Objective The study aims to optimize the conditions for constructing orthotopic nude mouse models of liver cancer by injecting human liver tumor cell lines and to explore appropriate timings for drug administration. Methods Human hepatocellular carcinoma Hep3B and hepatoblastoma HepG2 cell lines, which stably expressing the luciferase reporter gene (LUC), were selected. The linear correlation between the luciferase luminescence intensity and the number of liver tumor cells was analyzed using a Small Animal In Vivo Imaging system to verify the luminescent efficiency of the human liver tumor cells. Different concentrations (8×10⁶, 2.4×10⁷, 7.2×10⁷ cells/mL) and resuspension media (PBS, Matrigel) of human liver tumor cell suspensions HepG2-LUC and Hep3B-LUC were orthotopically inoculated into the liver lobes of 5-week-old female BALB/c nude mice (12 groups, 7 mice each) to construct human liver tumor nude mouse orthotopic cancer models. Every 7 days, the weights of mice were recorded, and the growth of orthotopic tumors was monitored using the Small Animal In Vivo Imaging system. On day 35 post-cell inoculation, mouse livers were dissected, and pathological slices were prepared for HE staining to observe histopathological changes in liver tissues. Results The luminescence intensity of human liver tumor cell lines was positively correlated with the number of cells (R²=0.983 1, R²=0.970 5), indicating their suitability for orthotopic model construction. Successful modeling was achieved in the high-concentration groups of HepG2-LUC, the low-, medium-, and high-concentration groups of HepG2-LUC+Matrigel, the medium- and high-concentration groups of Hep3B-LUC, and the low-, medium-, and high-concentration groups of Hep3B-LUC+Matrigel. For both HepG2-LUC+Matrigel and Hep3B-LUC+Matrigel groups, mice in the high-concentration groups exhibited significantly reduced body weight compared to the low- and medium-concentration groups (both with P<0.05). The luminescence intensity of successfully modeled mice increased exponentially over time (R²>0.950 0), and reached a minimum of 1.0×10⁷ p/(s·cm²·sr) by day 14 post-transplantation. Mice in the low- and medium-concentration groups of HepG2-LUC and the low-concentration group of Hep3B-LUC showed no significant pathological changes, while the other groups exhibited evident liver tumors and hepatocyte lesions. Conclusion For the HepG2-LUC cell line, the recommended injection volume is 50 µL with a cell density of 2.4×10⁷ cells/mL, resuspended with Matrigel, followed by drug administration or prognostic measures on day 7 post-modeling. For the Hep3B-LUC cell line, the recommended injection volume is 50 µL with a cell density of 7.2×10⁷ cells/mL, not resuspended with Matrigel, with administration or prognostic measures on day 14 post-modeling.

Objective:To establish a method for detecting the binding affinity of peptides to HLA-B*1301 by HLA molecular reconstitution after weak acid treatment,and its practicality was evaluated.Methods:C1R cells overexpressing HLA-B*1301 were treated with citrate buffer of different pH for different time.After neutralization of pH,cells were resuspended in culture medium con-taining β2m and brefeldin A.Cells were incubated at 37℃with peptide(peptide group)or without peptide(blank control),after addition of anti-HLA antibody,the cells were detected by flow cytometry.Ratio of fluorescence intensity between peptide group and blank control group was used as an index to measure the level of HLA molecular remodeling,and then represented the interactions between peptide and HLA-B*1301 molecule.Results:HLA-polypeptide complex was denatalized and dissociated after being treated with pH3.0 buffer for 1 min,and only HLA heavy chain molecules were retained on the cell surface.The addition of peptides with binding force could significantly improve the level of HLA molecular remodeling,and the binding force of peptides with HLA-B*1301 could be evaluated by this method.Conclusion:HLA molecular reconstitution assay is a simple and reliable method to detect the binding of peptides to HLA-B*1301,which can also provide reference for the study of other low frequency HLA molecular antigen pre-sentation patterns.

