ObjectiveA multiplex amplification system was constructed based on the capillary electrophoresis platform for simultaneous detection of saliva, semen, and vaginal secretions using tissue-specific RNA markers. The aim of this study is to identify the tissue origin of suspicious body fluid stains found at crime scenes and determine whether the body fluid stains at the crime scene are one or several types among saliva, semen, and vaginal secretions. MethodsThirty saliva samples, forty semen samples, and forty vaginal secretion samples (half from 2015 and half from 2024) were collected from healthy adult volunteers. Through primer designing, system formulation, and PCR condition optimization, a multiplex fluorescent amplification system was constructed. The specificity, sensitivity, and detection ability for mixed samples of this system were investigated, and it was tested using real crime scene materials. In the primer design stage, to reduce the requirements for RNA template quality, the amplification products were set within 80-300 bp. In the system formulation stage, dominant and subordinate primers were mainly considered. By reducing the concentration of dominant primers and increasing that of subordinate primers, a capillary electrophoresis spectrum with an appropriate peak height ratio was finally obtained. Additionally, gradient experiments were designed to adjust the concentrations of PCR reagents and PCR amplification conditions, and multiple versions of DNA amplification enzymes were optimized to achieve the best experimental results. ResultsThrough statistical analysis, there was no significant difference in the capillary electrophoresis of the 3 types of body fluid samples from the two years (2015 and 2024), demonstrating that the sample preservation method in this study can preserve samples for a relatively long time. The composite amplification system constructed in this study exhibited high specificity for all 3 types of body fluid, with no cross-reactions between the markers of each type of body fluid. The minimum detection thresholds for the 3 types of body fluid reached 0.002 9, 0.001 5, and 0.42 mg/L, respectively. This system also had a high degree of discrimination for mixed samples, especially for semen-saliva mixtures, where each body fluid marker could still be successfully detected when the concentration ratio of semen to saliva was 100:1. Meanwhile, in the two actual cases presented in this article, the application of this composite amplification system performed outstandingly. ConclusionThe composite amplification detection system constructed in this study can achieve the correct screening of saliva, semen, and vaginal secretions, overcoming the problems such as low specificity and sensitivity of marker tests and unbalanced RFU values of each marker in previous studies. The specificity and sensitivity meet the practical work requirements, and the operation is simple. It provides an analytical and identification method for body fluid stains in actual case and is applicable to the identification of the tissue origin of biological evidence at crime scenes involving sexual assault, indecent assault, and other criminal acts. In the future, more types of body fluid markers will be screened to expand the types of body fluids detected by the system, and body fluid-specific cSNP and cInDel genetic markers will be introduced to infer the sources (individuals and types) of mixed and complex stains more accurately.

Objective To explore the effectiveness and safety of percutaneous coronary artery shock wave balloon angioplasty(IVL)under the guidance of intravascular ultrasound(IVUS)for the treatment of severe calcification lesions in the left main artery(LM).Methods A total of 26 patients with severe LM(mouth,body,bifurcation)calcification admitted to Jiangnan University Affiliated Hospital from October 2022 to April 2024 were included,with an average age of 72.0(61.8,75.4)years.Under the guidance of IVUS,IVL was used for pre-treatment of calcified lesions,followed by percutaneous coronary intervention(PCI)with stent/drug balloon implantation.All patients were evaluated using IVUS before and after the use of IVL and after PCI.And compare the IVUS intracavity related data before and after treatment[plaque burden(PB)、minimum lumen area(MLA)、minimum lumen diameter(MLD)]and calcification fracture number,minimum stent area(MSA),stent expansion coefficient(expansion,EXP),etc.Results There were 26 patients(2 with opening lesions,7 with body lesions,and 17 with bifurcation lesions at the end of the main trunk),including 7 with stable angina pectoris(SAP),10 with unstable angina(UA),4 with acute ST-segment elevation myocardial infarction(STEMI),and 5 with non ST-segment elevation myocardial infarction(NSTEMI).The PB at the most severe site of calcification decreased by 79.50(76.00,83.75)％compared to 80.00(76.00,83.75)％after IVL(P=0.001),MLA increased by 3.39(3.14,3.68)mm2 compared to 3.38(3.14,3.67)mm2 after IVL(P=0.039),MLD increased by 3.21(3.07,3.30)mm compared to 3.20(3.07,3.30)mm after IVL(P=0.024),and there was 100％calcification rupture(1/2 cases,2/9 cases,≥3/15 cases).The stent/drug ball was successfully implanted 100％,with EXP of(89.15±4.42)％and an MSA of 7.20(6.46,7.45)mm2.No adverse events such as death,angina or recurrent myocardial infarction occurred during the 3 months follow-up after surgery.Conclusions After evaluation by IVUS and pre-treatment with IVL,PCI was successfully completed for severe calcification lesions in LM,and IVL can be used as an option for the treatment of severe calcification in LM.

