Background and purpose:Intracellular deubiquitylating enzymes,such as ubiquitin-specific peptidase 2(USP2),play a pivotal role in regulating protein degradation and cellular homeostasis by modulating protein ubiquitin deconjugation,which have been implicated in the proliferation and survival of multiple myeloma(MM)cells.Targeting the inhibition of USP2 activity in MM cells might modulate their biological behavior.This study aimed to investigate regulatory effects of the leukotriene receptor antagonist montelukast sodium on USP2 in MM cells and its subsequent biological effects.Methods:An in vitro deubiquitination reaction system was established using purified USP2 protein and its substrate,the glutathione S-transferase(GST)tagged ubiquitin A-52 residue ribosomal protein fusion product(UbA52),known as GST-UbA52 protein.This system was used to characterize inhibitory effects of montelukast sodium on USP2 deubiquitinase activity.The MM cell lines MM1.S and H929 were used as in vitro models.Cellular thermal shift assay(CETSA)was subsequently employed to test interaction mode between montelukast sodium and USP2 in MM cells.Western blot assay was applied to detect expression levels of USP2 and its targeting regulators,including cell cycle supervisors cyclin D1(CCND1)and cyclin A1(CCNA1),classical signaling transducer KRAS and glucose regulated protein 78kD(GRP78),as well as apoptotic molecule C/EBP-homologous protein(CHOP)in MM1.S and H929 cells before and after the treatment with different concentrations of montelukast sodium.MM cells with either overexpression(H929-OE,MM1.S-OE)or knockdown(H929-LE,MM1.S-LE)of USP2 were generated using a lentiviral vector.Cell counting kit-8(CCK-8)and flow cytometry were utilized to detect the proliferation and apoptotic rates of H929-OE,MM1.S-OE,H929-LE and MM1.S-LE cells treated with montelukast sodium.Results:Montelukast sodium was found to inhibit USP2 mediated degradation of GST-UbA52 protein in a concentration-dependent manner,with a half inhibitory concentration(IC50)of 3.814 μmol/L.Additionally,montelukast sodium significantly enhanced the thermal stability of USP2 at temperatures of 49.1,53.2 and 56.4℃.It was also shown that montelukast sodium could down-regulate expressions of CCND1,CCNA1 and KRAS,while increase levels of GRP78 and CHOP in MM1.S and H929 cells.Furthermore,after treating with 40 μmol/L montelukast sodium for 24 h,the proliferation inhibition and apoptotic rate of H929-OE cells reached to(37.68±1.10)%and(18.99±0.26)%,while the proliferation inhibition and apoptotic rate of MM1.S-OE cells reached to(24.48±0.49)%and(33.29±0.75)%,which were significantly lower than those in H929 and MM1.S cells[H929:(57.19±1.93)%and(45.65±0.24)%;MM1.S:(50.04±0.53)%and(40.25±0.91)%;P＜0.05,n=3].Conversely,the proliferation inhibition and apoptotic rates of H929-LE and MM1.S-LE cells were significantly higher[H929-LE-1#:(80.70±1.60)%and(89.08±0.49)%;H929-LE-2#:(75.30±3.80)%and(82.41±1.07)%;MM1.S-LE-1#:(70.64±0.84)%and(67.63±0.21)%;MM1.S-LE-2#:(68.47±1.32)%and(85.90±0.18)%;P＜0.05,n=3].Conclusion:Montelukast sodium can target ubiquitin proteasome regulator USP2 and inhibit its deubiquitylating activity,which may modulate USP2 directing protein and trigger endoplasmic reticulum stress to induce cell cycle arrest and apoptosis in MM cells.

