Objective:To construct a full-length infectious clone of enterovirus D68（EV-D68）expressing enhanced green fluorescent protein（EGFP）by reverse genetics in order to provide an efficient tool for studying the biological characteristics and screening antiviral drugs for EV-D68.Methods:Gene synthesis and overlap PCR techniques were used to construct the full-length clone plasmid pUC57-EV-D68 of EV-D68. The full-length viral sequence was then transferred into the pCAGGS plasmid to obtain the pCAGGS-EV-D68 plasmid. The EGFP gene was amplified and inserted into the pCAGGS-EV-D68 plasmid to construct the pCAGGS-EGFP-EV-D68 plasmid. Then，the two constructed plasmids were transfected into human rhabdomyosarcoma（RD）cells to rescue recombinant viruses RV-EV-D68 and RV-EGFP-EV-D68. The rescued viruses were identified using PCR，Western blot，and immunofluorescence techniques. The antiviral effect of doxycycline was evaluated using the rescued RV-EGFP-EV-D68. Statistical analysis was performed using the two independent samples t-test. Results:The recombinant virus RV-EGFP-EV-D68 capable of expressing EGFP was successfully rescued. Even after 15 serial passages，the virus retained EGFP expression with no significant difference in viral titers compared to the parental virus，indicating its stable passage in RD cells. Besides，the rescued strains exhibited similar replication characteristics to the parental virus. While at 24 and 36 h after infection，the titers of the rescued strains were significantly lower than that of the parental strain（both P<0.05）. This study demonstrated that doxycycline significantly reduced the fluorescence intensity of RV-EGFP-EV-D68-infected RD cells（ P<0.01）. Meanwhile，a negative correlation was observed between the doxycycline concentration and the fluorescence intensity，indicating that the rescued virus could be used for antiviral drug evaluation. Conclusions:This study successfully constructs an infectious clone of EV-D68 expressing EGFP. The rescued recombinant virus RV-EGFP-EV-D68 has been verified to be applicable for the evaluation of antiviral drugs.

Objective:To compare the clinical efficacy of unilateral puncture and bilateral puncture vertebroplasty in osteoporotic compression fracture patients.Methods:A regression comparative analysis was performed using case-control study to collect clinical data of 372 patients with osteoporosis compression fractures admitted to Juxian People′s Hospital from January 2022 to July 2023, including 213 males and 159 females, and the age was (59.8±6.2) years old, aged range of 50 to 70 years old. 194 patients treated with unilateral puncture vertebroplasty were included in the unilateral group, and 178 patients treated with bilateral puncture vertebroplasty were included in the bilateral group. The intraoperative indicators, vertebral changes before surgery and 6 months after surgery, and the occurrence of adverse reactions after surgery of the two groups of patients were compared. The visual analogue scale (VAS), activities of daily living (ADL) score, oswestry disability index (ODI), and quality of life (QOL) scale were used to compare the pain degree, activity ability, improvement of functional disability, and changes in quality of life and clinical efficacy of the two groups of patients before the operation and at 3 and 6 months after the operation. SPSS 21.0 software was conducted to analyze data. Measurement data with normal distribution were represented as mean±standard deviation( ± s), and the comparison between groups was conducted using the t-test. The comparison of count data were represented as n(%) and was conducted by χ2 test or Fisher exact probability. Between-group comparisons of different time intervals were performed with repeated-measures analysis of variance test. Results:The surgical time, cement dosage, bleeding amount, and fluoroscopy of patients in the unilateral group were (29.86±5.87) min, (2.58±0.37) mL, (22.65±4.83) mL, and 12 times, respectively, and (42.63±4.55) min, (7.12±0.61) mL, (50.31±5.84) mL, 25 times in the bilateral group, respectively. The difference between the two groups was statistically significant ( P<0.05). Preoperatively, the anterior height ratio of the vertebral body, sagittal concavity angle, and kyphosis angle of the affected vertebra in the unilateral group were (59.83±5.55)%, 13.04°±1.14°, and 18.93°±2.56°, respectively, while in the bilateral group, there were (60.28±5.79)%, 13.08°±1.13°, and 18.67°±2.69°, respectively. After the operation, the unilateral group were (90.20±5.52), 3.95°±0.57°, and 11.03°±1.21°, respectively. The bilateral group were (90.58±6.00), 3.99°±0.59°, and 