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.

The inference of tissue origin of body fluid stains is crucial for case investigation and court proceedings.However,traditional methods for identification of body fluid stains,such as morpho-logical,chemical,and immunoassay identifications have certain limitations,and there is an urgent need for more efficient methods for confirmatory experiments.In recent years,the rapid development of tran-scriptomics technology has provided new means for the identification of tissue origin of body fluid stains.Different types of RNA in the transcriptome have their own advantages.This paper elaborates in detail on the application of different types of RNA,such as mRNA,miRNA,circRNA,lncRNA,piRNA and microbial transcriptomics in body fluid identification,and summarizes their respective ad-vantages and limitations,in order to provide a reference for related research.

Objective To investigate the effect of miR-185-5p-mediated targeted negative regulation of transmembrane 9 superfamily member 1(TM9SF1)on proliferation,migration and autophagy in lung adenocarcinoma cells.Methods The expression of miR-185-5p in lung adenocarcinoma tissues was analyzed using dataset GSE51853 downloaded from the Gene Expression Omnibus(GEO)database.Potential target proteins of miR-185-5p were predicted using online databases(miRTargetLink,miRTarbase,and DIANA-microT-CD),and autophagy-related proteins were obtained from HADb.The intersected results from these four databases was identified,and survival curves of vascular endothelial growth factor A(VEGFA)and TM9SF1 within the overlapping candidates were analyzed using the StarBase database.TM9SF1 3'UTR wild-type(WT)or TM9SF1 3'UTR mutant(MUT)reporter plasmids were separately co-transfected with miR-185-5p control plasmid(CON)or miR-185-5p overexpression plasmid(over-miR-185-5p)into HEK-293T cells.A dual-luciferase reporter gene assay was employed to assess the binding interaction between miR-185-5p and TM9SF1 and quantify the subsequent luciferase activity.Western blotting was used to assess TM9SF1 protein expression levels in A549 cells transfected with over-miR-185-5p.A549 cells were divided into three groups:(1)CON+NC group,co-transfected with miR-185-5p control plasmid and TM9SF1 control plasmid;(2)over-miR-185-5p+NC group,co-transfected with over-miR-185-5p and TM9SF1 control plasmid;(3)over-miR-185-5p+over-TM9SF1 group,co-transfected with both miR-185-5p and TM9SF1 overexpression plasmids.EdU cell proliferation assay,wound healing assay,and Transwell migration assay were performed to validate the effects of miR-185-5p targeted binding to TM9SF1 on proliferation and migration capacities in lung adenocarcinoma.Changes in autophagic flux and mitochondrial membrane potential(MMP)of lung adenocarcinoma cells were detected using stubRFP-sensGFP-LC3 lentivirus and JC-1 assays,respectively.Results In the GSE51853 dataset,miR-185-5p expression level was significantly lower in lung adenocarcinoma tissues compared with normal lung tissues(P<0.01).qRT-PCR analysis revealed that miR-185-5p expression was downregulated in lung adenocarcinoma cell lines NCI-H1299 and A549 compared with normal lung epithelial cells BEAS-2B(P<0.01).Bioinformatics predictions using miRTargetLink,miRTarbase,DIANA-microT-CD,and HADb databases indicated that miR-185-5p could target and regulate the autophagy-related protein TM9SF1.Dual-luciferase reporter assays and Western blotting demonstrated that miR-185-5p directly bound to the 3'UTR region of TM9SF1 mRNA,and overexpression of miR-185-5p significantly reduced the expression of target protein TM9SF1(P<0.05).EdU cell proliferation,wound healing,and Transwell migration assays demonstrated that miR-185-5p overexpression inhibited proliferation and migration capacities of lung adenocarcinoma cells,whereas TM9SF1 overexpression could attenuate this inhibition effect(P<0.05).Results of stubRFP-sensGFP-LC3 for autophagic flux analysis demonstrated that overexpression of miR-185-5p enhanced autophagic flux in A549 cells,whereas co-overexpression of miR-185-5p and TM9SF1 suppressed autophagic flux.JC-1 assays showed a decreased MMP level in A549 cells after miR-185-5p overexpression,with higher MMP level observed when miR-185-5p and TM9SF1 were co-overexpressed.Conclusion miR-185-5p may suppress proliferation,migration,and autophagy capacities in lung adenocarcinoma cells by targeting TM9SF1 through negative regulation.