Objective:To investigate the efficacy and safety of lacrimal sac massage, lacrimal duct lavage, and dacryocystorhinostomy in combination for the treatment of congenital dacryocystitis.Methods:This study used a prospective study design, enrolling 150 children diagnosed with congenital dacryocystitis who received treatment at the Department of Ophthalmology, Shaoxing Maternity and Child Healthcare Hospital between January 2021 and December 2023. The children were randomly assigned to either a control group or an observation group using a random number table method. The control group ( n = 75) received lacrimal sac massage and lacrimal duct lavage, while the observation group ( n = 75) underwent a combination of lacrimal sac massage, lacrimal duct lavage, and dacryocystorhinostomy. Tear secretion score, level of inflammatory factor C-reactive protein, clinical efficacy, and complications (false passages, eyelid edema, and damage to the lacrimal duct mucosa) were compared between the two groups. Results:At 3 months after treatment, tear secretion score in the observation group was significantly lower than that in the control group [(1.55 ± 0.39) vs. (2.62 ± 0.58), t = 13.26, P < 0.05). At 3 months after treatment, the C-reactive protein level in the observation group was significantly lower than that in the control group [(1.38 ± 1.10) mg/L vs. (3.14 ± 1.27) mg/L, t = 9.07, P < 0.05). The cure rate in the observation group was significantly higher than that in the control group [97.33% (73/75) vs. 88.00% (66/75), χ2 = 4.81, P < 0.05]. There was no significant difference in the incidence of complications between the observation and control groups [9.33% (7/75) vs. 6.67% (5/75), χ2 = 0.36, P > 0.05]. Conclusions:The combination of lacrimal sac massage, lacrimal duct irrigation, and lacrimal duct probing has shown good efficacy in treating congenital dacryocystitis. This approach can decrease the severity of tearing, reduce the levels of inflammatory factors, achieve a high overall response rate, and lead to fewer complications.

Objective To investigate the effect of orthosis design parameters on correction of scoliosis and orthosis-trunk interface pressure.Methods A finite element model of scoliosis was constructed to simulate the assembly effect of the orthosis.The orthosis was divided into four loading areas(left rib,right rib,anterior-left and posterior-right area)to simulate six modification conditions.In Models 1,2 and 3,a fixed modification of 20 mm was applied on the anterior left and posterior right areas,while the displacement loads of 20,25 and 30 mm were applied on both the left rib and right rib areas.In Models 4,5 and 6,a fixed modification of 25 mm was applied on left rib and right rib areas,with the displacement loads of 15,20 and 25 mm applied on both anterior left and posterior right areas.The Cobb angle,apical vertebral rotation(AVR)and interface pressure were calculated.Results The correction of Cobb angle in Models 1,2 and 3 was 8.94°,15.62° and 17.91°,respectively,with AVR correction of 7.53°,6.69° and 5.87°,respectively.In Models 4,5 and 6,the correction of Cobb angle was 14.55°,15.62° and 16.09°,with AVR correction of 5.25°,6.69° and 8.63°,respectively.In Model 6,the correction rate of Cobb angle and AVR was 45.48%and 41.22%,respectively,with a maximum pressure of 26.51 kPa on orthosis-trunk interface,achieving the most significant outcome.Conclusions The modification of orthosis has a significant effect on the correction of Cobb and AVR angles.The loading on the left rib and right rib areas mainly affect the Cobb angle,while the loading on the anterior left and posterior right areas mainly affect the spinal axial-rotation.A modification of 25 mm on all loading areas achieves the optimal spinal correction.This study provides the quantitative data for orthosis design.