Objective To explore association between childhood trauma,resilience and non-suicidal self-injury in adolescents.Methods One hundred and fifty-eight first-episode adolescent patients with mood disorders were selected and divided into NSSI group(n=94)and non-NSSI group(n=64)based on presence or absence of NSSI.The Hamilton depression scale(HAMD),Hamilton anxiety scale(HAMA),childhood trauma questionnaire(CTQ-SF)and Connor-Davidson resilience scale(CD-RISC)were used to evaluate the depression and anxiety symptoms,childhood trauma and resilience.Results There were more cases of younger age(16.17±1.67 vs.16.73±1.37),lower education level(30.9% vs.15.6% ),left behind experience(48.9% vs.29.7% ),school bullying(46.8% vs.25.0% ),suicide ideation(85.1% vs.37.5% )and history of attempted suicide(29.8% vs.6.3% )in the group with NSSI compared to those without NSSI.The HAMD score(27.99±5.94 vs.24.19±5.19),HAMA score(18.02±5.94 vs.15.45±4.99),CTQ total score(48.43±15.40 vs.41.97±9.75),emotional abuse score(12.77±6.06 vs.10.19±4.06),and emotional neglect score(11.40±5.34 vs.9.14±3.55)were higher in the group with NSSI,and the differences between the two groups were significant(P<0.05).The total scores of psychological resilience(39.83±10.27 vs.28.66±12.75),resilience(19.59±4.92 vs.12.28±6.47),strength(12.03±3.98 vs.9.99±4.67),and optimism(8.98±2.97 vs.6.47±3.73)in the group without NSSI were higher than those in the group with NSSI(P<0.05).Logistic regression analysis showed that left behind experience(OR=4.494,95% CI:1.192-16.940),school bullying(OR=5.983,95% CI:1.329-26.945),suicidal ideation(OR=13.225,95% CI:2.908-60.146),history of attempted suicide(OR=16.769,95% CI:1.845-152.379),HAMD(OR=1.264,95% CI:1.046-1.626),emotional abuse(OR=1.327,95% CI:1.093-1.612),and resilience(OR=0.468,95% CI:0.266-0.823)were significantly associated with adolescent mood disorders with NSSI(P<0.05).Conclusion Left behind experience,campus bullying,suicidal ideation and attempted suicide,emotional abuse,degree of depression,and psychological resilience may be associated with NSSI behavior in adolescents with mood disorders.

To establish a simple,convenient,sensitive,and specific method for rapid detection of Zi-ka virus(ZIKV),the whole genome sequences of ZIKV isolated from different times and regions were analyzed.The specific primers and probes were designed based on the screened target se-quences located in the conserved region of the ZIKV NS5 gene.By combining RT-LAMP isother-mal amplification technology and immunochromatography technology,a reverse transcription loop mediated isothermal amplification nucleic acid and flow visualization strip(RT-LAMP-VF)detec-tion method for ZIKV was established.The results showed that the method had good specificity and sensitivity.When the ratio of inner,outer,and ring primers(FIP∶LF∶F3)was 4∶2∶1,the detection method can specifically detect 102 copies/pL RNA transcripts or 2.15 pfu ZIKV at 61 ℃for 45 minutes,with no cross reaction with other flaviviruses such as Japanese encephalitis virus and classical swine fever virus.Other RNAs in blood tissue samples did not affect the sensitivity and specificity of RT-LAMP-VF,indicating that the method can be applied to clinical practice.The ZIKV RT-LAMP-VF detection method established in this study is easy to perform and does not require special instruments and equipment.It is particularly suitable for the rapid detection of ZIKV in grassroots units,providing technical support and material support for the establishment of on-site rapid detection and early warning and prediction systems for ZIKV disease.