10.91°±1.14°, respectively. Comparison of data before and after surgery between the two groups, the difference was statistically significant ( P<0.05). The overall complication rate of unilateral group was 13 cases (6.70%), and the overall complication rate of bilateral group was 26 cases (14.61%). The difference between the two groups was statistically significant ( P=0.013). The VAS, ADL, ODI, and QOL of the unilateral group were (3.49±1.10), (66.10±13.02), (18.22±4.81) and (62.10±10.00) points, the two-sided groups were (5.49±1.13), (56.19±10.27), (24.76±5.81) and (52.13±9.56) points, respectively. The differences between the two groups were statistical significance ( P<0.05), the overall treatment efficiency of patients in the unilateral group was 90.72%, and the overall treatment efficiency of patients in the bilateral group was 83.15%, and the difference between the two groups was statistically significant( P<0.05). Conclusions:Unilateral puncture vertebroplasty has the advantages of short operation time, less bone cement usage, low blood loss, fewer fluoroscopy times, low complication rate, better postoperative pain relief and functional recovery, and high overall treatment efficiency in the treatment of patients with osteoporotic compression fractures. It has obvious advantages over bilateral puncture vertebroplasty.

ObjectiveThis study aimed to investigate the intervention effect of Renqing Mangjue on the malignant transformation of gastric mucosal epithelial cells induced by N-methyl-N′-nitro-N-nitrosoguanidine （MNNG） and to explore its molecular mechanism in preventing precancerous lesions of gastric cancer based on the cyclic guanosine monophosphate （cGMP）/protein kinase G （PKG）/mitogen-activated protein kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodsHuman gastric mucosal epithelial cells （GES-1） were initially induced by MNNG to establish a precancerous cell model （MC cells）. The effective concentration of MNNG for inducing malignant transformation in GES-1 cells was screened using the cell proliferation activity decection （CCK-8） assay， and the effective concentration of Renqing Mangjue for inhibiting the proliferation of transformed GES-1 cells was also determined. GES-1 cells were divided into a blank control group， a model group， and treatment groups with Renqing Mangjue at concentrations of 1， 3， 10， and 30 mg·L^-1. Furthermore， the effects of Renqing Mangjue on the migratory ability and epithelial-mesenchymal transition （EMT） characteristics of GES-1 malignant transformed cells were evaluated using Transwell migration assays， wound healing assays， and real-time quantitative reverse transcription polymerase chain reaction （Real-time PCR）. Additionally， candidate chemical components and target sites of Renqing Mangjue were obtained from the TCMIP v2.0 database， and disease targets at various stages of gastric cancer precursors were sourced from the Gene Expression Omnibus （GEO） database. Pathway enrichment analysis was performed using the Metascape database to predict the potential mechanisms of action of Renqing Mangjue. Finally， the protective mechanism of Renqing Mangjue against gastric cancer precursors was validated through Western blot analysis. ResultsAt a concentration of 20 μmol·L^-1， MNNG exhibited an inhibition rate of approximately 50% on GES-1 cells （P<0.01）， and at this concentration， the GES-1 cells displayed biological characteristics indicative of malignant transformation. In contrast， Renqing Mangjue had no significant effect on the proliferation of normal GES-1 cells， but significantly inhibited the proliferation of MC cells （P<0.01） and markedly reduced their migratory capacity （P<0.01）. Moreover， it also increased the mRNA expression level of E-cadherin during the EMT process （P<0.05）， while inhibiting the expression of both N-cadherin and the transcription factor Snail mRNA （P<0.05， P<0.01）. Network predictions suggested that Renqing Mangjue may prevent gastric cancer precursors through modulating the cGMP/PKG and MAPK/ERK signaling pathways. Furthermore， Western blot results indicated that Renqing Mangjue upregulated the expression of PKG and NPRB （B-type natriuretic peptide receptor） proteins in the cGMP/PKG pathway （P<0.01）， while downregulating the expression of the downstream proteins MEK and ERK （P<0.05， P<0.01）. ConclusionIn summary， Renqing Mangjue can prevent gastric cancer precursors by inhibiting the proliferation and migration of malignant transformed GES-1 cells， thereby delaying the EMT process. The underlying mechanisms may be related to the activation of the cGMP/PKG pathway and the inhibition of the MEK/ERK signaling pathway.