Objective:To explore the evaluation and relationship of hepatitis B virus(HBV)surface antigen(HBsAg)in clinical laboratory in different detection systems,further scientifically and reasonably to explain the test results for serving clinical practices.Methods:During the period from June 2021 to July 2022,100 425 specimens of patients with screened,suspected and confirmed HBV infections were collected from the clinical departments(mainly infectious hepatology)of Nanyang Central Hospital.Detection methodology included quantitative(electrochemiluminescence and chemiluminescence),qualitative(gold standard),semi-quantita-tive(ELISA),and highly sensitive HBV-DNA(RT-PCR)methods,and then analyzed the strengths and weaknesses and closeness be-tween each methodology.The relationship between the two Roche HBsAg detection systems was analyzed by correlation analysis.The HBsAg efficacy analysis was validated using Cut/Off value setting and detection limit,which in turn analyzed the distribution of false-positive and false-negative reporting models.Results:Detection results for low-and medium-concentration HBsAg showed a correla-tion between the electrochemical luminescence semi-quantitative method and ELISA method.ELISA method still had advantages in terms of sensitivity and specificity when detecting HBsAg,and there were no significant differences compared to domestic and interna-tional HBsAg quantitative detection systems.Performance validation conducted in accordance with the CNAS-GL038 document showed that the minimum detection limit for HBsAg calculated using the ELISA method in this laboratory was 0.1 U/ml.When the ROC curve Cut/Off value was set to 0.105,the area under the curve was the largest(AUC=0.986).Based on Roche's semi-quantitative electroche-miluminescence detection and patient medical history,in the common reporting model for hepatitis B five-item detection using ELISA method,HBsAg false positives occurred most frequently when HBsAg was positive alone,and HBsAg occurred most frequently in the false positive range when the OD value was less than 0.5.In ELISA method for detecting HBsAg,as the OD value increased from 0.01 to 0.10,the number of false-negative results also increased.Roche Elecsys HBsAg Ⅱ Quant Ⅱ and Elecsys HBsAg Ⅱ testing systems exhibited good linearity under certain conditions,with a ratio of approximately 1/0.18.In Elecsys HBsAg Ⅱ Quant Ⅱ detection sys-tem,the test results of HBsAg samples diluted 400 times were highly consistent with the original test results with a coefficient of deter-mination R2=0.993 8.Conclusion:There was a certain relationship between various detection systems of HBsAg at a suitable concen-tration.The detection of HBsAg by ELISA can meet the needs of clinical detection.

Objective To investigate the impacts of long non coding RNA(LncRNA)-PH domain containing 5 antisense RNA 1(FGD5-AS1)on the proliferation,migration,and invasion of colorectal cancer(CRC)cells by regulating the miR-299-5p/recombinant lysine specific demethylase 4B(KDM4B)axis.Methods The expressions of LncRNA FGD5-AS1,miR-299-5p,and KDM4B were detected by qRT-PCR in the surgically resected CRC tissues and adjacent tissues of 34 CRC patients CRC cells HCT116 were separated into si-NC group,si-FGD5-AS1 group,si-FGD5-AS1+inhibitor NC group,and si-FGD5-AS1+miR-299-5p inhibitor group.The levels of mRNA in each group were detected.CCK8,scratch assay,and Transwell assay were ap[plied to detect the proliferation,migration,and invasion of HCT116 cells in each group.Western blot was applied to detect the expression of proteins.Dual luciferase reporter gene assay verified the interaction mechanism between LncRNA FGD5-AS1 and miR-299-5p,and between miR-299-5p and KDM4B.Results LncRNA FGD5-AS1 and KDM4B were highly expressed in CRC tissue,while miR-299-5p was low expressed in CRC tissue(P＜0.05).The expression of LncRNA FGD5-AS1 mRNA,KDM4B mRNA,OD450,scratch healing rate,invasion number,PCNA,MMP-2,MMP-9,KDM4B in the si-FGD5-AS1 group were lower than those in the si-NC group,the expression of miR-299-5p mRNA was higher than that in the si-NC group(P＜0.05).Compared with the si-FGD5-AS1 group and si-FGD5-AS1+inhibitor NC group,the expression of miR-299-5p in the si-FGD5-AS1+miR-299-5p inhibitor group decreased(P＜0.05),the KDM4B mRNA,OD450,scratch healing rate,number of invasions,and expression of PCNA,MMP-2,MMP-9,KDM4B increased(P＜0.05).LncRNA FGD5-AS1 targeted negative regulation of miR-299-5p,while miR-299-5p targeted negative regulation of KDM4B.Conclusion Knocking down LncRNA FGD5-AS1 may inhibit the proliferation,migration,and invasion of CRC cells,and its mechanism may be achieved by regulating the miR-299-5p/KDM4B signaling axis.

