A quadruplex RT-qPCR method was developed for rapid identification and diagnosis of transmissible gastroenteritis virus(TGEV)of swine,porcine epidemic diarrhea virus(PEDV),porcine deltacorona virus(PDCoV),and porcine rotavirus type A(PoRVA).The full-length sequences of PEDV(77 strains),TGEV(63 strains),PDCoV(17 strains)that are prevalent in China,as well as the 85 VP6 gene sequences of PoRVA,were downloaded from the NCBI database for homology analysis.Based on the relatively conserved sequences,the corresponding primers and probes for each virus were designed and used to establish the quadruplex RT-qPCR method.After optimization of the probes and the reaction conditions,the specificity,sensitivity,and repeatability were determined.Using the established method,109 clinical samples of diarrhea were tested and further compared with the results by standard method.The results showed that the quadruplex RT-qPCR method established in this experiment has good amplification effect,with the C,value linearly correlated with the copies of templates(R2＞0.99).Specificity assay demonstrated that the quadruplex RT-qPCR method can identify TGEV,PEDV,PDCoV,PRoVA strains,and do not de-tect African swine fever virus(ASFV),porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV)and other epidemic viruses.Sensitivity assay showed that the detection limits for TGEV,PEDV,PDCoV and PoRVA were 10,20,20 and 50 copies/μL,respectively.The method exhibits excellent reproducibility,with coefficients of variation(Cv)for both intra-and inter-assay repli-cates being less than 1％.Detection of 109 samples of diarrhea by this method yielded the coinci-dence rate of 100％with the industry standard,indicating high practical applicability.The devel-oped method possesses the advantages of strong strain compatibility,high sensitivity,strong speci-ficity,good repeatability and stability.It is suitable for virus diagnosis and large-scale clinical sam-ple testing,providing technical support for disease prevention and control as well as epidemiologi-cal investigation.

Objective To establish a pediatric rare disease catalog,analyze the current status of therapeutic drugs and their coverage of the medical insurance in China,and propose strategies to enhance drug accessibility.Methods Pediatric rare diseases were identified from China's two national rare disease catalogs combined with the EU Orphanet database,US FDA orphan drug database,and the Diagnosis and Treatment Standards for Rare Diseases in Children.We created a specialized drug catalog for pediatric rare diseases,then analyzed drug types(ATC classification),pricing,and medical insurance coverage using descriptive statistics based on Yaozhi.com drug bidding prices and the 2024 Drug of List National Basic Medical Insurance(NBMIDL).Drug affordability was assessed through annual treatment cost calculations.Results The national catalogs included 151 pediatric rare diseases(72.95%of listed conditions),spanning 13 disease systems.We identified 94 dedicated orphan drugs(by generic name)for these conditions,among which 43 were approved internationally but unavailable in China.The average unit price per package was 6 113.53 yuan.Overall NBMIDL coverage was 68.83%,but drugs priced above 7 000 yuan per unit had only 7.69%coverage.Annual treatment costs reached 4.54 million for laronidase(mucopolysaccharidosis).Conclusions Critical gaps persist in China's pediatric rare disease treatment landscape,including catalog deficiencies,inadequate coverage for high-cost drugs and insufficient domestic innovation.It is recommended to establish a list of orphan drugs for pediatric rare diseases,accelerate the import of foreign drugs and the local innovative drugs through policy incentives,optimizing medical insurance reimbursement mechanisms for pediatric rare disease drugs to comprehensively improve therapeutic accessibility.