The diagnosis of hematological disorders is currently established from the combined results of different tests, including those assessing morphology (M), immunophenotype (I), cytogenetics (C), and molecular biology (M) (collectively known as the MICM classification). In this workflow, most of the results are interpreted manually (i.e., by a human, without automation), which is expertise-dependent, labor-intensive, time-consuming, and with inherent interobserver variability. Also, with advances in instruments and technologies, the data is gaining higher dimensionality and throughput, making additional challenges for manual analysis. Recently, artificial intelligence (AI) has emerged as a promising tool in clinical hematology to ensure timely diagnosis, precise risk stratification, and treatment success. In this review, we summarize the current advances, limitations, and challenges of AI models and raise potential strategies for improving their performance in each sector of the MICM pipeline. Finally, we share perspectives, highlight future directions, and call for extensive interdisciplinary cooperation to perfect AI with wise human-level strategies and promote its integration into the clinical workflow.

Objective:To investigate the clinical and genetic characteristics of a Henan Han family with pontine autosomal dominant microangiopathy and leukoencephalopathy (PADMAL), aiming to enhance understanding of this disease.Methods:The proband was first admitted to the Department of Neurology, Henan Provincial People′s Hospital, Fuwai Central China Cardiovascular Hospital in December 2019 due to cerebral infarction and unilateral limb numbness and weakness. Detailed medical history collection, pedigree mapping, whole-exome sequencing screening, and Sanger sequencing validation were performed for the proband and family members. The patients′ clinical manifestations, imaging features, neuropsychological scale assessment results, and pathological changes were summarized, and genetic analysis was conducted on the gene variant site. Relevant literature was reviewed to summarize the characteristics of PADMAL.Results:The proband was a 47-year-old female, with 3 generations of family members affected, including 7 patients, 3 of whom had died. The clinical features of the patients were similar, with the first stroke occurring around the age of 40, without vascular risk factors such as hypertension or diabetes. The main clinical manifestation was unilateral limb numbness and weakness. The proband and her niece sought medical attention due to stroke symptoms. Brain magnetic resonance imaging revealed acute infarct lesions located in the pons, accompanied by multiple oval infarct foci (the "raisin bread sign") and white matter hyperintensity changes. Genetic testing showed that 4 patients carried a heterozygous c. *34GT mutation in the 3′-untranslated region (3′-UTR) of the COL4A1 gene, while the other 4 unaffected family members did not carry this variant, consistent with genotype- phenotype co-segregation in the family. Conclusions:PADMAL is an extremely rare monogenic cerebral small vessel disease caused by pathogenic variants in the 3′-UTR of the COL4A1 gene. The "raisin bread sign" in the pons is a relatively specific imaging feature that distinguishes it from other cerebral small vessel diseases. For patients with this sign, genetic testing for PADMAL should be considered.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a non-atherosclerotic, non-amyloidogenic inherited cerebral small vessel disease caused by mutations in the NOTCH3 gene, typically manifesting in middle age. The diagnosis can be confirmed through the detection of granular osmiophilic material in skin biopsies and NOTCH3 gene mutations identified by genetic testing. However, the pathogenesis of this disease remains unclear, and treatment options are limited. A major contributing factor is that currently available preclinical animal models fail to adequately recapitulate the characteristic pathological features and clinical phenotypes of the disease. Therefore, a comprehensive review of recent advances in CADASIL animal models was conducted, and the advantages and limitations of existing models were systematically analyzed, aiming to offer guidance for selecting appropriate animal models in fundamental research and to propose future directions for developing experimental models.