Objective To compare the diagnostic value of ultrasonography and CT in acute appendicitis.Methods A retrospective analysis was conducted on 279 patients who were diagnosed with acute appendicitis and followed emergency surgery.Patients were divided into different subgroups based on postoperative pathological results and body mass index(BMI),and the pathological results were used as the gold standard to analyze whether there were differences in the diagnostic accuracy of ultrasonography and CT examination for acute appendicitis.Results A total of 279 patients with confirmed acute appendicitis,with 64 cases of simple appendicitis,127 cases of suppurative appendicitis,and 88 cases of gangrenous appendicitis according to pathological classification.The diagnostic accuracy of ultrasonography was 68.75%(44/64),73.22%(93/127),and 81.81%(72/88),respectively.The diagnostic accuracy of CT was 71.87%(46/64),82.67%(105/127),and 90.90%(80/88),respectively.There was no statistically significant difference in diagnostic accuracy between the two examinations(P>0.05).Subgroup analysis based on patient BMI showed that there was no difference in diagnostic accuracy of the two examinations for patients with normal BMI(P>0.05),while for overweight and obese patients,the diagnostic accuracy of CT was better than that of ultrasonography,with a statistical difference(P<0.05).Conclusion There is no difference in the diagnostic accuracy of ultrasonography and CT examinations for acute appendicitis of different pathological types.But for overweight and obese acute appendicitis patients,the diagnostic accuracy of CT examination is superior to ultrasonography.

Humans ; Diabetes Mellitus, Type 2/epidemiology* ; Air Pollution/adverse effects* ; Air Pollutants/toxicity* ; Risk Assessment ; Particulate Matter ; Environmental Exposure

Objective:To analyze effects of ozone bath, 1∶8 000 potassium permanganate bath and 1∶5 000 potassium permanganate bath on the recovery of skin lesions in patients with pemphigus.Methods:Patients with pemphigus, who received immersion bath treatment during their hospitalization, were retrospectively collected from Department of Dermatology, Peking University First Hospital from January 2016 to June 2021. The efficacy and safety of different immersion bath methods including ozone bath, 1∶8 000 potassium permanganate bath and 1∶5 000 potassium permanganate bath were compared. Categorical variables were compared using chi-square test or Fisher′s exact test, and univariate and multivariate logistic regression models were used to analyze relationships between candidate variables and patients′ condition at discharge.Results:A total of 74 patients with pemphigus were included, including 45 (60.81%) males and 29 (39.19%) females, their age ( M[ Q1, Q3]) was 52 (41, 60) years, and the median percentage of primary skin lesion area in body surface area was 40%. There were no significant differences in hospital stays, disease duration (time from onset to admission) , gender ratio or skin lesion areas among patients receiving ozone bath (32 cases) , patients receiving 1∶8 000 potassium permanganate bath (25 cases) and those receiving 1∶5 000 potassium permanganate bath (17 cases, all P > 0.05) . These patients still received other treatments, including glucocorticoids, gamma globulin, plasma transfusion, rituximab, immunosuppressants, topical antibiotics, etc., and there was no significant difference in the proportions of patients using the above therapies among the three groups (all P > 0.05) , while the ozone bath group showed a lower proportion of patients using systemic antibiotics compared with the two potassium permanganate bath groups (both P < 0.01) . At discharge, in the ozone bath group, 1 case was improved, 21 were nearly cured, and 10 were cured; in the 1∶8 000 potassium permanganate bath group, 4 cases were improved, 13 were nearly cured, and 8 were cured; in the 1∶5 000 potassium permanganate bath group, 5 were improved, 8 were nearly cured, and 4 were cured. The proportion of well-recovered patients was significantly higher in the ozone bath group (31/32 cases) than in the potassium permanganate bath group (33/42 cases, P = 0.036) . Univariate and multivariate analyses showed that ozone bath significantly affected the patients′ condition at discharge ( P < 0.05) , and was an independent predictor of "good recovery" at discharge ( HR = 8.455, 95% CI: 1.011 - 70.672, P = 0.049) . Conclusion:Ozone bath therapy can facilitate recovery of skin lesions in patients with pemphigus.

