Objective:To investigate the dynamic changes of cellular miRNA expression profiles in EBV latently infected Raji cells upon reactivation with Phorbol ester (TPA).Methods:Total RNA was extracted using TRIzol reagent from Raji cells treated with TPA at different time points (0 h, 24 h, 48 h). Small RNA libraries were constructed and sequenced on an Illumina SE50 platform. Differentially expressed miRNAs were identified, and their target genes were predicted and functionally annotated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out through online tools. Additionally, miRanda and RNAhybrid software were used to predict cellular miRNAs targeting the EBV genome. Real-time RT-qPCR was employed to validate the expression levels of differentially expressed novel miRNAs.Results:High-throughput sequencing identified 1 301 celluar miRNAs, comprising 1 189 known and 112 novel miRNAs. A total of 264 known differentially expressed cellular miRNAs and 13 novel miRNAs were identified through high-throughput miRNA sequencing. Secondary structure prediction revealed that the novel miRNAs exhibited typical pre-miRNA hairpin structures. Stem-loop quantitative real-time PCR (RT-qPCR) validation of Novel_miR_183 and Novel_miR_242 did not exhibit a statistically significant difference ( F=1.407, P=0.370 7 for Novel_miR_183; F=1.277, P=0.397 0 for Novel_miR_242) between the TPA-stimulated and untreated groups. Target gene prediction analysis revealed that the differentially expressed cellular miRNAs were involved in various important biological processes and signaling pathways. Furthermore, 1 189 known cellular miRNAs and 108 novel miRNAs were predicted to target the EBV genome. Conclusions:Treatment of Raji cells with TPA stimulation successfully reactivated Raji cells and significantly altered their miRNA expression patterns. Differentially expressed miRNAs were identified, suggesting that these miRNAs probably play crucial roles in regulating EBV infection and replication by directly targeting the EBV genome.

Objective:By analyzing the disease spectrum and composition of inpatients in general surgery department of a children′s hospital in Beijing from January 2018 to June 2024, to understand the impact of the COVID-19 on the number of inpatients in children′s hospitals and the composition of disease types, and to provide scientific evidence for hospital capacity building and medical response after the outbreak of the epidemic.Methods:The hospitalization medical records of children in the general surgery ward of the children′s hospital from January 1, 2018 to June 30, 2024 were collected. SPSS 22.0 software and EXCEL tools were used to analyze the ranking and composition of general surgery diseases in the hospital from three dimensions: different years, different genders, and different age groups. At the same time, the source of children in the hospital was counted by province.Results:Overall, the total number of hospitalized children has shown a significant decrease since 2020, and has since slowly rebounded; Acute appendicitis, inguinal hernia, and common bile duct cyst rank among the top three diseases annually; From a gender perspective, inguinal hernia and acute appendicitis are the most common in male children, while acute appendicitis and common bile duct cysts are the most common in female children; In terms of age, the 1-year-old group has the highest number of patients with inguinal hernia, while the 5-year-old and 10-18-year-old groups have the highest number of patients with acute appendicitis; Compared with 2018-2019, the number of hospitalized children with inguinal hernia decreased sharply in the first half of 2020-2024, and the first priority disease changed from inguinal hernia to acute appendicitis.Conclusions:This study analyzed the disease spectrum and composition of the children′s hospital before and after the COVID-19 epidemic, which has important reference value for the capacity building, resource allocation and epidemic response of the children′s hospital.

Objective:To investigate the effect of nuclear factor erythroid-2 related factor (Nrf2) overexpression on high-risk human papillomavirus type 16 (HPV16) E6 protein.Methods:The pRK5-FLAG-NFE2L2 plasmid was constructed, and pRK5-FLAG-NFE2L2 and pRK5-HA-HPV16E6 plasmids were co-transfected in HEK293T cells, and the expression of the two proteins was detected by Western blotting (WB). Next, pRK5-HA-HPV16E6 and pRK5-FLAG-Nrf2 plasmids were expressed using an in vitro transcription system to observe the expression of both. Finally, pEGFP-HPV16E6 and pLVX-mCherry-Nrf2 plasmids were co-transfected in HEK293T cells, and the cellular localization of the E6 protein and the Nrf2 protein was observed using fluorescence microscopy.Results:Nrf2 protein was successfully overexpressed in HEK293T cells, and the WB result showed that Nrf2 inhibited HPV16 E6 protein expression in a significant dose-dependent manner. The expression of HPV16 E6 protein in the TNT Quick Coupled Transcription/Translation Systems was affected by Nrf2, while the expression of HPV16 E6 in TnT SP6 High-Yield Wheat Germ Protein Expression System was no longer inhibited by Nrf2. Fluorescence imaging further showed no intracellular co-localization of Nrf2 and HPV16 E6.Conclusions:Overexpression of the nuclear transcription factor Nrf2 reduces HPV16 E6 protein expression, but there is no intracellular co-localization of them.