The PCR-amplified severe fever with thrombocytopenia syndrome virus(SFTSV)Gn-DⅢ-Ⅲ gene was inserted into the pET-30a(+)prokaryotic expression vector to generate the re-combinant plasmid pET-SFTSV-Gn-D Ⅲ-Ⅲ.The plasmid was transformed into E.coli BL21(DE3)for Gn-DⅢ-m protein expression and the expression conditions were optimized.The Gn-DⅢ-Ⅲ protein purified with Ni-NTA column affinity chromatography was applied as the captured antigen to establish an indirect ELISA method for the detection of SFTSV antibody.The results demonstrated that the recombinant plasmid pET-SFTSV-Gn-D Ⅲ-Ⅲ was successfully constructed as identified by PCR and sequencing.The recombinant protein SFTSV Gn-D m-Ⅲ was soluble ex-pression in E.coli under the optimal induction conditions of 0.4 mmol/L IPTG at 25 ℃ for 4 h,and the protein purity was 91.77％after purification by Ni-NTA column.The optimal reaction con-ditions for the indirect ELISA of SFTSV antibody were as follows:coating antigen concentration(5 μg/mL),primary antibody(incubation at 37 ℃ for 1.5 h),and secondary antibody(diluted 1:10 000 and incubated at 37 ℃ for 1 h).The established method had no cross-reactivity with Rift Valley fever virus(RVFV),Ebola virus(EBOV),and tick-borne encephalitis virus(TBEV)posi-tive sera.The method had a high sensitivity,with P/N＞2.1 for SFTSV-positive sera diluted to 81920.Coefficients of variation for intra-and inter-batch reactions were less than 10％.Detection of four SFTSV-infected human clinical serum samples showed the serum samples from patients in re-mission were tested as positive(P/N＞2.1),while serum samples from patients with multiple or-gan failure were detected as negative(P/N＜2.1).The results indicated that the SFTSV Gn-D Ⅲ-Ⅲ protein was successfully expressed and purified,and it was used as the coating protein to estab-lish an indirect ELISA assay for SFTSV antibody,which possesses good specificity,sensitivity and reproducibility.This method might be applied to detect human SFTSV clinical serum samples.

Objective To discover the host factor interacting with severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)papain-like protease(PLpro)and explore the potential mechanism.Methods The second-generation proximity-dependent biotin identification(BioID2)approach combined with mass spectrometry analysis was used to search for the potential host factors.Immunofluorescence and co-immunoprecipitation(Co-IP)assay were used to verify the interactions between DEAD-box helicase 3(DDX3)and PLpro.The influence of PLpro on DDX3-inhibitor of kappa B kinase ε(IKKε)-TANK-binding kinase 1(TBK1)and DDX3-mitochondrial antiviral signaling protein(MAVS)complexes was also investigated by Co-IP.The effect of PLpro on interferon-β(IFN-β)immune pathway and the protease activity on substrates were studied via luciferase activity assay.Results DDX3 could co-locate and interact with PLpro intracellularly.PLpro might possibly inhibit both the formation of DDX3-MAVS complex and the interactions between DDX3-IKK-ε-TBK1.PLpro could negatively regulate type Ⅰ interferon pathway.Overexpression of DDX3 could lead to a significant increase in the cleavage activity of PLpro/PLP-TM that might be significantly decreased in case of inventions with DDX3 expressions.Conclusion DDX3 may be one of the host factors that interact with SARS-CoV-2 PLpro.PLpro negatively regulates IFN-β immune pathway induced by DDX3,which may provide a favorable immune environment for virus replication.