Objective To establish a pediatric rare disease catalog,analyze the current status of therapeutic drugs and their coverage of the medical insurance in China,and propose strategies to enhance drug accessibility.Methods Pediatric rare diseases were identified from China's two national rare disease catalogs combined with the EU Orphanet database,US FDA orphan drug database,and the Diagnosis and Treatment Standards for Rare Diseases in Children.We created a specialized drug catalog for pediatric rare diseases,then analyzed drug types(ATC classification),pricing,and medical insurance coverage using descriptive statistics based on Yaozhi.com drug bidding prices and the 2024 Drug of List National Basic Medical Insurance(NBMIDL).Drug affordability was assessed through annual treatment cost calculations.Results The national catalogs included 151 pediatric rare diseases(72.95%of listed conditions),spanning 13 disease systems.We identified 94 dedicated orphan drugs(by generic name)for these conditions,among which 43 were approved internationally but unavailable in China.The average unit price per package was 6 113.53 yuan.Overall NBMIDL coverage was 68.83%,but drugs priced above 7 000 yuan per unit had only 7.69%coverage.Annual treatment costs reached 4.54 million for laronidase(mucopolysaccharidosis).Conclusions Critical gaps persist in China's pediatric rare disease treatment landscape,including catalog deficiencies,inadequate coverage for high-cost drugs and insufficient domestic innovation.It is recommended to establish a list of orphan drugs for pediatric rare diseases,accelerate the import of foreign drugs and the local innovative drugs through policy incentives,optimizing medical insurance reimbursement mechanisms for pediatric rare disease drugs to comprehensively improve therapeutic accessibility.

A quadruplex RT-qPCR method was developed for rapid identification and diagnosis of transmissible gastroenteritis virus(TGEV)of swine,porcine epidemic diarrhea virus(PEDV),porcine deltacorona virus(PDCoV),and porcine rotavirus type A(PoRVA).The full-length sequences of PEDV(77 strains),TGEV(63 strains),PDCoV(17 strains)that are prevalent in China,as well as the 85 VP6 gene sequences of PoRVA,were downloaded from the NCBI database for homology analysis.Based on the relatively conserved sequences,the corresponding primers and probes for each virus were designed and used to establish the quadruplex RT-qPCR method.After optimization of the probes and the reaction conditions,the specificity,sensitivity,and repeatability were determined.Using the established method,109 clinical samples of diarrhea were tested and further compared with the results by standard method.The results showed that the quadruplex RT-qPCR method established in this experiment has good amplification effect,with the C,value linearly correlated with the copies of templates(R2＞0.99).Specificity assay demonstrated that the quadruplex RT-qPCR method can identify TGEV,PEDV,PDCoV,PRoVA strains,and do not de-tect African swine fever virus(ASFV),porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV)and other epidemic viruses.Sensitivity assay showed that the detection limits for TGEV,PEDV,PDCoV and PoRVA were 10,20,20 and 50 copies/μL,respectively.The method exhibits excellent reproducibility,with coefficients of variation(Cv)for both intra-and inter-assay repli-cates being less than 1％.Detection of 109 samples of diarrhea by this method yielded the coinci-dence rate of 100％with the industry standard,indicating high practical applicability.The devel-oped method possesses the advantages of strong strain compatibility,high sensitivity,strong speci-ficity,good repeatability and stability.It is suitable for virus diagnosis and large-scale clinical sam-ple testing,providing technical support for disease prevention and control as well as epidemiologi-cal investigation.

Objective To investigate the role of Toll-like receptor 4(TLR4)in hepatocyte regeneration after acet-aminophen(APAP)-induced injury in human normal liver cell(L02)and its possible mechanism.Methods L02 cells were cultured in vitro,and cell viability was detected by CCK-8 assay.The optimal concentration and duration of APAP and the concentration of TLR4 inhibitor(TAK-242)were determined.The protein expression levels of nu-clear factor-κB(NF-κB),microtubule-associated protein light chain 3(LC3),p62,receptor interacting protein kinase 1(RIP1),receptor interacting protein kinase 3(RIP3),signal transducer and activator of transcription 3(STAT3),phosphorylation of STAT3(p-STAT3),proliferating cell nuclear antigen(PCNA)and Cyclin D1 were detected by Western blot.The mRNA expression levels of TLR4,NF-κB,tumor necrosis factor-α(TNF-α),inter-leukin-6(IL-6),interleukin-1 β(IL-1 β),PCNA,Cyclin D1 and Ki67 were detected by qRT-PCR.Results Ac-cording to the results of CCK-8,L02 cells were treated with 5 mmol/L APAP for 24,36,48 h to simulate liver in-jury and regeneration model in vitro,and TAK-242 100 nmol/L was pretreated 2 ht before APAP to inhibit TLR4.Compared with the control group,the protein levels of NF-κB,RIP1,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TNF-α,IL-1 β and PCNA increased in the APAP 24 h group;the protein levels of NF-κB,RIP1,RIP3,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1 β,PCNA and Cyclin D1 in-creased in the APAP 36 h group;the protein levels of NF-κB,RIP1,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6,PCNA,Cyclin D1 and Ki67 increased in the APAP 48 h group.The protein levels of NF-κB,RIP1,RIP3,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6,PCNA,Cyclin D1,Ki67 significantly decreased in APAP+TAK-242 24 h and 48 h group than the APAP group at the same time point;the protein levels of NF-κB,PCNA and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6,PCNA and Ki67 in APAP+TAK-242 36 h group were also significantly lower than those in APAP 36 h group.Compared with the control group,autophagy was activated in the APAP group,while autophagy was inhibited in the APAP+TAK-242 group.Conclusion TLR4 may affect the TLR4/NF-κB pathway,up-regulate the levels of inflammatory factors and autophagy,and promote hepatocyte regeneration after APAP-in-duced liver injury in L02 cells.