Objective:To investigate the value of ultra micro angiography（UMA）imaging in evaluating the inflammation activity of pediatric Crohn's disease（CD）patients.Methods:In this prospective study，71 pediatric CD patients confirmed by clinical symptoms，laboratory and ileocolonoscopical results who planed to accept infliximab injection treatments in Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine from April to December 2024 were included. The CD patients were included and divided into 3 groups according to the pediatric Crohn's disease activity index（PCDAI）score：inactive CD group（ n=44），mild CD group（ n=17），and moderate-to-severe CD group（ n=10）. Before treatment，pediatric CD patients underwent B mode ultrasound（BMUS），the parameters such as bowel wall thickness（BWT），bowel wall stratification，mesenteric lymph nodes（MLNs），and existence of mesenteric fat hypertrophy（MFH）were recorded. Color Doppler flow imaging（CDFI）and UMA were used to evaluate the blood flow in the most thickened diseased bowel wall using Limberg scores. The diffierences of the above parameters among the 3 groups were compared. The diagnostic performance of UMA and CDFI in assessing the inflammatory activity of pediatric CD was evaluated. Results:There were statistically significant differences in BWT among the 3 groups（all P<0.05）. However，there were no statistically significant differences in MLNs among the 3 groups（all P>0.05）. Compared with the inactive CD，there were statistically significant differences in bowel wall stratification and MFH in both the mild and moderate-to-severe CD groups（all P<0.05）. When assessing bowel wall blood flow signals using CDFI，there was no statistically significant difference in blood flow signals in the bowel wall of the mild CD compared with the inactive CD（ P>0.05）. However，the blood flow signals in the bowel wall of the moderate-to-severe CD was significantly increased（ P<0.05）. There was also statistically significant difference in blood flow signals in the bowel wall between the mild and moderate-to-severe CD groups（ P<0.05）. When assessing bowel wall blood flow using UMA，there were statistically significant differences in blood flow signals in the bowel wall among all 3 groups（all P<0.05）. As the degree of inflammation increased，the blood flow signals in the bowel wall also increased significantly. ROC analysis revealed that diagnostic performance of the UMA-BMUS model was significantly better than that of the CDFI-BMUS model（AUC 0.945 vs. 0.873，sensitivity 88.9% vs. 74.1%，specificity 97.7% vs. 95.5%；all P<0.05）. Conclusions:By sensitively depicting the low-velocity and tiny blood flow signals of the inflamed bowel wall，UMA is an effective noninvasive imaging method to make semi-quantitative evaluation of the activity of pediatric Crohn's disease.

