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 investigate the efficacy of the therapy of soothing liver and strengthening spleen(shortened as Shugan Jianpi therapy)in the treatment of active thyroid-associated ophthalmopathy(TAO)complicated with dry eye,and to provide a reference basis for clinical treatment.Methods A total of 108 patients with active TAO complicated with dry eye of liver depression and qi stagnation type were randomly divided into observation group and control group,54 patients in each group.Both groups were given conventional treatment for intervention of Graves'disease,and additionally the control group was given hormone shock therapy by intravenous injection of Methylprednisolone Sodium Succinate,and the observation group was treated with Chinese medicine prescription for soothing liver and strengthening spleen orally and intravenous injection of Methylprednisolone Sodium Succinate.The treatment period lasted for 12 weeks,and then the patients were followed up till to the 6th month.The changes of clinical activity score(CAS),proptosis,ocular surface disease index(OSDI),corneal fluorescein staining(FL),Schirmer I test(SIT)and tear film break-up time(BUT)in the two groups were observed before and after the treatment.After treatment,the clinical efficacy of the two groups was evaluated.Results(1)After 6 months of treatment,the total effective rate in the observation group was 94.44%(51/54)and that in the control group was 74.07%(40/54),and the intergroup comparison(tested by chi-square test)showed that the efficacy of the observation group was significantly superior to that of the control group(P＜0.05).(2)After treatment,the CAS,OSDI score and proptosis of the patients in the two groups were all lower than those before treatment(P＜0.01),and the reduction in the observation group was significantly superior to that in the control group(P＜0.01).(3)After treatment,the indicators of tear secretion function such as SIT,FL score and BUT of patients in the two groups were improved compared with those before treatment(P＜0.01),and the improvement in the observation group was significantly superior to that in the control group,the differences being all statistically significant(P＜0.01).Conclusion Shugan Jianpi therapy exerts certain clinical efficacy in treating patients with active TAO complicated with dry eye of liver depression and qi stagnation type,which can effectively relieve the proptosis,prolong the tear film break-up time,promote the secretion of tears and the repair of corneal epithelium,improve the visual function,and enhance the quality of life of the patients.

Aim To investigate the mechanism by which formononetin (FN) inhibits mitochondrial dynamic-related protein 1 (DRP1) -NLRP3 axis via intervening the generation of ROS to reduce allergic airway inflammation. Methods In order to establish allergic asthma mouse model, 50 BALB/c mice aged 8 weeks were divided into the control group, model group, FN treatment group and dexamethasone group after ovalbumin (OVA) induction. Airway inflammation and collagen deposition were detected by HampE and Masson staining. Th2 cytokines and superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and IgE levels in bronchoalveolar lavage fluid (BALF) were measured by ELISA, ROS in BEAS-2B cells was assessed by DCFH-DA staining, DRP1 expression in lung tissue and BEAS-2B cells was detected by immunohistochemistry and immunofluorescence, and the DRP1-NLRP3 pathway was analyzed by immunoblotting. Results FN treatment could effectively ameliorate the symptoms of asthmatic mouse model, including reducing eosinophil accumulation, airway collagen deposition, decreasing Th2 cytokine and IgE levels, reducing ROS and MDA production, increasing SOD and CAT activities, and regulating DRP1-NLRP3 pathway-related protein expression, thereby relieving inflammation. Conclusion FN ameliorates airway inflammation in asthma by regulating DRP1-NLRP3 pathway.

Human exhaled breath has great application prospects,e.g.,monitoring pharmacokinetics,disease diagnosis,due to its advantages such as non-invasive and high-frequency sampling.Breath samples can be collected from the oral and nasal cavity.However,the oral and nasal environment affect the chemical composition of breath sample.Therefore,the investigation on the chemical composition of mouth-exhaled breath and nose-exhaled breath is crucial for selection of appropriate sampling strategy for individual studies.In this work,secondary electrospray ionization-high resolution mass spectrometry(SESI-HRMS)was applied to analysis of respiratory metabolomics in real time.A quantitative analysis approach was established for 9 kinds of volatile organic compounds(VOCs)e.g.2-butanone,2-pentanone,ethyl acetate,methyl methacrylate,toluene,styrene,mesitylene,isoprene and limonene.The limit of detection was 2.3?240.8 ng/m3.The intra-day(n=6)and inter-day(n=18)relative standard deviations were 0.6%?4.6%and 4.3%?12.2%,respectively.Nine healthy subjects were recruited to investigate the chemical composition of mouth-exhaled and nose-exhaled breath.The results showed the good performance in quantitative analysis of 9 VOCs in breath air.It was found that the number of unique component(m/z)detected in mouth-exhaled breath(167)was 2.2 times greater than that detected in nose-exhaled breath(76),which might result from the complex environment in oral cavity.The signal intensity of commun component(163)was significantly different between mouth-exhaled breath and nose-exhaled breath.Additionally,the elemental composition analysis showed that the proportion of polar compounds detected in nose-exhaled breath was higher than that in mouth-exhaled breath.This study demonstrated that there was significant differences in the chemical composition between mouth-exhaled and nose-exhaled breath,which provided a theoretical basis for selection of exhalation mode.