Objective:To investigate the clinical effect of extraperitoneal robot-assisted laparoscopic modified Y-V plasty(LMYVP)in the treatment of refractory bladder neck contracture(BNC).Methods:We retrospectively analyzed the clinical data on 10 cases of refractory BNC after transurethral resection of the prostate between September 2020 and January 2023,all with a history of recurrent urethral dilatation and at least two failures in transurethral surgical treatment of scarring.After definite diagnosis and re-moval of some of the scar tissues to flatten the elevated bladder neck under the cystoscope,we performed robot-assisted LMYVP using the da Vinci Si robotic system and a four-port extraperitoneal approach.The surgical procedure involved an inverted T-shaped incision in the bladder neck and urethral stricture ring,an inverted V-shaped excision of the scar area at the 3-9 o'clock position on the ventral side of the prostatic urethra,continuous full-layer suturing of the bladder neck and inverted V-shaped urethra with 3-0 barbed thread,and indwelling of an F20 silicone catheter for 2 weeks.At 3 months after surgery,we performed cystoscopic examination,measured the maximum urinary flow rate(Qmax),and obtained the IPSS and quality of life(QOL)scores of the patients.Results:Operations were successfully completed in all the cases.At 3 months after surgery,the patients showed significantly increased Qmax([3.65±1.27]vs[20.3±1.77]ml/s,P＜0.05),IPSS(5.9±2.02 vs 30±1.15,P＜0.05)and QOL score(1.3±0.95 vs 5.2±0.79,P＜0.05)compared with the baseline.Cystoscopy revealed a wide and flat bladder neck with good survival and hemody-namics of the bladder flap.All the patients met the criteria for clinical cure at a median follow-up of 13.2 months.Conclusion:Extraper-itoneal robot-assisted LMYVP provides a new strategy for urinary tract reconstruction in the management of refractory BNC,with the ad-vantages of minimal invasiveness,high efficiency and high success rate.

Objective: To explore the efficacy of chemotherapy re-challenge in the third-line setting for patients with metastatic colorectal cancer (mCRC) in the real world. Methods: The clinicopathological data, treatment information, recent treatment efficacy, adverse events and survival data of mCRC patients who had disease progression after treatment with oxaliplatin-based and/or irinotecan-based chemotherapy and received third-line chemotherapy re-challenge from January 2013 to December 2020 at Tianjin Medical University Cancer Institute and Hospital were retrospectively collected. Survival curves were plotted with the Kaplan-Meier method, and the Cox proportional hazard model was used to analyze the prognostic factors. Results: A total of 95 mCRC patients were included. Among them, 32 patients (33.7%) received chemotherapy alone and 63 patients (66.3%) received chemotherapy combined with targeted drugs. Eighty-three patients were treated with dual-drug chemotherapy (87.4%), including oxaliplatin re-challenge in 35 patients and irinotecan re-challenge in 48 patients. The remaining 12 patients were treated with triplet chemotherapy regimens (12.6%). Among them, as 5 patients had sequential application of oxaliplatin and irinotecan in front-line treatments, their third-line therapy re-challenged both oxaliplatin and irinotecan; 7 patients only had oxaliplatin prescription before, and these patients re-challenged oxaliplatin in the third-line treatment. The overall response rate (ORR) and disease control rate (DCR) reached 8.6% (8/93) and 61.3% (57/93), respectively. The median progression free survival (mPFS) and median overall survival (mOS) were 4.9 months and 13.0 months, respectively. The most common adverse events were leukopenia (34.7%) and neutropenia (34.7%), followed by gastrointestinal adverse reactions such as nausea (32.6%) and vomiting (31.6%). Grade 3-4 adverse events were mostly hematological toxicity. Cox multivariate analysis showed that gender (HR=1.609, 95% CI: 1.016-2.548) and the PFS of front-line treatments (HR=0.598, 95% CI: 0.378-0.947) were independent prognostic factors. Conclusion: The results suggested that it is safe and effective for mCRC patients to choose third-line chemotherapy re-challenge, especially for patients with a PFS of more than one year in front-line treatments.
Humans ; Irinotecan/therapeutic use* ; Oxaliplatin/therapeutic use* ; Colorectal Neoplasms/pathology* ; Retrospective Studies ; Fluorouracil ; Colonic Neoplasms/chemically induced* ; Rectal Neoplasms/drug therapy* ; Antineoplastic Combined Chemotherapy Protocols/adverse effects* ; Camptothecin/adverse effects*

