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 concept of "qi deficiency with stagnation" refers to a pathological state characterized by the depletion of primordial qi， impaired qi transformation， and the development of internal stagnation. Under the cyclic chemotherapy regimen in oncology， chemotherapy-induced myelosuppression follows a progressive pathological course from qi deficiency to increasing stagnation. This sequential evolution from mild to severe myelosuppression closely aligns with the dynamic syndrome differentiation and treatment framework of "qi deficiency with stagnation". "Qi deficiency" reflects the gradual depletion of qi， blood， and essence， while "stagnation" refers to the accumulation of phlegm， turbid dampness， and blood stasis. These two components interact reciprocally， forming a vicious cycle where deficiency leads to stagnation， and stagnation further damages the healthy qi. In the early stage of mild myelosuppression， chemotoxicity begins to accumulate in the bone marrow， leading to qi consumption， blood deficiency， yin injury， and the gradual formation of turbid phlegm and damp stagnation. In the advanced stage of severe myelosuppression， the accumulation of toxicity causes qi sinking， exhaustion of essence， and marrow depletion， along with blood stasis obstructing the collaterals. Treatment strategies should be based on syndrome differentiation， with an emphasis on assessing the severity of the condition， balancing deficiency and excess， and achieving both symptomatic relief and root cause resolution.

ObjectiveBy observing the effect of Qianyang Yuyin granules on the phenotype of renal tubule epithelial cells， the intervention of Qianyang Yuyin granule on renal interstitial fibrosis was investigated. MethodThe renal tubular epithelial cells （HK-2） were treated with different concentrations of transforming growth factor （TGF）-β₁ （5， 10， 15， 20， 25 μg·L^-1） for 24 hours， and cell morphology and growth state were observed with an inverted phase contrast microscope. The 20 μg·L^-1 was selected as the most appropriate concentration of TGF-β₁ according to Western blot results for subsequent experiments. HK-2 cells were divided into six groups： blank group， TGF-β₁ group （concentration of 20 μg·L^-1）， low， medium， and high dose Qianyang Yuyin granule groups （concentration of 0.5， 1， 2 g·L^-1）， and valsartan group （1 × 10^-5 mol·L^-1）. The cell activity was measured by cell proliferation and cell counting kit-8 （CCK-8）. The cell migration ability was detected by scratch test. The Transwell method was used to detect the invasiveness of cells. Western blot was used to detect levels of fibronectin （FN）， E-cadherin， α-smooth muscle activator （α-SMA）， Vimentin， collagen type Ⅰ（Col Ⅰ）， collagen type Ⅳ（Col Ⅳ）， and other related proteins. ResultTGF-β₁ stimulating epithelial-mesenchymal transition （EMT） in renal tubular epithelial cells was time- and concentration-dependent. Compared with the blank group， higher concentration in the TGF-β₁ group indicates longer intervention time and more obvious long spindle change of cells， and the migration and invasion ability of the cells was significantly enhanced. The protein expression level of FN， α-SMA， Vimentin， Col Ⅰ， and Col Ⅳ increased significantly （P<0.05， P<0.01）， while the expression level of E-cadherin protein decreased （P<0.05）. Compared with the TGF-β₁ group， Qianyang Yuyin granule groups could maintain normal cell morphology， and the migration and invasion ability of the cells was inhibited. The protein expression level of FN， α-SMA， Vimentin， Col Ⅰ， and Col Ⅳ decreased （P<0.05， P<0.01）， and the expression of E-cadherin protein was significantly restored （P<0.05）. ConclusionQianyang Yuyin granule can reverse TGF-β₁-induced interstitial transformation of renal tubular epithelial cells by reducing the phenotypic expression of mesenchymal cells and increasing the phenotypic expression of epithelial cells.