Objective:To summarize the clinical manifestations, diagnosis, treatment and prognosis of acute flaccid myelitis (AFM) in children.Methods:Clinical characteristics of 4 AFM cases from Department of Neurology, Children′s Hospital Affiliated to Capital Institute of Pediatrics, from September 2018 to November 2022, were analyzed retrospectively.Results:The age of 4 children with AFM was 7 years, 4 years and 3 months, 7 years and 1 month, 6 years and 5 months, respectively. There were 2 boys and 2 girls. Prodromal infection status showed 3 children of respiratory tract infection and 1 child of digestive tract infection. The main manifestation was asymmetrical limb weakness after infection, and the affected limb range was from monoplegia to quadriplegia. Cranial nerve injury was involved in 1 child, no encephalopathy. Magnetic resonance imaging in the spinal cord of all 4 children showed long T1 and T2 signals, mainly involving gray matter. Cerebrospinal fluid cell-protein separation was observed in 2 children. Pathogen detected in 1 child pharyngeal swab was enterovirus D68. Antibody IgM to adenovirus was positive in the blood of 1 child. Antibody IgG against Echo and Coxsackie B virus were positive in the blood of another child. After glucocorticoid, human immunoglobulin or simple symptomatic treatment and at the same time under later rehabilitation training, muscle strength recovered to different degrees, but there were disabilities left in 3 children.Conclusions:AFM should be considered in children with acute and asymmetrical flaccid paralysis accompanied by abnormal magnetic resonance imaging signal in the central region of spinal cord, especially post-infection. The effective treatment is limited and the prognosis is poor.

Objective:To investigate the protective effects of a sunscreen lotion containing Calendula extracts on children′s skin against sun exposure, as well as to evaluate its safety and tolerability when applied to children.Methods:A single-center, randomized, split-body/face study was conducted on 200 healthy children aged 3 - < 18 years, who were enrolled from Beijing Children′s Hospital, Capital Medical University from July to August 2022. The participants were randomly and equally divided into Group A (the left side of the body/face topically treated with the test sunscreen, and the right side with the control sunscreen) and Group B (the right side of the body/face topically treated with the test sunscreen, and the left side with the control sunscreen) at a ratio of 1∶1. After applying the sunscreen, they were engaged in outdoor activities under sunlight. Skin tests were conducted on the temporal area, the extensor aspect of the upper arm and forearm before and after sun exposure. The test product was a mild sunscreen lotion containing Calendula extracts with the sun protection factor (SPF) being 50+ and the long-wave ultraviolet protection factor (PA) being +++, and the control product was a baby sunscreen containing licorice extracts (SPF35, PA++). Bilateral differential scales were used to assess clinical symptoms after sun exposure, erythema values to clinically evaluate erythema after sun exposure, and the multifunctional skin testing platform MPA10 to measure melanin and erythema values, stratum corneum hydration, and transepidermal water loss (TEWL) at the tested sites. Related adverse events were observed and recorded during the study. The paired t-test or Wilcoxon signed-rank test was used for the comparison of quantitative data, and chi-square test (Fisher′s exact test) for the comparison of count data. Results:Totally, 198 children completed the study and visits, including 100 males (50.5%) and 98 females (49.5%), aged from 3 to 17 years (8.11 ± 0.23 years), and there were 99 cases each in the Group A and Group B. The numbers of participants with more obvious sunburn symptoms after sun exposure in the 3 tested areas were all higher on the control side than on the test side (the temporal area: 11 cases vs. 4 cases; the extensor aspect of the upper arm: 16 cases vs. 2 cases; the extensor aspect of the forearm: 33 cases vs. 3 cases), with significant differences between the bilateral sides (all P<0.001). No significant differences were observed in the erythema values between the test side and control side in the 3 tested areas (all P > 0.05). In the extensor aspect of the upper arm and forearm, the difference in the melanin value before and after sun exposure was significantly smaller on the test side (3.57 ± 2.41, 1.74 ± 1.68, respectively) than on the control side (9.50 ± 2.21, 8.13 ± 1.87, respectively, both P < 0.001) ; in the temporal region and the extensor aspect of the upper arm and forearm, the difference in the stratum corneum hydration level before and after sun exposure was significantly greater on the test side (7.72[-2.19, 19.44], 9.56 ± 1.37, 9.05 ± 1.37, respectively) than on the control side (-3.25[-13.54, 9.94], 3.63 ± 1.32, 3.73 ± 1.31, respectively, all P < 0.001) in the temporal region and the extensor aspect of the upper arm and forearm. However, there were no significant differences in the changes in the erythema or TEWL values before and after sun exposure between the test side and control side in either of the 3 tested areas (all P > 0.05). During this study, 1 case (0.51%) experienced transient urticaria on the control side, and no serious adverse events occurred. Conclusion:The mild sunscreen lotion containing Calendula extracts demonstrated superior efficacy to the control product in improving skin symptoms after sun exposure such as hyperpigmentation among healthy children aged 3 - < 18 years, with good tolerability and a relatively low incidence of adverse reactions.