Objective:To investigate how age and nail-tract volumetric bone mineral density (vBMD) are associated with postoperative mechanical performance of proximal femoral nail anti-rotation (PFNA-Ⅱ) fixation for geriatric intertrochanteric fractures using a finite-element analysis.Methods:Fifteen elderly patients with intertrochanteric fracture of the femur were selected for this study. They were 11 females and 4 males and divided into 5 groups based on their ages ( n=3): 55-year-old, 65-year-old, 75-year-old, 85-year-old, and 95-year-old groups. After three-dimensional models of the proximal femur were constructed using the preoperative CT data of their healthy contralateral hip, 31-A1.2 fractures of the AO/OTA type were created and PFNA-Ⅱ fixations simulated. Two loading schemes were created: graded quasi-static axial loads (700 N, 1,400 N, 2,100 N, and 2,800 N) were applied to compute equivalent plastic strain volumes in the femoral head region; displacement-controlled loading was applied to failure to derive load-displacement curves for stiffness and the maximum failure load. Nail-tract vBMD and regional hip vBMDs were measured by quantitative CT. Pearson correlation analysis was conducted to investigate the associations of age and nail-tract vBMD with the aforementioned mechanical indicators. Results:Under the same load, compared with the 55-year-old, 65-year-old, and 75-year-old groups, the plastic strain unit volumes of the fracture models in the 85-year-old and 95-year-old groups increased significantly. Under a load of 700 N, no plastic strain was observed in the fracture models in the 55-year-old, 65-year-old, and 75-year-old groups, while an average plastic strain of approximately 50 mm 3 was observed in the fracture models in the 85-year-old group. Under a load of 2,800 N, the high strain areas in the fracture models in the 85-year-old and 95-year-old groups were mainly concentrated at the tip of the head nail and the junction between the head nail and the main nail. Load-displacement curves showed a marked reduction in the failure load in patients aged ≥85 years. Under loads of 1,400 N, 2,100 N, and 2,800 N, there was a strong association between the nail-tract vBMD and the volume of the plastic strain unit ( r=-0.82, -0.88, -0.89, respectively), which was stronger than those for total-hip vBMD. Conclusions:Finite-element analysis indicates that age and nail-tract vBMD are closely related to local plastic strain and overall stiffness of the proximal femur after PFNA-Ⅱ fixation for the geriatric intertrochanteric fractures. Patients aged ≥85 years old are more prone to plastic yielding, which compromises fixation stability.

Objective To investigate the capacity of low-intensity pulsed ultrasound(LIPUS)for promoting osteogenic differentiation of senescent bone marrow mesenchymal stem cell(BMMSC)in mice,also the potential mechanism of involving myocardin-related transcription factor-A(MRTF-A)during this process.Methods Five aged mice received LIPUS irradiation on the left femur for eight weeks.Micro CT and HE staining were employed to evaluate bone status of bilateral femur.Bone marrow cells were isolated from mouse long bones,BMMSC were cultured,and the cells were divided into H2O2+LIPUS group,H2O2 group and control group.Mouse BMMSC were subjected to H2O2 for 3 days,followed by reactive oxygen species fluorescence staining and senescence-associated β-galactosidase staining to assess cellular senescence.Immunofluorescence and phalloidin staining were performed to examine MRTF-A distribution and cytoskeletal status.Based on H2O2-induced senescence model,MRTF-A nuclear translocation inhibitor CCG-100602 was applied,and the cells were further divided into H2O2+CCG-100602+LIPUS group,H2O2+CCG-100602 group,H2O2+LIPUS group and H2O2 group.Immunofluorescence staining was used to assess MRTF-A distribution.Alkaline phosphatase(ALP)and alizarin red staining were conducted to evaluate osteogenic differentiation capacity.Results LIPUS resulted in increased trabecular bone in the left femur of aged mice compared to the right femur,with increased bone volume to total volume and trabecular number and decreased trabecular spacing(both P＜0.05).Compared with H2O2 group,H2O2+LIPUS group showed increased nuclear expression of MRTF-A and increased intracellular rigid actin filament density.The nuclear expression of MRTF-A in H2O2+CCG-100602+LIPUS group was significantly lower than that in H2O2+LIPUS group.Compared with the H2O2+CCG-100602+LIPUS group and H2O2 group,the ALP and alizarin red staining positive areas in H2O2+LIPUS group were significantly larger.Conclusion LIPUS could improve osteoporosis in aged mice.Nuclear translocation of MRTF-A was a key regulator to LIPUS for promoting osteogenic differentiation of senescent BMMSC.