Objective To establish a high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)for the simultaneous determination of gefitinib,erlotinib,nillotinib and imatinib plasma concentrations and analyze the results.Methods The plasma samples were treated with acetonitrile precipitation and separated by Diamonsil C18 column(150 mm ×4.6 mm,3.5 μm)with mobile phase of 0.1％formic acid water(A)-0.1％formic acid acetonitrile(B).The flow rate of gradient elution was 0.7 mL·min-1,and the column temperature was 40 ℃ and the injection volume was 3 μL.Using arotinib as the internal standard,the scanning was carried out by using electrospray ionization source in positive ionization mode with multi-reaction monitoring.The specificity,standard curve,lower limit of quantitation,precision,accuracy,recovery rate,matrix effect and stability of the method were investigated.The concentrations of imatinib and erlotinib in 20 patients with chronic myelogenous leukemia(CML)and gefitinib and erlotinib in 3 patients with non-small cell lung cancer were measured.Results The standard curves of the four drugs were as follows,gefitinib:y=2.536 × 10-3x+9.362 × 10-3(linear range 20-2 000 ng·mL-1,R2=0.996 6);erlotinib:y=3.575× 10-3x+7.406 × 10-3(linear range 50-5 000 ng·mL-1,R2=0.994 9);nilotinib:y=1.945 x 10-3x+0.015 643(linear range 50-5 000 ng·mL-1,R2=0.990 6);imatinib:y=4.56 x 10-3x+0.010 451(linear range 100～104 ng·mL-1,R2=0.9963).RSD of intra-day and inter-day were less than 10％,and the accuracy ranged from 90％to 110％,and the recovery rates were 91.35％to 98.93％(RSD＜10％);the matrix effect ranged from 91.64％to 107.50％(RSD＜10％).Determination of 23 patients showed that the blood concentration of nilotinib ranged from 623.76 to 2 934.13 ng·mL-1,and the blood concentration of imatinib ranged from 757.77 to 2 637.71 ng·mL-1,and the blood concentration of gefitinib ranged from 214.76 to 387.40 ng·mL-1.The serum concentration of erlotinib was 569.57 ng·mL-1.Conclusion The method of this research is simple,fast,sensitive and dedicated,which can be monitored by the concentration of clinical blood.

BACKGROUND:Superovulation is a common therapy in assisted reproductive technology.In clinical practice,some patients experience repeated superovulation to get pregnant. OBJECTIVE:To explore the effect of repeated superovulation on the developmental potential of oocytes in mice and humans. METHODS:Both animal experiments and retrospective clinical research were conducted.The animal study involved 90 SPF grade ICR 8-week-old female mice,who were randomly divided into three groups for 1,3,and 5 superovulations,respectively.The clinical study involved 306 patients who had undergone three consecutive in vitro fertilization cycles.The number of ovules obtained and embryonic development in different cycles were compared. RESULTS AND CONCLUSION:(1)The animal study indicated that repeated superovulation did not affect the embryonic development or developmental speed of mouse embryos.Similarly,there was no significant difference in the mouse blastocyst apoptosis,DNA damage,or the formation of inner cell mass and trophectoderm(P>0.05).(2)The clinical study also revealed no significant differences in the number of retrieved oocytes(8.60±5.04,8.58±4.87,and 8.38±4.63,P=0.81)and transferable embryos(2.42±1.99,2.40±1.92,and 2.64±2.00,P=0.26)over the three cycles.(3)In both the young group(<35 years)and the old group(≥35 years),the embryo quality was not affected by repeated superovulation(P>0.05).(4)These findings show that repeated superovulation does not affect the developmental potential of oocytes in mice and humans.

Objective To explore the application of metagenomic next-generation sequencing(mNGS)technology in the investigation of healthcare-associated infection(HAI)outbreaks of carbapenem-resistant Acinetobacter bau-mannii(CRAB).Methods Pathogenic detection by mNGS and conventional pathogen culture were performed on 5 patients in the intensive care unit(ICU)of a hospital from June 8 to 22,2023 from whom CRAB were detected.Microbial sampling was carried out in potentially contaminated environment.Bacterial culture,identification,and antimicrobial susceptibility testing were conducted.Comprehensive control measures were taken,and the effect was evaluated.Results The time required for reporting results by mNGS was shorter than the culture time([3.92± 1.05]days vs[6.24±0.25]days,P＜0.001).CRAB was isolated from the specimens of 5 patients.mNGS de-tected OXA-23 resistance genes from all patients.After comprehensive assessment by experts,4 patients were HAI and 1 patient was due to specimen contamination.According to the definition from Guidelines for HAI outbreak control,this event was considered an outbreak of HAI.The monitoring results of environmental hygiene showed that the detection rate of CRAB in the environment during the outbreak was 51.30％(59/115),mainly from the hands of health care workers and the surface of ventilators.After implementing multidisciplinary infection control measures,clinicians'hand hygiene compliance rate and implementation rate of ventilator disinfection increased from 40.83％(49/120)and 33.33％(16/48)to 82.61％(95/115)and 83.33％(30/36),respectively.The prognosis of patients was good,and no new case emerged during subsequent monitoring.The outbreak of HAI in this hospital has been effectively controlled.Conclusion mNGS is characterized by high precision,less time consumption,and high accuracy,and can be applied to the prevention and control of HAI outbreak and the study of antimicrobial-re-sistant genomes.It is of great significance for the anti-infection treatment of patients with multidrug-resistant orga-nism infection as well as the formulation of HAI prevention and control measures.Continuous improving disinfec-tion effectiveness and hand hygiene compliance is important for preventing and controlling CRAB infection.