Objective:To construct a full-length infectious clone of enterovirus D68（EV-D68）expressing enhanced green fluorescent protein（EGFP）by reverse genetics in order to provide an efficient tool for studying the biological characteristics and screening antiviral drugs for EV-D68.Methods:Gene synthesis and overlap PCR techniques were used to construct the full-length clone plasmid pUC57-EV-D68 of EV-D68. The full-length viral sequence was then transferred into the pCAGGS plasmid to obtain the pCAGGS-EV-D68 plasmid. The EGFP gene was amplified and inserted into the pCAGGS-EV-D68 plasmid to construct the pCAGGS-EGFP-EV-D68 plasmid. Then，the two constructed plasmids were transfected into human rhabdomyosarcoma（RD）cells to rescue recombinant viruses RV-EV-D68 and RV-EGFP-EV-D68. The rescued viruses were identified using PCR，Western blot，and immunofluorescence techniques. The antiviral effect of doxycycline was evaluated using the rescued RV-EGFP-EV-D68. Statistical analysis was performed using the two independent samples t-test. Results:The recombinant virus RV-EGFP-EV-D68 capable of expressing EGFP was successfully rescued. Even after 15 serial passages，the virus retained EGFP expression with no significant difference in viral titers compared to the parental virus，indicating its stable passage in RD cells. Besides，the rescued strains exhibited similar replication characteristics to the parental virus. While at 24 and 36 h after infection，the titers of the rescued strains were significantly lower than that of the parental strain（both P<0.05）. This study demonstrated that doxycycline significantly reduced the fluorescence intensity of RV-EGFP-EV-D68-infected RD cells（ P<0.01）. Meanwhile，a negative correlation was observed between the doxycycline concentration and the fluorescence intensity，indicating that the rescued virus could be used for antiviral drug evaluation. Conclusions:This study successfully constructs an infectious clone of EV-D68 expressing EGFP. The rescued recombinant virus RV-EGFP-EV-D68 has been verified to be applicable for the evaluation of antiviral drugs.

Objective:To investigate the clinical and genetic characteristics of a Henan Han family with pontine autosomal dominant microangiopathy and leukoencephalopathy (PADMAL), aiming to enhance understanding of this disease.Methods:The proband was first admitted to the Department of Neurology, Henan Provincial People′s Hospital, Fuwai Central China Cardiovascular Hospital in December 2019 due to cerebral infarction and unilateral limb numbness and weakness. Detailed medical history collection, pedigree mapping, whole-exome sequencing screening, and Sanger sequencing validation were performed for the proband and family members. The patients′ clinical manifestations, imaging features, neuropsychological scale assessment results, and pathological changes were summarized, and genetic analysis was conducted on the gene variant site. Relevant literature was reviewed to summarize the characteristics of PADMAL.Results:The proband was a 47-year-old female, with 3 generations of family members affected, including 7 patients, 3 of whom had died. The clinical features of the patients were similar, with the first stroke occurring around the age of 40, without vascular risk factors such as hypertension or diabetes. The main clinical manifestation was unilateral limb numbness and weakness. The proband and her niece sought medical attention due to stroke symptoms. Brain magnetic resonance imaging revealed acute infarct lesions located in the pons, accompanied by multiple oval infarct foci (the "raisin bread sign") and white matter hyperintensity changes. Genetic testing showed that 4 patients carried a heterozygous c. *34GT mutation in the 3′-untranslated region (3′-UTR) of the COL4A1 gene, while the other 4 unaffected family members did not carry this variant, consistent with genotype- phenotype co-segregation in the family. Conclusions:PADMAL is an extremely rare monogenic cerebral small vessel disease caused by pathogenic variants in the 3′-UTR of the COL4A1 gene. The "raisin bread sign" in the pons is a relatively specific imaging feature that distinguishes it from other cerebral small vessel diseases. For patients with this sign, genetic testing for PADMAL should be considered.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a non-atherosclerotic, non-amyloidogenic inherited cerebral small vessel disease caused by mutations in the NOTCH3 gene, typically manifesting in middle age. The diagnosis can be confirmed through the detection of granular osmiophilic material in skin biopsies and NOTCH3 gene mutations identified by genetic testing. However, the pathogenesis of this disease remains unclear, and treatment options are limited. A major contributing factor is that currently available preclinical animal models fail to adequately recapitulate the characteristic pathological features and clinical phenotypes of the disease. Therefore, a comprehensive review of recent advances in CADASIL animal models was conducted, and the advantages and limitations of existing models were systematically analyzed, aiming to offer guidance for selecting appropriate animal models in fundamental research and to propose future directions for developing experimental models.