Background and purpose:Intracellular deubiquitylating enzymes,such as ubiquitin-specific peptidase 2(USP2),play a pivotal role in regulating protein degradation and cellular homeostasis by modulating protein ubiquitin deconjugation,which have been implicated in the proliferation and survival of multiple myeloma(MM)cells.Targeting the inhibition of USP2 activity in MM cells might modulate their biological behavior.This study aimed to investigate regulatory effects of the leukotriene receptor antagonist montelukast sodium on USP2 in MM cells and its subsequent biological effects.Methods:An in vitro deubiquitination reaction system was established using purified USP2 protein and its substrate,the glutathione S-transferase(GST)tagged ubiquitin A-52 residue ribosomal protein fusion product(UbA52),known as GST-UbA52 protein.This system was used to characterize inhibitory effects of montelukast sodium on USP2 deubiquitinase activity.The MM cell lines MM1.S and H929 were used as in vitro models.Cellular thermal shift assay(CETSA)was subsequently employed to test interaction mode between montelukast sodium and USP2 in MM cells.Western blot assay was applied to detect expression levels of USP2 and its targeting regulators,including cell cycle supervisors cyclin D1(CCND1)and cyclin A1(CCNA1),classical signaling transducer KRAS and glucose regulated protein 78kD(GRP78),as well as apoptotic molecule C/EBP-homologous protein(CHOP)in MM1.S and H929 cells before and after the treatment with different concentrations of montelukast sodium.MM cells with either overexpression(H929-OE,MM1.S-OE)or knockdown(H929-LE,MM1.S-LE)of USP2 were generated using a lentiviral vector.Cell counting kit-8(CCK-8)and flow cytometry were utilized to detect the proliferation and apoptotic rates of H929-OE,MM1.S-OE,H929-LE and MM1.S-LE cells treated with montelukast sodium.Results:Montelukast sodium was found to inhibit USP2 mediated degradation of GST-UbA52 protein in a concentration-dependent manner,with a half inhibitory concentration(IC50)of 3.814 μmol/L.Additionally,montelukast sodium significantly enhanced the thermal stability of USP2 at temperatures of 49.1,53.2 and 56.4℃.It was also shown that montelukast sodium could down-regulate expressions of CCND1,CCNA1 and KRAS,while increase levels of GRP78 and CHOP in MM1.S and H929 cells.Furthermore,after treating with 40 μmol/L montelukast sodium for 24 h,the proliferation inhibition and apoptotic rate of H929-OE cells reached to(37.68±1.10)%and(18.99±0.26)%,while the proliferation inhibition and apoptotic rate of MM1.S-OE cells reached to(24.48±0.49)%and(33.29±0.75)%,which were significantly lower than those in H929 and MM1.S cells[H929:(57.19±1.93)%and(45.65±0.24)%;MM1.S:(50.04±0.53)%and(40.25±0.91)%;P＜0.05,n=3].Conversely,the proliferation inhibition and apoptotic rates of H929-LE and MM1.S-LE cells were significantly higher[H929-LE-1#:(80.70±1.60)%and(89.08±0.49)%;H929-LE-2#:(75.30±3.80)%and(82.41±1.07)%;MM1.S-LE-1#:(70.64±0.84)%and(67.63±0.21)%;MM1.S-LE-2#:(68.47±1.32)%and(85.90±0.18)%;P＜0.05,n=3].Conclusion:Montelukast sodium can target ubiquitin proteasome regulator USP2 and inhibit its deubiquitylating activity,which may modulate USP2 directing protein and trigger endoplasmic reticulum stress to induce cell cycle arrest and apoptosis in MM cells.