Nucleic acid-based molecular diagnostic methods are considered the gold standard for detecting infectious pathogens.However,when applied to portable or on-site rapid diagnostics,they still face various limitations and challenges,such as poor specificity,cumbersome operation,and portability difficulties.The CRISPR(Clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein(Cas)-fluorescence detection method holds the potential to significantly enhance the specificity and signal-to-noise ratio of nucleic acid detection.In this study,we developed a portable grayscale reader detection system based on loop-mediated isothermal amplification(LAMP)-CRISPR/Cas.On one hand,in the presence of CRISPR RNA(crRNA),the CRISPR/Cas12a system was employed to achieve precise fluorescent detection of self-designed LAMP amplification reactions for influenza A and influenza B viruses.This further validated the high selectivity and versatility of the CRISPR/Cas system.On the other hand,the accompanying independently developed portable grayscale reader allowed for low-cost collection of fluorescence signals and high-reliability visual interpretation.At the end of the detection process,it directly provided positive or negative results.Practical sample analyses using this detection system have verified its reliability and utility,demonstrating that this system can achieve highly sensitive and highly specific portable analysis of influenza viruses.

【Objective】 To understand the prevalence of overweight/obesity among school-age children in Pudong New Area of Shanghai, and to explore the influence of gestational weight gain and pre-pregnancy body mass index (BMI) on weight status of school-age children. 【Methods】 From November to December 2020,a stratified cluster sampling method was adopted to select first-grade students from 13 primary schools in Pudong New Area of Shanghai.After matching with the birth monitoring database, 755 students with complete birth information were selected as the study subjects.The relevant information of mothers before and during pregnancy was retrospectively collected, and the effects of pregnancy weight gain combined with pre-pregnancy BMI on overweight/obesity in school-age children were analyzed. 【Results】 1) The prevalence rates of overweight and obesity of first-grade children were 15.89% and 18.41%, respectively.2) Maternal excessive weight gain during pregnancy (OR=1.678) and overweight/obesity before pregnancy (OR=2.315,2.412) were risk factors for overweight/obesity of the offspring at school age(P<0.05).3) For mothers who were underweight before pregnancy, excessive weight gain during pregnancy was associated with overweight/obesity in school-age children in their offspring (OR=7.436, 95%CI: 1.489 - 37.143,P<0.05).4) Excessive weight gain during pregnancy combined with overweight/obesity before pregnancy significantly increased the risk of overweight/obesity in offspring (OR=3.606, 95%CI: 2.030 - 6.405, P<0.05). Mothers who gained a moderate amount of weight during pregnancy and were emaciated before pregnancy had a significantly lower risk of overweight/obesity in their school-age children (OR=0.217, 95%CI: 0.049 - 0.967, P<0.05). 【Conclusion】 Excessive weight gain during pregnancy increases the risk of overweight/obesity in school-age children in their offspring, strengthening pregnancy health education and perinatal care to help pregnant women maintain appropriate weight gain during pregnancy may be an important and novel strategy to prevent childhood obesity.