Objective:To establish a protocol for virus isolation using the mixed method, and evaluate the efficacy of the suspended method and the mixed method in isolating herpes simplex virus (HSV).Methods:Simulated HSV-infected clinical samples were prepared using HSV-1 F strain and CDC-P1 strain. Both the suspended method and the mixed method were used to isolate HSV-1 from these samples. The virus isolation efficiency of the mixed method under various conditions was assessed. These conditions included different multiplicity of infection (MOI), cell seeding densities, and virus adsorption times.The 50% tissue culture infective dose (TCID 50) assay was used for the assessment. The positive rates of virus detection under low viral load conditions were compared between the two methods. Results:Under the conditions of a MOI of 0.005, a virus adsorption time of 15 min, and a cell seeding density of 1×10 6 cell/ml, the mixed method achieved effective isolation of HSV-1. When the virus titer of the sample was 100 TCID 50/ml, the positivity rate of the mixed method reached 100.0%, while the positivity rates of the suspended method were 50.7% (38/75) and 52.0% (39/75) after cultured for 72 h and 96 h, respectively. When the virus titer of the sample was 10 TCID 50/ml, the positivity rate of the mixed method was 100.0%, while the positivity rate of the suspension method was 0. Conclusions:The mixed method exhibits significantly higher efficiency in HSV isolation compared with the suspended method. Under the conditions of high viral load, both the suspended method and the mixed method can be effective in isolating HSV-1. For clinical samples with low viral loads, the mixed method has greater applicability.

Objective:To investigate the correlation between mutations in KRAS, NRAS, BRAF, and PIK3 CA and the clinical characteristics of elderly colorectal cancer(CRC)patients. Methods:Paraffin-embedded tissue samples were obtained from 191 elderly CRC patients who consulted at Huadong Hospital, affiliated to Fudan University, between January 2022 and July 2023.Following deoxyribonucleic acid(DNA)extraction, the amplification refractory mutation system polymerase chain reaction(ARMS-PCR)was employed to detect the mutation profiles of KRAS, NRAS, BRAF, and PIK3 CA.Concurrently, serum samples collected prior to radical resection were analyzed for carcinoembryonic antigen(CEA), carbohydrate antigen 19-9(CA19-9), and carbohydrate antigen 72-4(CA72-4)using electrochemical luminescence.A comparative analysis of the clinical characteristics and preoperative serological tumor marker concentrations among patients with different gene mutations was conducted to elucidate their correlation. Results:A total of 191 elderly CRC patients were enrolled in the study, with ages ranging from 60 to 94 years(mean age 72.1±7.8 years), including 112 males.The mutation rate of KRAS, NRAS, BRAF, and PIK3 CA, as determined by combined detection, was found to be 49.21%(94/191)among elderly CRC patients. KRAS exhibited the highest mutation rate at 35.08%, with statistically significant differences observed in gender, primary site, degree of differentiation, and neurovascular invasion between patients with and without KRAS mutations( P<0.05 for all comparisons).The BRAF mutation rate was 8.90%, and significant differences in gender, age, primary site, and degree of differentiation were also noted between patients with and without BRAF mutations( P<0.05 for all).The mutation rates for NRAS and PIK3 CA were 2.62% and 5.24%, respectively, with no statistically significant differences in the clinical characteristics of patients across different groups( P>0.05 for all).Additionally, the proportion of patients over the age of 90 in the double mutation group was significantly higher( P<0.01).Significant differences in serum CA19-9 concentrations were observed among the various mutation types( P<0.05). Conclusions:There are notable differences in age, gender, primary site, degree of differentiation, and neurovascular invasion among elderly CRC patients with varying mutation statuses of KRAS, NRAS, BRAF, and PIK3 CA.Patients with double mutations exhibited higher concentrations of CA19-9 in preoperative serum.

Sacroiliac complex injuries are commonly seen in high-energy pelvic fractures. The injuries make a big difference in treatment patterns due to the diverse injury types, posing considerable challenges in formulating optimal treatment strategies, and hence are persistent clinical difficulties in orthopedic trauma. The clinical management of sacroiliac complex injuries presents several key challenges such as a non-negligible rate of missed diagnoses in associated vascular and visceral injuries, absence of standardized protocols for surgical approaches and reduction-fixation strategies across different injury patterns, and ongoing controversies regarding surgical indications and optimal timing for patients combined with concomitant lumbosacral plexus injuries. Currently, no systematic clinical guidelines are available for the diagnosis and treatment of sacroiliac complex injuries both domestically and internationally. To this end, the Pelvic and Acetabular Surgery Group, Orthopedic Branch, China International Exchange and Promotive Association for Medical and Health Care and Orthopedic Physician Branch, Chinese Medical Doctor Association organized a panel of domestic experts in the field to develop the Clinical guideline for the diagnosis and treatment of sacroiliac complex injuries ( version 2025), based on evidence-based medicine and adhering to the principles of scientific rigor, clinical applicability, and innovation. These guidelines provided 11 recommendations covering diagnosis, therapeutic principles and techniques, management protocols for lumbosacral plexus injuries, outcome evaluation, and postoperative rehabilitation pathways, etc., aiming to standardize the clinical management of sacroiliac complex injuries.