ObjectiveTo investigate the effect of Shugan Quyu Jiedu prescription （SGQYJDF） on inducing ferroptosis in hepatocellular carcinoma cells based on the tumor protein 53 （p53）/solute carrier family 7 member 11 （SLC7A11）/glutathione peroxidase 4 （GPX4） pathway. MethodMHCC97H cells were divided into the blank serum group （10% blank serum medium）， SGQYJDF-containing serum low concentration group （5% SGQYJDF-containing serum and 5% blank serum medium）， SGQYJDF-containing serum medium concentration group （7.5% SGQYJDF-containing serum and 2.5% blank serum medium）， SGQYJDF-containing serum high concentration group （10% SGQYJDF-containing serum medium） and sorafenib group （sorafenib concentration of 10 μmol·L^-1 in 10% blank serum medium）. After 24 hours of intervention， the cell survival rate was detected by cell counting kit-8 （CCK-8） assay. The cell proliferation ability was detected by 5-ethynyl-2′-deoxyuridine （EdU） staining. The intracellular ferrous ion （Fe²⁺） level was detected by ferrous ion fluorescent probe （FerroOrange） staining. The intracellular malondialdehyde （MDA） and glutathione （GSH） levels were detected by colorimetric assays. The ultrastructure of mitochondria was observed by transmission electron microscopy. The expression levels of ferroptosis-related proteins p53， SLC7A11 and GPX4 were detected by Western blot. ResultIn terms of cell viability， compared with the blank serum group， the SGQYJDF group showed a dose-dependent decrease in the survival rate of MHCC97H cells. Effect of the medium and high concentrations of SGQYJDF on the survival rate of MHCC97H cells were significantly decreased （P<0.01）. Additionally， the results of the EdU assay showed that both the medium and high concentrations of SGQYJDF were able to inhibit the proliferation ability of MHCC97H cells （P<0.05， P<0.01）. Regarding the biochemical indicators of ferroptosis， compared to the blank serum group， the medium and high concentrations of SGQYJDF were able to dose-dependently increase the intracellular Fe²⁺ level （P<0.01）. The low， medium， and high concentrations of SGQYJDF were able to dose-dependently decrease the level of GSH in MHCC97H cells （P<0.01） and increase the level of MDA in the cells （P<0.05， P<0.01）. In terms of pathway-related protein expression， compared to the blank serum group， the medium and high concentrations of SGQYJDF could significantly increase the expression of p53 （P<0.01）. The low， medium， and high concentrations of SGQYJDF could significantly decrease the expression of GPX4 （P<0.01）. The high concentration of SGQYJDF could decrease the expression of SLC7A11 （P<0.01）. In terms of the cell morphology of ferroptosis， compared with the blank serum group， transmission electron microscopy revealed that the low concentration of SGQYJDF caused mitochondrial deformation， while the medium and high concentrations of SGQYJDF resulted in reduced mitochondrial volume， increased double-layer membrane density， and decreased mitochondrial cristae. These features were similar to those of sorafenib-induced ferroptosis. Furthermore， compared with the sorafenib group， the high concentration of SGQYJDF showed no statistically significant differences in cell survival rate， proliferation ability， Fe²⁺ level， MDA level， and GSH level. ConclusionThe results suggest that SGQYJDF may induce ferroptosis and inhibit proliferation in hepatocellular carcinoma MHCC97H cells by upregulating the expression of p53， suppressing the expressions of GPX4 and SLC7A11， downregulating the level of GSH， and leading to the accumulation of intracellular Fe²⁺ and MDA.