Objective To investigate the effect of orthosis design parameters on correction of scoliosis and orthosis-trunk interface pressure.Methods A finite element model of scoliosis was constructed to simulate the assembly effect of the orthosis.The orthosis was divided into four loading areas(left rib,right rib,anterior-left and posterior-right area)to simulate six modification conditions.In Models 1,2 and 3,a fixed modification of 20 mm was applied on the anterior left and posterior right areas,while the displacement loads of 20,25 and 30 mm were applied on both the left rib and right rib areas.In Models 4,5 and 6,a fixed modification of 25 mm was applied on left rib and right rib areas,with the displacement loads of 15,20 and 25 mm applied on both anterior left and posterior right areas.The Cobb angle,apical vertebral rotation(AVR)and interface pressure were calculated.Results The correction of Cobb angle in Models 1,2 and 3 was 8.94°,15.62° and 17.91°,respectively,with AVR correction of 7.53°,6.69° and 5.87°,respectively.In Models 4,5 and 6,the correction of Cobb angle was 14.55°,15.62° and 16.09°,with AVR correction of 5.25°,6.69° and 8.63°,respectively.In Model 6,the correction rate of Cobb angle and AVR was 45.48%and 41.22%,respectively,with a maximum pressure of 26.51 kPa on orthosis-trunk interface,achieving the most significant outcome.Conclusions The modification of orthosis has a significant effect on the correction of Cobb and AVR angles.The loading on the left rib and right rib areas mainly affect the Cobb angle,while the loading on the anterior left and posterior right areas mainly affect the spinal axial-rotation.A modification of 25 mm on all loading areas achieves the optimal spinal correction.This study provides the quantitative data for orthosis design.

Objective:To investigate the methodological differences in the detection, the inflammatory markers and the pathogenic epidemiological characteristics of influenza A virus in clinical laboratories, in order to provide more diagnostic and epidemiological data for diagnosis and prevention for children with influenza A.Methods:A retrospective cross-sectional study was conducted to collect 96 731 patients with suspected influenza A from January 2016 to October 2024 in Nanyang City Center Hospital from the Clinical Laboratory Testing Information System, including 5 731 patients with confirmed influenza A, aged 5.2 (2.8, 43.7) years old. We analyzed the distribution of influenza A patients from age and mixed infections, the relationship between patient age and positive detection rate by restricted cubic spline (RCS), analyzed differences in testing methods used Kappa consistency testing and receiver operating characteristic (ROC) curves, established a model of inflammatory markers by logistic regression, as well as developed a prediction model and also the mutation of the hemagglutinin (HA) sequence of the influenza A subtype H3N2 virus using evolutionary tree analysis.Results:RCS analysis showed an inverted 'S' shaped non-linear relationship between the positive detection rate of influenza A and the age groups of the patients. Among the mixed infections, 1.43%(1 352/94 867) of the cases were combined with Mycoplasma pneumoniae infection. The Kappa values of reverse transcription PCR (RT-PCR) and serological indirect immunofluorescence assay (IFA) for detecting influenza A in nasopharyngeal swabs and alveolar lavage fluid in clinical laboratories were 0.632 and 0.809, respectively, and those of magnetic particle chemiluminescence assay were 0.614 and 0.668, respectively, and the area under curves in ROC curve of IFA and RT-PCR were 0.869 and 0.792, respectively. The inflammatory indexes were usually elevated in severe children compared with mild children. By binary logistic regression model analysis, neutrophil-to-lymphocyte ratio, D-dimer/fibrinogen and prognosis nutrition index were the risk factors and serum amyloid A/C reactive protein ratio was the protective factor for severe children with influenza A, and the OR values of the above factors were 1.760, 7.076, 1.045, and 0.719, respectively, and P<0.01. By the Bayesian Interdiction Criterion, the optimal seasonal autoregressive moving average mixed model for influenza A epidemics was ARIMA (1, 1, 1) (2, 1, 2) 12 with the highest prediction accuracy of 98.63%. The seven strains of H3N2 all belonged to the same isoforms, with nucleotide similarity of the HA gene ranging from 99.5% to 99.9%, and the glycosylation site, receptor-binding site, and the conserved amino acid residue Glycosylation sites, receptor binding sites and conserved amino acid residues remained unchanged. HA sequence analysis showed that the prevalent strains in Nanyang had undergone mutation to different degree compared with the vaccine strains. Conclusion:Scientific and rational testing and characteristic inflammatory markers in the clinical laboratory are of great clinical value in the diagnosis of children with severe influenza A. At the same time, the epidemiological monitoring of influenza A variants should be strengthened.