Objective To explore the correlation between sleep status and traditional Chinese medicine(TCM)constitution types of sub-healthy population in Lingnan area.Methods From May 2022 to May 2023,an investigation was carried out in a sub-healthy population of 688 persons who lived in Shenzhen for more than 5 years,had no serious cardiovascular disease and were confirmed by the sub-health rating scale.The questionnaire survey of Pittsburgh Sleep Quality Index(PSQI)scale and the TCM constitution classification rating scale was conducted,and multiple linear regression analysis was performed to explore the correlation between the sleep status and TCM constitution types.Results(1)A total of 688 questionnaires were released,of which 19 were lost to follow-up,and 669 were responded.After weeding out the 29 invalid questionnaires,640 valid questionnaires were finally included for the analysis.(2)According to the PSQI score,the population was divided into excellent sleep group(total PSQI score＜8)of 350 cases and poor sleep group(total PSQI score≥8)of 290 cases.The item scores of time for falling asleep,sleep time and sleep disorder as well as the total PSQI score of the excellent sleep group were significantly lower than those of the poor sleep group(P＜0.05 or P＜0.01).(3)The persons with excellent sleep usually had the balanced constitution(86.86%),while the persons with poor sleep often had the biased constitution(79.64%).The top three biased constitution types in the persons with poor sleep were damp-heat constitution(15.52%),phlegm-damp constitution(13.79%)and qi-stagnation constitution(13.10%).(4)Multiple linear regression analysis showed that the time for falling asleep was positively correlated with phlegm-damp constitution and qi-stagnation constitution(P＜0.01),and was negatively correlated with balanced constitution(P＜0.05);the sleep efficiency was positively correlated with damp-heat constitution and inherited special constitution(P＜0.05 or P＜0.01),and was negatively correlated with balanced constitution(P＜0.05);sleep disorder was positively correlated with qi-stagnation constitution and yin-deficiency constitution(P＜0.01);hypnotic drugs were positively correlated with yin-deficiency constitution(P＜0.01);daytime dysfunction was positively correlated with phlegm-damp constitution(P＜0.05).Conclusion The sleep status of sub-healthy population in Lingnan area has a certain correlation with TCM constitution types.Damp-heat constitution,phlegm-damp constitution,qi-stagnation constitution,yin-deficiency constitution,balanced constitution and inherited special constitution are the six constitution types that affect sleep status.It is of great significance to guide the sub-healthy population to achieve the purpose of prevention before illness by improving their constitution after the identification of TCM constitution types.

Objective To observe the therapeutic effect and mechanism of Baoyuan Decoction for chronic heart failure model rat.Methods SD rats were randomly divided into blank group,model group,Baoyuan Decoction group,Captopril group,Baoyuan Decoction+CCT020312[protein kinase R-like endoplasmic reticulum kinase(PERK)activator]group,15 rats in each group.Except for the blank group,the rats in the other groups were induced by Adriamycin to construct a chronic heart failure model.After corresponding drug intervention,cardiac function indexes[left ventricular end-diastolic diameter(LVEDD),left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),brain natriuretic peptide(BNP),cardiac troponin I(cTnI)],inflammation-related factors[tumor necrosis factor α(TNF-α),interleukin 1β(IL-1β)],oxidative stress factors[malondialdehyde(MDA),superoxide dismutase(SOD)]were detected in each group.Changes in apoptosis-related indicators[B-cell lymphoma 2(Bcl-2),B-cell lymphoma 2-associated X protein(Bax),Caspase-3]and PERK/transcription activator 4(ATF4)signaling pathway-related proteins glucose-regulated protein 78(GRP78),PERK,ATF4,C/EBP homologous protein(CHOP)levels.Results Compared with the blank group,LVEDD,BNP,cTnI,TNF-α,IL-1β,MDA levels,protein expression levels of Bax,Caspase-3,GRP78,PERK,ATF4,CHOP in the model group were significantly increased,LVEF,LVFS,SOD levels and Bcl-2 protein expression level were significantly decreased(all P＜0.05).Compared with the model group and Baoyuan Decoction+CCT020312 group,LVEDD,BNP,cTnI,TNF-α,IL-1β,MDA levels,protein expression levels of Bax,Caspase-3,GRP78,PERK,ATF4,CHOP in Baoyuan Decoction group and Captopril group were significantly decreased,LVEF,LVFS,SOD levels and Bcl-2 protein expression level were significantly increased(all P＜0.05).Compared with the Captopril group,there was no significant change in the above indexes(except CHOP protein expression level)in the Baoyuan Decoction group(P＞0.05).Conclusion Baoyuan Decoction can delay the progression of chronic heart failure rats,and its mechanism may be related to inhibiting the PERK/ATF4 signaling pathway to alleviate cardiomyocyte apoptosis,further reducing the degree of inflammatory response and oxidative stress,thereby promoting the repair of cardiac function and myocardial injury.