Objective Atrial fibrillation(AF)is a disease of high heterogeneity,and the association between AF phenotypes and the outcome of different catheter ablation strategies remains unclear.Conventional classification of AF(e.g.according to duration,atrial size,and thromboembolism risk)fails to provide reference for the optimal stratification of the prognostic risks or to guide individualized treatment plan.In recent years,research on machine learning has found that cluster analysis,an unsupervised data-driven approach,can uncover the intrinsic structure of data and identify clusters of patients with pathophysiological similarity.It has been demonstrated that cluster analysis helps improve the characterization of AF phenotypes and provide valuable prognostic information.In our cohort of AF inpatients undergoing radiofrequency catheter ablation,we used unsupervised cluster analysis to identify patient subgroups,to compare them with previous studies,and to evaluate their association with different suitable ablation patterns and outcomes.Methods The participants were AF patients undergoing radiofrequency catheter ablation at West China Hospital between October 2015 and December 2017.All participants were aged 18 years or older.They underwent radiofrequency catheter ablation during their hospitalization.They completed the follow-up process under explicit informed consent.Patients with AF of a reversible cause,severe mitral stenosis or prosthetic heart valve,congenital heart disease,new-onset acute coronary syndrome within three months prior to the surgery,or a life expectancy less than 12 months were excluded according to the exclusion criteria.The cohort consisted of 1 102 participants with paroxysmal or persistent/long-standing persistent AF.Data on 59 variables representing demographics,AF type,comorbidities,therapeutic history,vital signs,electrocardiographic and echocardiographic findings,and laboratory findings were collected.Overall,data for the variables were rarely missing(<5% ),and multiple imputation was used for correction of missing data.Follow-up surveys were conducted through outpatient clinic visits or by telephone.Patients were scheduled for follow-up with 12-lead resting electrocardiography and 24-hours Holter monitoring at 3 months and 6 months after the ablation procedure.Early ablation success was defined as the absence of documented AF,atrial flutter,or atrial tachycardia>30 seconds at 6-month follow-up.Hierarchical clustering was performed on the 59 baseline variables.All characteristic variables were standardized to have a mean of zero and a standard deviation of one.Initially,each patient was regarded as a separate cluster,and the distance between these clusters was calculated.Then,the Ward minimum variance method of clustering was used to merge the pair of clusters with the minimum total variance.This process continued until all patients formed one whole cluster.The"NbClust"package in R software,capable of calculating various statistical indices,including pseudo t2 index,cubic clustering criterion,silhouette index etc,was applied to determine the optimal number of clusters.The most frequently chosen number of clusters by these indices was selected.A heatmap was generated to illustrate the clinical features of clusters,while a tree diagram was used to depict the clustering process and the heterogeneity among clusters.Ablation strategies were compared within each cluster regarding ablation efficacy.Results Five statistically driven clusters were identified:1)the younger age cluster(n=404),characterized by the lowest prevalence of cardiovascular and cerebrovascular comorbidities but the highest prevalence of obstructive sleep apnea syndrome(14.4% );2)a cluster of elderly adults with chronic diseases(n=438),the largest cluster,showing relatively higher rates of hypertension,diabetes,stroke,and chronic obstructive pulmonary disease;3)a cluster with high prevalence of sinus node dysfunction(n=160),with patients showing the highest prevalence of sick sinus syndrome and pacemaker implantation;4)the heart failure cluster(n=80),with the highest prevalence of heart failure(58.8% )and persistent/long-standing persistent AF(73.7% );5)prior coronary artery revascularization cluster(n=20),with patients of the most advanced age(median:69.0 years old)and predominantly male patients,all of whom had prior myocardial infarction and coronary artery revascularization.Patients in cluster 2 achieved higher early ablation success with pulmonary veins isolation alone compared to extensive ablation strategies(79.6% vs.66.5%;odds ratio[OR]=1.97,95% confidence interval[CI]:1.28-3.03).Although extensive ablation strategies had a slightly higher success rate in the heart failure group,the difference was not statistically significant.Conclusions This study provided a unique classification of AF patients undergoing catheter ablation by cluster analysis.Age,chronic disease,sinus node dysfunction,heart failure and history of coronary artery revascularization contributed to the formation of the five clinically relevant subtypes.These subtypes showed differences in ablation success rates,highlighting the potential of cluster analysis in guiding individualized risk stratification and treatment decisions for AF patients.