Animal experiments are an essential component of life sciences and medical research. However, the external validity and reliability of individual animal studies are frequently challenged by inherent limitations such as small sample sizes, high design heterogeneity, and poor reproducibility, which impede the effective translation of research findings into clinical practice. Systematic reviews and meta-analysis represent a key methodology for integrating existing evidence and enhancing the robustness of conclusions. Currently, however, the application of systematic reviews and meta-analysis in the field of animal experiments lacks standardized guidelines for their conduct and reporting, resulting in inconsistent quality and, to some extent, diminishing their evidence value. To address this issue, this paper aims to systematically delineate the reporting process for systematic reviews and meta-analysis of animal experiments and to propose a set of standardized recommendations that are both scientific and practical. The article's scope encompasses the entire process, from the preliminary preparatory phase [including formulating the population， intervention， comparison and outcome （PICO） question, assessing feasibility, and protocol pre-registration] to the key writing points for each section of the main report. In the core methods section, the paper elaborates on how to implement literature searches, establish eligibility criteria, perform data extraction, and assess the risk of bias, based on the Preferred Reporting Items for Systematic Reviews and Meta-analysis （PRISMA) statement, in conjunction with relevant guidelines and tools such as Animal Research： Reporting of in Vivo Experiments （ARRIVE) and a risk of bias assessment tool developed by the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE). For the presentation of results, strategies are proposed for clear and transparent display using flow diagrams and tables of characteristics. The discussion section places particular emphasis on how to scientifically interpret pooled effects, thoroughly analyze sources of heterogeneity, evaluate the impact of publication bias, and cautiously discuss the validity and limitations of extrapolating findings from animal studies to clinical settings. Furthermore, this paper recommends adopting the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology to comprehensively grade the quality of evidence. Through a modular analysis of the entire reporting process, this paper aims to provide researchers in the field with a clear and practical guide, thereby promoting the standardized development of systematic reviews and meta-analysis of animal experiments and enhancing their application value in scientific decision-making and translational medicine.

Objective : To investigate the regulatory mechanism of nucleotide excision repair(NER) in hepatocellular carcinoma(HCC). Methods : Based on the expression levels of genes in the NER pathway, we performed molecular typing of HCC using the TCGA database. HCC cell lines were constructed through the knockdown of RNA binding motif protein 39(RBM39) using siRNA. HCC cell lines were constructed through the overexpression ofRBM39usingRBM39plasmid. Cells were treated with Indisulam, a reagent that induces RBM39 protein degradation. Western blot and real-time fluorescence quantitative PCR were used to detect the expression levels and changes of mRNA and protein of RBM39 and excision repair cross complementation group 1(ERCC1); flow cytometry was used to detect NER efficiency; CCK-8 assay was used to detect cell viability. Results : HCC patients were categorized into three types—C1, C2, and C3—based on NER activity, with the C3 subtype showing the highest NER activity(P<0.000 1). In the groups transfected with RBM39 siRNA or treated with Indisulam, the NER repair efficiency decreased compared to the control group(P<0.01), the cell survival rate decreased(P<0.01), and both the mRNA and protein expression of ERCC1 were reduced(P<0.01). In contrast, in the RBM39 overexpression group, the mRNA and protein expression of ERCC1 were enhanced compared to the control group(P<0.01). Conclusion RBM39 may influence NER repair efficiency by regulating ERCC1 expression in HCC.