Objective:We aimed to develop a novel visualized model based on nomogram to predict postoperative overall survival.Methods:This was a multicenter, retrospective, observational cohort study, including participants with histologically confirmed gastric and colorectal cancer who underwent radical surgery from 11 medical centers in China from August 1, 2015 to June 30, 2018. Baseline characteristics, histopathological data and nutritional status, as assessed using Nutrition Risk Screening 2002 (NRS 2002) score and the scored Patient-Generated Subjective Global Assessment, were collected. The least absolute shrinkage and selection operator regression and Cox regression were used to identify variables to be included in the predictive model. Internal and external validations were performed.Results:There were 681 and 127 patients in the training and validation cohorts, respectively. A total of 188 deaths were observed over a median follow-up period of 59 (range: 58 to 60) months. Two independent predictors of NRS 2002 and Tumor-Node-Metastasis (TNM) stage were identified and incorporated into the prediction nomogram model together with the factor of age. The model's concordance index for 1-, 3- and 5-year overall survival was 0.696, 0.724, and 0.738 in the training cohort and 0.801, 0.812, and 0.793 in the validation cohort, respectively.Conclusions:In this study, a new nomogram prediction model based on NRS 2002 score was developed and validated for predicting the overall postoperative survival of patients with gastric colorectal cancer. This model has good differentiation, calibration and clinical practicability in predicting the long-term survival rate of patients with gastrointestinal cancer after radical surgery.

Objective    To investigate the use of opioid analgesics during the postoperative hospitalization in patients undergoing lung resection and analyse its influencing factors. Methods    The clinical data of 450 patients undergoing lung resection in Sichuan Cancer Hospital among a multicenter symptom research database (CN-PRO-Lung 1) between November 2017 and January 2020 were analyzed. There were 248 males and 202 females with an average age of 54.7±10.3 years. Results    A total of 448 (99.6%) patients used opioid analgesics. The average daily morphine equivalent dose during the postoperative hospitalization was 48.9 mg. There were statistical differences in postoperative morphine equivalent dose across patients with different sex, age, highest level of education, Charlson Comorbidity Index score, surgical approach, surgical type, operative time, postoperative hospital stay and grade of postoperative complications (all P<0.05). Multivariate analysis showed that sex, surgical approach and postoperative hospital stay were independent influencing factors for morphine use during the postoperative hospitalization in patients undergoing lung resection (all P<0.05). Conclusion    In clinical practice, attention should be paid to postoperative pain for male patients, as well as to promote the application of minimally invasive surgery, and to shorten the length of postoperative hospital stay, in order to ultimately reduce the use of opioids.