Objective To establish a method and study the pharmacokinetics for concentration determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma By LC-MS/MS. Methods The mobile phase was methanol-water (0.1% formic acid) system under the positive ion mode of C18 chromatographic column, gradient elution was adopted, and the flow rate was 0.3 ml/min. In the negative ion mode, the mobile phase was acetonitrile-water (0.1% formic acid) system, gradient elution, with a flow rate of 0.4 ml/min. Caffeic acid, rosmarinic acid, syringic acid, rutin in positive ion mode and Atractylodes lactone II and Atractylodes lactone III in negative ion mode were respectively determined. Normal rats were intragastrically given Xiakucao Xiaoliu Mixture 7.8 ml/kg, and blood was taken from the orbit at different time points after the administration. The blood concentration was determined by the validated LC-MS/MS method and the non-standard DAS2.0 software was used. The pharmacokinetic parameters of rats after administration were calculated by the compartment model. Results The pharmacokinetic parameters belonged to non atrioventricular model. The pharmacokinetic characteristics of the four main anti-cancer active ingredients of Caffeic acid, Rosmarinic acid, Syringic acid and Atractylodes Ⅲ in rats after administration of Xiakucao Xiaoliu Mixture were significantly different from those reported in the literature after the administration of monomers. Conclusion The established method was simple, accurate and sensitive, which could be suitable for the content determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma, which would be a valuable information for the study on main anticancer active substances.

ObjectiveWe conducted a two-sample Mendelian randomization （MR） study to assess the causal relationship between circulating isoleucine， leucine and valine levels and the risk of peripheral atherosclerosis. MethodsBased on the large-scale genome-wide association study （GWAS） database， single nucleotide polymorphisms （SNPs） closely related to the circulating levels of isoleucine， leucine and valine were identified as instrumental variables （IVs）. Two-sample MR analysis applying the inverse variance weighted （IVW） method and the weighted median estimator （WME） method were performed to estimate the causal relationship between the risk of peripheral atherosclerosis and the exposure with more than three SNPs that were available as IVs. The pleiotropy was evaluated by using the MR-Egger regression and MR-PRESSO method， and the leave-one-out method was used in sensitivity analysis. ResultsFour， one and one SNPs were identified as IVs for circulating isoleucine， leucine and valine levels， respectively. For isoleucine， the IVW model demonstrated there was no evidence of heterogeneity among the IVs （P=0.715）， and there was a significant causal relationship between the increase of circulating isoleucine level and a higher risk of peripheral atherosclerosis risk. Per every 1 elevated standard deviation （SD） of circulating isoleucine level resulted in increasing 31% of peripheral atherosclerosis risk （OR=1.31， 95%CI： 1.07‒1.61）. Similarly， the OR（95%CI） was 1.33 （1.04‒1.71） in the WME model. The MR-Egger regression and MR-PRESSO analysis indicated no evidence of pleiotropy in IVs （all P>0.05）. The result of the leave-one-out sensitivity analysis was stable. The Wald ratio model displayed that the causal relationship between circulating leucine and valine levels and the risk of peripheral atherosclerosis was not statistically significant. The OR （95%CI） for leucine and valine was 1.13 （0.78‒1.63） and 1.11 （0.82‒1.50）， respectively. ConclusionThere is a significant causal relationship between the increase of circulating isoleucine level and a higher peripheral atherosclerosis risk. The causal relationships between circulating leucine and valine levels and the risk of peripheral atherosclerosis need to be further confirmed in future studies.