Aim To develop an UPLC-MS/MS method to determine the concentration of lorcaserin hydrochloride in beagle plasma, and study the pharmacokinetics of osmotic pump controlled-release tablets of lorcaserin hydrochloride. Methods A randomized crossover design was used, carbamazepine as the internal standard(IS), and plasma protein precipitation with acetonitrile. The chromatographic was Phenomenex Polar C18 column(100 mm×2. 1 mm, 3 μm), and acetonitrile - water(containing 10 mmol·L-1 ammonium acetate and 0.1% formic acid)(40:60, V/V)was mobile phase. Multiple reaction monitoring mode and electrospray positive ionization were used to detect lorcaserin hydrochloride. The MS/MS ion transitions were monitored at m/z 196.2→129.2 for lorcaserin hydrochloride and m/z 237→194.1 for carbamazepine, respectively. Results The linear range was 1 to 500 μg·L-1(r=0.999 2), the extraction recovery rate ranged from 87.70% to 89.70%, the precision RSD was 9.7%. The accuracy and matrix effect met the requirements, and the stability of lorcaserin hydrochloride was good in -20 ℃ refrigerator for 45 d, repeated freezing and thawing for three times, placed at room temperature for 24 h, and the disposed samples placed in automatsampler for 6 h were stable. The main pharmacokinetic parameters of the controlled-release tablet and immediate-release tablet were as follows:Tmax was(8.00±1.27)h and(1.00±0.13)h, Cmax was(70.56±3.73)μg·L-1 and(176.33±16.73)μg·L-1, and AUC0-t was(966.33±7.56)μg·h·L-1 and(973.05±69.09)μg·h·L-1, respectively. Conclusions The established UPLC-MS/MS method can be used to study the pharmacokinetics of lorcaserin hydrochloride in the plasma of beagle dogs, and osmotic pump controlled-release tablets has sustained release effect.

OBJECTIVES: To establish the menstrual blood identification model based on Naïve Bayes and multivariate logistic regression methods by using specific mRNA markers in menstrual blood detection technology combined with statistical methods, and to quantitatively distinguish menstrual blood from other body fluids. METHODS: Body fluids including 86 menstrual blood, 48 peripheral blood, 48 vaginal secretions, 24 semen and 24 saliva samples were collected. RNA of the samples was extracted and cDNA was obtained by reverse transcription. Five menstrual blood-specific markers including members of the matrix metalloproteinase (MMP) family MMP3, MMP7, MMP11, progestogens associated endometrial protein (PAEP) and stanniocalcin-1 (STC1) were amplified and analyzed by electrophoresis. The results were analyzed by Naïve Bayes and multivariate logistic regression. RESULTS: The accuracy of the classification model constructed was 88.37% by Naïve Bayes and 91.86% by multivariate logistic regression. In non-menstrual blood samples, the distinguishing accuracy of peripheral blood, saliva and semen was generally higher than 90%, while the distinguishing accuracy of vaginal secretions was lower, which were 16.67% and 33.33%, respectively. CONCLUSIONS The mRNA detection technology combined with statistical methods can be used to establish a classification and discrimination model for menstrual blood, which can distignuish the menstrual blood and other body fluids, and quantitative description of analysis results, which has a certain application value in body fluid stain identification.
Female ; Humans ; RNA, Messenger/metabolism* ; Bayes Theorem ; Logistic Models ; Menstruation ; Body Fluids ; Saliva ; Semen ; Forensic Genetics/methods*

