dose of 100U demonstrated durable efficacy and safety in the management of overactive bladder.

The clinical data of 1 patient with long-term survival metastatic prostate cancer were analyzed retrospectively, and the related literature was reviewed and discussed. The patient, male, 70 years old, was admitted to the hospital in 2009 due to dysuria with lower abdominal pain for one month.Blood PSA>1 000 ng/ml. The pathology of prostate biopsy was prostatic adenocarcinoma, Gleason score was 8 points (4+ 4), and was diagnosed as prostate cancer (T 4N 0M 1b) with bone metastasis. The patient underwent combined androgen-blocked treatment(castration and bicalutamide 50mg) for four years, then progressed to mCRPC. The initial treatment was continued in the fifth year due to the absence of novel therapeutic agents, and then symptoms progressed. The regimens were adjusted successively to increased anti-androgen (castration and bicalutamide 150 mg) from Jan 2015, then switch to another anti-androgen (Flutamide 250 mg) from Aug 2015, and then withdraw the anti-androgens from Feb 2016. All these treatments showed limited benefit for a relatively short time. The t-PSA increased steadily to over 1 000 ng/ml with persistent symptoms. In April 2017, he started the treatment with the original abiraterone acetate and underwent a PSA flare-up in the following month.tPSA decreased sharply since May 2017, less than 0.02ng/ml in Aug 2017. Meanwhile, the regimen relieved the ostealgia. He could take care of himself in daily life. raditional CAB therapy can maintain PSA-free progression and symptom-free progression for several years for some metastatic prostate cancer patients. After disease progression, the increased dosage of anti-androgens, the substitution of anti-androgen, and the withdrawal of anti-androgens showed limited benefit within a short time. However, the novel hormone therapy is still effective in relieving clinical symptoms and prolonging patients' survival time.