Objective To analyze the short- and long-term effect of chemoradiotherapy combined with rhAd-p53 on locally advanced cervical cancer (LACC). Methods A total of 51 patients with stage ⅡA-ⅣA LACC were divided into experimental group (chemoradiotherapy+rhAd-p53 gene therapy, RCT-p53) and control group (chemoradiotherapy, RCT). Short-term effect, long-term effect and early side-effect were evaluated. Results ORR of RCT-p53 group and RCT group were 91.7% and 62.9%, respectively (P=0.037); CR rates were 66.7% and 40.7%, respectively. There was no significant difference of early side-effects between two groups, in terms of hematologic toxicity, digestive toxicity, hepatotoxicity or renal toxicity. Five-year PFS of RCT-p53 group and RCT group were 79.8% and 51.3%, respectively (P=0.028); 5-year OS were 81.6% and 60.3%, respectively (P=0.061). Conclusion rhAd-p53 treatment has better short-term effect and long-term effect than concurrent chemoradiotherapy, without increasing early side effects.

Objective To evaluate the efficacy and safety of bronchoalveolar lavage( BAL) in the treatment of neonatal atelectasis with fiberoptic bronchoscopy under ultrasound monitoring. Methods From June 2018 to December 2018,29 children were diagnosed as atelectasis by lung ultrasound. After conventional mechanical vibration and sputum ineffective,BAL was treated with fiberoptic bronchoscopy. All patients be-fore operation were monitored by ultrasound to find the lung segment where the atelectasis was located. 0. 9%NaCl solution was injected by fibrobronchoscope(1～2 ml/kg),and then sucked to ensure the recovery rate of the lavage fluid was more than 50%. After each lavage,ultrasound was immediately used to monitor the recovery of atelectasis to determine whether or not to continue the lavage. One course of treatment could be continuously performed BAL 1 to 3 times a day,1 course per day,and up to 3 courses of lavage. We analyzed the efficacy,adverse reactions and complications of BAL in the treatment of neonatal atelectasis under ultra-sound monitoring. Results Twenty-nine patients underwent BAL treatment with fiberoptic bronchoscopy,25 cases (86. 2%) were cured,3 cases (10. 3%) were effective,and 1 case (3. 4%) was ineffective. All chil-dren had stable vital signs during treatment. Among them,11 cases (37. 9%) had transient hypoxemia, 3 ca-ses (10. 3%) had tracheal mucosal injury, and 2 cases (6. 9%) had hoarseness. There were no serious com-plications such as pulmonary hemorrhage,pneumothorax,and cardiac arrest. Conclusion BAL treatment of atelectasis under lung ultrasound monitoring has obvious effect,easy to operate,no radiation,no obvious ad-verse reactions and complications,which is worthy of clinical application.

Objective: Short-course neoadjuvant radiotherapy (SCRT) combined with delayed surgery seems to be safer than SCRT in combination with immediate surgery. However, the clinical efficacy between SCRT and long-course neoadjuvant radiotherapy (LCRT) combined with delayed surgery has not been compared. Therefore, this meta-analysis was performed to compare the safety and efficacy between SCRT and LCRT followed by delayed surgery in patients with locally advanced rectal cancer. Methods: Relevant literatures were searched using relevant databases. Baseline characteristics and treatment results of patients were extracted. The included studies were subject to bias risk assessment. Evidence assessment and data analysis were conducted. Results: A total of 7 studies with 4967 patients were included. Meta-analysis results illustrated no statistical significance between two groups in terms of sphincter preservation rate, R₀ resection rate, postoperative complications, local recurrence rate (LRR), distant metastasis, recurrence-free survival (RFS), overall survival (OS), length of hospital stay and acute radiotherapy toxicity (all P>0.05). Compared with SCRT with delayed surgery, LCRT with delayed surgery was associated with a significant increase in the tumor downstaging rate (RR=0.84, 95%CI=0.76-0.93, P<0.05) and a considerable increase in pathologically complete remission rate (RR=0.46, 95%CI=0.34-0.61, P<0.05). Conclusions SCRT with delayed surgery is as effective as LCRT with delayed surgery in terms of sphincter preservation rate, R₀ resection rate, postoperative complications, LRR, RFS, OS, grade Ⅲ-Ⅳ acute toxicity and length of hospital stay. However, LCRT in combination with delayed surgery enhances the tumor downstaging rate and pathologically complete remission rate.