Purpose: This study was aimed to investigate long-term survivals and toxicities of early-stage nasopharyngeal carcinoma (NPC) in endemic area, evaluating the role of chemotherapy in stage II patients. Materials and Methods: Totally 187 patients with newly diagnosed NPC and restaged American Joint Committee on Cancer/ International Union Against Cancer 8th T1-2N0-1M0 were retrospectively recruited. All received intensity-modulated radiotherapy (IMRT)±chemotherapy (CT) from 2001 to 2010. Results: With 15.7-year median follow-up, 10-year locoregional recurrence-free survival, distant metastasis-free survival (DMFS), disease-specific survival (DSS), and overall survival (OS) were 93.3%, 93.5%, 92.9% and 88.2%, respectively. Multivariable analyses showed cervical lymph nodes positive and pre-treatment prognostic nutritional index ≥ 52.0 could independently predict DMFS (p=0.036 and p=0.011), DSS (p=0.014 and p=0.026), and OS (p=0.002 and p < 0.001); Charlson comorbidity index < 3 points could predict DSS (p=0.011); age > 45 years (p=0.002) and pre-treatment lactate dehydrogenase ≥ 240 U/L (p < 0.001) predicted OS. No grade 4 late toxicity happened; grade 3 late toxicities included subcutaneous fibrosis (4.3%), deafness or otitis (4.8%), skin dystrophy (2.1%), and xerostomia (1.1%). No differences on survivals were shown between IMRT+CT vs. IMRT alone in stage II patients, even in T2N1M0 (p > 0.05). Unsurprising, patients in IMRT+CT had more acute gastrointestinal reaction, myelosuppression, mucositis, late ear toxicity, and cranial nerve injury (all p < 0.05) than IMRT alone group. Conclusion Superior tumor control and satisfying long-term outcomes could be achieved with IMRT in early-stage NPC with mild late toxicities. As CT would bring more toxicities, it should be carefully performed to stage II patients.

Objective:To analyze the occurrence of and risk factors for skin and mucosal infections after primary tumor resection in patients with paraneoplastic pemphigus, and to summarize relevant nursing experience.Methods:Clinical characteristics and postoperative skin and mucosal infections were retrospectively analyzed in patients with confirmed paraneoplastic pemphigus, who underwent primary tumor resection in Department of Dermatology, Peking University First Hospital between January 1999 and January 2021. Common infectious agents were analyzed, and infection-related risk factors were identified by logistic regression analysis.Results:A total of 44 patients with paraneoplastic pemphigus were included in this study, including 25 (56.8%) males and 19 (43.2%) females, and their age were 33.8 ± 15.4 years. Postoperatively, 21 (47.73%) patients developed skin and mucosal infections, and their postoperative hospital stay (median [ Q1, Q3]) was 38 (25, 60) days, which was significantly longer than that in patients without skin and mucosal infections (21 [12, 23] days, Z = -4.08, P < 0.001) . The most common pathogen was methicillin-resistant Staphylococcus aureus (15 cases, 34.09%) . High glucocorticoid dosage per kilogram of body weight ( OR = 1.21, 95% CI: 1.00 - 1.46, P = 0.047) and receiving assisted ventilation therapy ( OR = 9.20, 95% CI: 2.01 - 42.13, P = 0.004) were independent risk factors for postoperative skin and mucosal infections. After active treatment and care, 37 (84.1%) patients recovered well at discharge. Conclusion:Skin and mucosal infections are a common postoperative complication in patients with paraneoplastic pemphigus, and the pathogens are mostly drug-resistant bacteria, which can lead to prolonged hospital stay, so attention should be paid to postoperative skin care; high postoperative glucocorticoid dosage per body weight and respiratory support may be associated with postoperative skin and mucosal infections.