Objective: To explore the efficacy of chemotherapy re-challenge in the third-line setting for patients with metastatic colorectal cancer (mCRC) in the real world. Methods: The clinicopathological data, treatment information, recent treatment efficacy, adverse events and survival data of mCRC patients who had disease progression after treatment with oxaliplatin-based and/or irinotecan-based chemotherapy and received third-line chemotherapy re-challenge from January 2013 to December 2020 at Tianjin Medical University Cancer Institute and Hospital were retrospectively collected. Survival curves were plotted with the Kaplan-Meier method, and the Cox proportional hazard model was used to analyze the prognostic factors. Results: A total of 95 mCRC patients were included. Among them, 32 patients (33.7%) received chemotherapy alone and 63 patients (66.3%) received chemotherapy combined with targeted drugs. Eighty-three patients were treated with dual-drug chemotherapy (87.4%), including oxaliplatin re-challenge in 35 patients and irinotecan re-challenge in 48 patients. The remaining 12 patients were treated with triplet chemotherapy regimens (12.6%). Among them, as 5 patients had sequential application of oxaliplatin and irinotecan in front-line treatments, their third-line therapy re-challenged both oxaliplatin and irinotecan; 7 patients only had oxaliplatin prescription before, and these patients re-challenged oxaliplatin in the third-line treatment. The overall response rate (ORR) and disease control rate (DCR) reached 8.6% (8/93) and 61.3% (57/93), respectively. The median progression free survival (mPFS) and median overall survival (mOS) were 4.9 months and 13.0 months, respectively. The most common adverse events were leukopenia (34.7%) and neutropenia (34.7%), followed by gastrointestinal adverse reactions such as nausea (32.6%) and vomiting (31.6%). Grade 3-4 adverse events were mostly hematological toxicity. Cox multivariate analysis showed that gender (HR=1.609, 95% CI: 1.016-2.548) and the PFS of front-line treatments (HR=0.598, 95% CI: 0.378-0.947) were independent prognostic factors. Conclusion: The results suggested that it is safe and effective for mCRC patients to choose third-line chemotherapy re-challenge, especially for patients with a PFS of more than one year in front-line treatments.
Humans ; Irinotecan/therapeutic use* ; Oxaliplatin/therapeutic use* ; Colorectal Neoplasms/pathology* ; Retrospective Studies ; Fluorouracil ; Colonic Neoplasms/chemically induced* ; Rectal Neoplasms/drug therapy* ; Antineoplastic Combined Chemotherapy Protocols/adverse effects* ; Camptothecin/adverse effects*

Objective To investigate the effect of microRNA (miR) - 140-3p targeting cell division cycle associated 8(CDCA8) on invasion and metastasis of lung adenocarcinoma cells. Methods The differentially expressed miRNAs were analyzed by GE02R in GEO database. The target genes of miR-140-3p were searched by TargetScan human7. 2 and miRWalk databases. The hub gene was screened by Cytoscape 3. 7. 2 software. GEPIA database was used to query the expression levels of target gene in lung adenocarcinoma tissues and normal lung tissues, the expression levels in different stages of lung adenocarcinoma, and the relationship between the expression levels of target gene and the overall survival rate of lung adenocarcinoma patients. The survival analysis of miR-140-3p in lung adenocarcinoma and the correlation between miR-140-3p and CDCA8 expression levels were searched in starBase database. Real-time PCR was used to detect the expression levels of miR-140-3p in normal lung epithelial cells BEAS-2B and lung adenocarcinoma cells A549, as well as the efficiency of infection. Expression levels of CDCA8 mRNA and protein were detected by Real-time PCR and Western blotting experiments after overexpression of miR-140-3p. Dual-luciferase reporter assay verified whether miR-140- 3p directly binds to CDCA8. Transwell invasion assay detected the effect of overexpression of miR-140-3p and CDCA8 on the invasiveness of lung adenocarcinoma cells. Results Analysis result from GEO and other databases showed that the expression level of miR-140-3p in normal lung tissues was significantly higher than that in lung adenocarcinoma, and its predicted target gene CDCA8 expression level in lung adenocarcinoma was significantly higher than that in normal lung tissues, and CDCA8 was negatively correlated with the expression level of miR-140-3p in lung adenocarcinoma. The experimental result showed that the expression of miR-140-3p in A549 cells was significantly lower than that in BEAS-2B cells (P<0.05). The expression level of miR-140-3p increased significantly after lentiviral infection (P<0.05). CDCA8 mRNA and protein expression levels were significantly down-regulated after overexpression of miR-140-3p (P<0.05). Dual-luciferase reporter assay result showed that miR-140-3p could directly bind to CDCA8 (P<0.05). Compared with the control group, overexpression of miR- 140-3p inhibited the invasion and metastasis of lung adenocarcinoma A549, while CDCA8 reversed the inhibition of miR-140-3p on the invasion and metastasis of lung adenocarcinoma A549 (P<0.05). Conclusion MiR-140-3p targeting CDCA8 inhibits the invasion and metastasis of lung adenocarcinoma cells.