Objective To investigate the correlation between the clinical characteristics of allergic rhinitis(AR)in adults located in Shenzhen area and the traditional Chinese medicine(TCM)constitutions.Methods A cross-sectional survey of adult AR patients in Shenzhen was conducted.From June 2022 to December 2023,adult AR patients admitted to the Department of Otolaryngology outpatient clinic of Bao'an Traditional Chinese Medicine Hospital Affiliated to Guangzhou University of Chinese Medicine(Shenzhen Bao'an District Hospital of Traditional Chinese Medicine)were selected as the study subjects.The clinical baseline data,Total Nasal Symptoms Score(TNSS),and Total Non-Nasal Symptoms Score(TNNSS)of the patients were collected for the analysis of clinical characteristics,and TCM constitutions were differentiated based on the information collected by TCM constitutions questionnaire.After that,the correlation between the biased TCM constitutions and clinical characteristics were explored by using analysis of variance,non-parametric test and Logistic regression analysis.Results(1)A total of 215 cases of adult AR patients from Shenzhen were included.Most of the patients were male(60.93％),and the patients were usually aged 18-29 years old(accounting for 48.84％).AR in adults often arose in the spring(33.49％)and fall(30.70％).(2)Lung qi deficiency-cold syndrome was the predominated TCM syndrome type of AR in adults,accounting for 40.93％,and then came kidney yang deficiency syndrome(26.98％),spleen qi deficiency syndrome(20.00％),and heat accumulation in lung meridian syndrome(12.09％).Lung qi deficiency-cold syndrome and heat accumulation in lung meridian syndrome were more common in young males,and kidney yang deficiency syndrome was frequently seen in middle-aged and elderly males.(3)The leading three TCM constitution types of AR in adults were qi deficiency constitution(30.23％),yang deficiency constitution(24.65％),and inherited special constitution(15.81％).In adult AR patients with lung qi deficiency-cold syndrome,the commonly-seen TCM constitution types were qi deficiency constitution,inherited special constitution,and balanced constitution.In adult AR patients with spleen qi deficiency syndrome,the commonly-seen TCM constitution types were inherited special constitution,phlegm-damp constitution,and yang deficiency constitution.In adult AR patients with kidney yang deficiency syndrome,the commonly-seen TCM constitution types were yang deficiency constitution,qi stagnation constitution,and inherited special constitution.In adult AR patients with heat accumulation in lung meridian syndrome,the commonly-seen TCM constitution types were damp-heat constitution,yin deficiency constitution,and blood-stasis constitution.The leading five TNSS scores were shown in adult AR patients with inherited special constitution,qi deficiency constitution,yang deficiency constitution,phlegm-damp constitution,and qi stagnation constitution,respectively.And the leading five TNNSS scores were shown in adult AR patients with yang deficiency constitution,inherited special constitution,qi stagnation constitution,yin deficiency constitution,and damp-heat constitution,respectively.(4)The results of statistical analysis showed that the differences in gender among the adult AR patients with various TCM constitution types were not statistically significant(P＞0.05),but the differences in age groups,TCM syndrome types,TNSS scores and TNNSS scores were statistically significant(P＜0.01 or P＜0.001).(5)The results of Logistic regression analysis showed that the TNSS scores and TNNSS scores were all positively correlated with qi deficiency constitution,yang deficiency constitution,and inherited special constitution,and spleen qi deficiency syndrome was negatively correlated with damp-heat constitution,the differences being all statistically significant(P＜0.01 or P＜0.001).Conclusion The onset of AR in adults from Shenzhen area is closely related to age,gender,season,and TCM constitutions.The adult AR patients usually have the biased constitutions,in particular qi deficiency constitution,yang deficiency constitution,and inherited special constitution.In adult AR patients,higher TNSS scores are correlated with inherited special constitution,higher TNNSS scores are correlated with yang deficiency constitution,and the primary TCM syndrome type of lung qi deficiency-cold syndrome is correlated with qi deficiency constitution.