Child ; Humans ; Mpox (monkeypox)/prevention & control* ; Disease Outbreaks

Objective Pulmonary oxygen poisoning resulting from hyperbaric oxygen,frequently occurs in specialized operations,without any current effective prevention or treatment measures.To elucidate the impact and mechanism of neostigmine(NEO)in combination with anisodamine(ANI)(neoscopolamine)on pulmonary oxygen toxicity.Methods The animal model of pulmonary oxygen poisoning was established.C57BL/6 mice were exposed to 2.5 ATA 99.9%oxygen for 6 h.The control group mice were injected with normal saline ip,while the treatment group mice received injections of ANI(25 mg/kg,ip)and NEO(50 μg/kg,ip).Lung tissues were collected and stained with HE to observe any pathological injuries after exposure.Evans blue stain was utilized to identify lung permeability,wet/dry lung ratio,and protein concentration in the bronchoalveolar lavage fluid(BALF)to assess the lung injury's severity.The modifications in inflammatory factors,oxidative stress indicators,and iron content in lung tissue were assessed.Results The results showed that the 2.5 ATA 99.9%oxygen-exposed group experienced a significant worsening of lung injury,as well as increased lung permeability,lung wet/dry ratio,and protein content in alveolar lavage fluid when compared to the control group.Moreover,mRNA levels of pro-inflammatory cytokines IL-1β,IL-6,TNF-α,and IFN-γ in the lung tissue of the model group were significantly elevated,while the levels of anti-inflammatory cytokines IL-4 and TGF-β were significantly reduced.The oxidative index MDA also significantly increased,while the antioxidant index GSH significantly decreased.Additionally,the expression of GPX4,a marker of ferroptosis,increased with an increase in iron content.Neoscopolamine treatment successfully reversed those effects.Conclusion The combined use of ANI and NEO had a protective effect on pulmonary oxygen poisoning.Neoscopolamine may inhibit inflammation and oxidative stress by activating the cholinergic anti-inflammatory pathway,thereby reducing the content of free iron in lung tissue and finally inhibiting cell ferroptosis.

Objective To explore the relationship between serum ferritin levels and body fat distribution in patients with type 2 diabetes mellitus(T2DM).Methods A retrospective analysis was conducted on 151 patients with T2DM who were hospitalized in the Department of Endocrinology of the First Hospital of Lanzhou University from June to November 2020,and all the patients were divided into high serum ferritin(n=50)and normal serum ferritin(n=101)groups according to their serum ferritin levels.The visceral fat area(VFA),subcutaneous fat area(SFA),liver fat,height,weight and waist circumference(WC)were measured,as well as blood glucose,lipid indexes,body mass index(BMI)and visceral adiposity index(VAI)were also calculated.t-test or nonparametric test was used to compare the differences between the two groups,and the relationship between serum ferritin levels and body fat distribution was analyzed by Pearson or Spearman correlation analysis,multiple linear regression and logistic regression.Results The VAI and WC were significantly higher in high serum ferritin group[3.13(2.16,4.58)and(96.66±7.78)cm]than in normal serum ferritin group[2.66(1.66,3.81)and(91.96±9.75)cm,P<0.05].The prevalence of central obesity and dyslipidemia was higher in high serum ferritin group(88.0%and 90.0%)than in normal serum ferritin group(68.3%and 75.2%);and the composition ratios of poor glycemic control and insulin resistance(96.0%and 62.0%)were also higher than in normal serum ferritin group(78.2%and 40.6%)(P<0.05),there were no statistically significant differences in BMI,VFA,and SFA levels,as well as antidiabetic drug use and chronic complications of diabetes mellitus between the two groups(P>0.05).Serum ferritin levels in T2DM patients were positively correlated with VAI,WC,triglyceride(TG),fasting blood glucose(FPG),HbA1c,dyslipidemia and serum creatinine(r=0.171,0.207,0.187,0.243,0.270,0.162,0.162;P<0.05),and negatively correlated with age,sex and diabetes course(r=-0.191,-0.434,-0.352;P<0.05).Multivariate linear regression analysis showed that in male T2DM patients,duration of diabetes and FPG were risk factors for increased levels of serum ferritin.However,WC and VAI did not significantly affect serum ferritin levels.In female patients with T2DM,the course of diabetes,TG and VAI were the factors influencing serum ferritin(P<0.05).Conclusion Dyslipidemia and visceral fat accumulation are risk factors for elevated serum ferritin levels in female T 2DM patients.