OBJECTIVE: To investigate the clinical features, accompanying gene mutation characteristics and prognostic factors of adult patients with acute myeloid leukemia with mutated NPM1 (NPM1AML). METHODS: Seventy-three patients with newly diagnosed adult NPM1AML were selected. The mutations of 22 genes were detected by second generation sequencing and 43 fusion genes of AML were detected by real-time fluorescent quantitative PCR. The Kaplan-Meier survival curve and Cox multivariate regression analysis were used to study the prognostic factors. RESULTS: A total of 74 NPM1 site mutations were detected in 73 patients with NPM1AML. The incidence rates were 92.0% L287fs, 2.7% Q289fs and W288fs, 1.4% L258fs and Q289H, among which 1 patient had 2 NPM1 mutations; the different mutation sites had no effect on the prognosis of NPM1AML. The median value of NPM1 variant allele frequency (VAF) was 35.4% (1.8%-56.6%). Based on the uppermost quartile of 38.4%, the patients were classified as NPM1 VAF>38.4% (NPM1AML) and NPM1 VAF≤38.4% (NPM1AML). Compared with NPM1AML, the early mortality rate was statistically significantly higher (33.3% vs 7.3%, P<0.05), and median EFS (148 d，95%CI 58-238 d vs 372 d，95%CI 264-480 d) (P<0.01) and median OS (179 d 95%CI 6-352 d vs 444 d) (P<0.01) were significantly shorter in NPM1 AML. A total of 126 accompanying gene mutation sites were detected in 87.7% of patients with NPM1AML. The patients with NRAS gene mutation displayed a higher rate of complete remission (100% vs 58%) (P<0.05) and longer median OS (not reached to 320 d, 95%CI 150-490 d) (P<0.05). The 43 fusion genes were examined in 65 out of 73 cases of NPM1AML, and in all the patients the fusion gene test was negative. Multivariate analysis showed that NPM1 VAF>38.4% was an independent prognostic factor for EFS (HR=3.1, 95% CI 1.6-6.4, P<0.01) and OS (HR=3.0, 95% CI 1.4-6.2, P<0.01). CONCLUSION The NPM1 gene mutation in AML patients often is accompanied by other gene mutations, while the coexistence of fusion genes is rare; high NPM1 mutant allele burden is an independent prognostic factor for adult AML patients with mutated NPM1.
Alleles ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation ; Nuclear Proteins ; genetics ; Prognosis ; fms-Like Tyrosine Kinase 3