Objective To observe the effect of berberine on the proliferation and activation of hepatic stellate cells induced by transforming growth factor β1(TGF-β1)via regulating Hedgehog signaling pathway.Methods HSC-T6 cells were cultured and divided into blank group,TGF-β1 group,berberine low-,medium-and high-dose groups,berberine high-dose+purmorphamine(Hedgehog signaling pathway activator)group.After treatment,the proliferation of HSC-T6 cells was detected by methyl thiazolyl tetrazolium(MTT)assay.The apoptosis of HSC-T6 cells was detected by flow cytometry.The expressions of collagen I and α-smooth muscle actin(α-SMA)were detected by immunofluorescence staining.The mRNA expression levels of proliferating cell nuclear antigen(PCNA),Bcl-2 and Bcl-2 associated X protein(Bax)were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).The protein expression levels of Hedgehog signaling pathway related factors Smo,Gli-1,Shh and Ptch were detected by Western Blot.Results Compared with the blank group,the proliferation rate of HSC-T6 cells,the positive protein expressions of Collagen Ⅰ and α-SMA,the mRNA expression levels of PCNA and Bcl-2,and the protein expression levels of Smo,Gli-1,Shh and Ptch in the TGF-β1 group were significantly increased,while the apoptosis rate and the mRNA expression level of Bax were significantly decreased(all P＜0.05).Compared with TGF-β1 group,the proliferation rate of HSC-T6 cells,the expression of Collagen I and α-SMA positive protein,the mRNA expression levels of PCNA and Bcl-2,and the protein expression levels of Smo,Gli-1,Shh and Ptch in berberine low-,medium-and high-dose groups were significantly decreased,while the apoptosis rate and the mRNA expression level of Bax were significantly increased(P＜0.05)in a dose-dependent manner.Compared with the high-dose berberine group,the proliferation rate of HSC-T6 cells,the positive proteins expression of Collagen Ⅰ and α-SMA,the mRNA expression levels of PCNA and Bcl-2,and the protein expression levels of Smo,Gli-1,Shh and Ptch in the high-dose berberine+purmorphamine group were significantly increased,while the apoptosis rate and the mRNA expression level of Bax were significantly decreased(all P＜0.05).Conclusion Berberine promotes TGF-β1-induced apoptosis of hepatic stellate cells and inhibits their proliferation and activation by regulating Hedgehog signaling pathway.

Objective:To investigate the influence of curative-intent resection with textbook outcomes of liver surgery (TOLS) on long-term prognosis of gallbladder carcinoma (GBC).Methods:The retrospective cohort study was conducted. The clinicopathological data of 824 patients with GBC in the national multicenter database of Biliary Surgery Group of Elite Group of Chinese Journal of Digestive Surgery, who were admitted to 15 medical centers from January 2014 to January 2021, were collected. There were 285 males and 539 females, aged (62±11)years. According to the evalua-tion criteria of TOLS, patients were divided into those who achieved TOLS and those who did not achieve TOLS. Measurement data with normal distribution were represented as Mean± SD, and com-parison between groups was conducted using the independent sample t test. Measurement data with skewed distribution were represented as M( Q1, Q3), and comparison between groups was conducted using the Mann-Whitney U test. Count data were described as absolute numbers, and comparison between groups was conducted using the chi-square test. Comparison of ordinal data were conduc-ted using the Mann-Whitney U test. The Kaplan-Meier method was used to calculate survival rate and draw survival curve, and the Log-rank test was used for survival analysis. The COX stepwise regression model with backward Wald method was used for univariate and multivariate analyses. Results:(1) Achievement of TOLS. Of the 824 patients undergoing curative-intent resection for GBC, there were 510 cases achieving TOLS and 314 cases not achieving TOLS. (2) Follow-up. Of the 824 patients undergoing curative-intent resection for GBC, after excluding 112 deaths within 90 days after discharge, 712 cases were included for the survival analysis. The median follow-up time, median overall survival time and 5-year overall survival rate of the 510 patients achieving TOLS were 22.1(11.4,30.1)months, 47.6(30.6,64.6)months and 47.5%. The median follow-up time, median overall survival time and 5-year overall survival rate of the 202 patients not achieving TOLS were 14.0(6.8,25.5)months, 24.3(20.0,28.6)months and 21.0%. There was a significant difference in overall survival between patients achieving TOLS and patients not achieving TOLS ( χ2=58.491, P<0.05). (3) Analysis of factors influencing prognosis of patients. Results of multivariate analysis showed that TOLS, carcinoembryonic antigen (CEA), CA19-9, poorly differentiation of tumor, T2 stage of eighth edition of American Joint Committee on Cancer (AJCC) staging, T3 and T4 stage of eighth edition of AJCC staging, N1 stage of the eighth edition of AJCC staging, N2 stage of the eighth edition of AJCC staging, adjuvant therapy were independent factors influencing overall survival time of patients undergoing curative-intent resection for GBC ( hazard ratio=0.452, 1.479, 1.373, 1.612, 1.455, 1.481, 1.835, 1.978, 0.538, 95% c onfidence interval as 0.352-0.581, 1.141-1.964, 1.052-1.791, 1.259-2.063, 1.102-1.920, 1.022-2.147, 1.380-2.441, 1.342-2.915, 0.382-0.758, P<0.05). Conclusion:Patients under-going curative-intent resection for GBC with TOLS can achieve better long-term prognosis.