Objective To investigate the molecular mechanisms of upregulated expression of cellular Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein(c-FLIP)by calreticulin(CRT)in patients with rheumatoid arthritis (RA). Methods The semi-quantitative analysis and localization of c-FLIP in RA and osteoarthritis (OA)synovium were detected by immunohistochemistry.The fibroblast-like synoviocytes(FLS)were isolated by enzymatic digestion of synovial tissue specimens obtained from RA and OA patients,and cultured as an in vitro experiment model.The expressions of c-FLIP in RA and OA synovial fibroblasts were detected by immunofluorescence and Western blot assay. Whether CRT influenced c-FLIP expression and its molecular mechanism were explored by Western blot assay. Results The high expression of c-FLIP was found in RA synovium, mainly in the lining and sublining areas of FLS and vascular endothelial cells detected by immunohistochemistry.Meanwhile,weak staining of c-FLIP was observed in OA synovium.The expression of c-FLIP was significantly higher in RA synovium than that of OA synovium(t=11.717,P<0.001).Results of immunofluorescence and Western blot assay showed that c-FLIP was mainly located in cytoplasm, and which was higher expressed in FLS of RA than that of OA. The increased c-FLIP expression and phosphorylation of NF-κB were detected after being co-incubated with exogenous CRT (0, 10, 50, 100 μg/L), in dose-dependent manner. The effect of CRT upregulating c-FLIP expression was blocked by NF-κB inhibitor BAY 11-7082.Conclusion CRT can increase c-FLIP expression at least partly through NF-κB pathway in RA,which may provide therapeutic target for the treatment of RA.

BACKGROUND: The application of mesenchymal stem cells (MSCs) in the treatment of cartilage damage has become a hot spot of research. Further studies on the distribution of MSCs in the body after injection and on the underlying mechanism of action are needed. OBJECTIVE: To observe the migration of bone marrow mesenchymal stem cells (BMSCs) after injection into the region of osteochondral defect. METHODS: Thirty Sprague-Dawley rats were randomized into two groups (n=15 per group). In the control group, the femoral tochlear was exposed but an osteochondral defect was not made; and after the suture, PKH26-labeled BMSCs were directly injected into the articular cavity of rats. In the experimental group, a cartilage defect of 1 mm in diameter and 1 mm in depth was made in the rat femoral trochlea, and 5×106PKH26-labeled BMSCs were injected into the defect after operation. At 1, 3 and 7 days after injection, the femoral condyle was taken to make frozen sections followed by DAPI staining. The distribution of BMSCs was observed under laser scanning confocal microscope. RESULTS AND CONCLUSION: In the control group, PKH26-labeled BMSCs were not transferred to the subchondral bone. In the experimental group, BMSCs were detected in the subchondral bone area at 1, 3 days after injection of PKH26-BMSCs in the bone cartilage defect area, and the BMSCs were also found in the bone marrow cavity at 7 days after injection. In conclusion, BMSCs in the articular cavity cannot migrate into the subchondral bone and bone marrow cavity unless the cartilage of the femoral condyle is damaged.

BACKGROUND: Preparing a scaffold with cartilage derived components and good initial mechanical strength is the direction of tissue engineering cartilage research. OBJECTIVE: To prepare porous acellular osteochondral scaffolds, and to explore their mechanical properties and cell compatibility. METHODS: Osteochondral bone from the porcine knee joint was taken, and then porous osteochondral scaffolds were made by laser microporation technology. Subsequently, the scaffolds were decellularized chemical methods. Scaffold structure was observed by scanning electron microscopy, and the compression modulus of the scaffolds was determined. Bone marrow mesenchymal stem cells were cultured in L-DMEM containing 10% fetal bovine serum (control group) and cultured in the medium extract of porous acellular osteochondral scaffolds (experimental group), respectively. Cell proliferation was detected by cell counting kit-8 method within 5 days of culture. Bone marrow mesenchymal stem cells were seeded on the porous acellular osteochondral scaffolds, and within 28 days of co-culture, cell growth was observed by hematoxylin-eosin staining and toluidine blue staining. RESULTS AND CONCLUSION: (1) Observation under scanning electron microscopy: The porous acellular osteochondral scaffolds had the smooth surface with evenly distributed pores. The pores of the scaffold extended longitudinally into the subchondral bone. (2) Mechanical properties: The average compressive modulus of porous acellular osteochondral scaffolds was 0.77 MPa, which was close to the compression modulus of the normal cartilage (1.15 MPa). (3) Cell counting kit-8 test: There were no differences in cell proliferation between the control and experimental groups at 1, 2, 3, 4 and 5 days of culture. (4) Cell-scaffold co-culture: A large amount of bone marrow mesenchymal stem cells were observed to be adherent to the scaffold after 1 day of culture through hematoxylin-eosin and toluidine blue staining. However, as time went on, a few cells adhered to the pore wall or grew into the pores at 7 and 21 days of culture. There were also some adherent cells but a large amount of cell masses formed in the pores at 28 days of culture. To conclude, the porous acellular osteochondral scaffold has good mechanical properties and cell compatibility.