ObjectiveThe malaria parasites remodel the host erythrocyte structure by exporting parasite proteins that interact with the membrane skeleton proteins of red blood cells (RBCs), facilitating their intracellular survival and pathogenicity. Skeleton-binding protein 1 (SBP1) is a conserved exported protein across Plasmodium species. In Plasmodium falciparum, SBP1 has been reported to interact with erythrocyte membrane skeleton proteins 4.1R and spectrin, while its contribution to erythrocyte remodeling and parasite virulence in Plasmodium berghei (Pb) remains unclear. This study aims to determine whether PbSBP1 associates with the host cytoskeletal protein 4.1R and to investigate its role in the remodeling of host RBCs and the pathogenicity of Plasmodium berghei. MethodsIn Plasmodium berghei, the relationship between PbSBP1 and the erythrocyte cytoskeletal protein 4.1R was examined using co-immunoprecipitation. A Pbsbp1 gene knockout mutant of Plasmodium berghei (Pbsbp1∆) was generated based on the principle of double crossover homologous recombination. The deformability of erythrocytes infected with Pbsbp1∆ parasites was assessed using microfluidic methods. Microchannels with an array of cylindrical pillars were used to detect modifications in infected RBC deformability. The infected RBCs were squashed between the rows and recovered between the columns and the transit velocity (μm/s) of infected RBCs travelling through the microchannel was recorded. The component of the erythrocyte membrane skeleton junctional complex, tropomodulin (TMOD), was fluorescently labeled, and the cytoskeletal network of infected erythrocytes was imaged using super-resolution stochastic optical reconstruction microscopy (STORM) to analyze ultrastructural changes in the cytoskeleton of wild-type (WT) and Pbsbp1∆-infected erythrocytes. Actin-based junctional complexes were displayed as individual clusters by the labeled TMOD in the STORM images, and the cluster densities and distances between adjacent clusters of infected RBCs were calculated. Additionally, rodent malaria models (BALB/c mice) and experimental cerebral malaria models (C57BL/6 mice) were employed to monitor the growth of Pbsbp1∆ and WT parasites during the intraerythrocytic stage and their capacity to induce cerebral malaria in mice. ResultsPbSBP1 may participate in the remodeling of infected erythrocytes through direct or indirect interaction with the erythrocyte cytoskeletal protein 4.1R. Microfluidic assays revealed that the deformability of erythrocytes infected with Pbsbp1∆ parasites was significantly enhanced compared to those infected with WT parasites. STORM imaging further demonstrated that the ultrastructure of the erythrocyte cytoskeleton in Pbsbp1∆-infected cells was altered relative to that in WT-infected erythrocytes. The distances between nearest neighbors of clusters had a tendency to increase while the cluster densities were decreased in Pbsbp1∆-infected RBCs compared to WT-infected RBCs. Subsequent phenotypic analysis indicated that the growth rate of Pbsbp1∆ parasites during the intraerythrocytic stage was significantly slower than that of WT parasites, and their ability to induce cerebral malaria in mice was also attenuated. These findings suggest that PbSBP1 is involved in the remodeling of the erythrocyte membrane skeleton, likely through its direct or indirect interaction with protein 4.1R, thereby regulating the deformability of infected erythrocytes and influencing the pathogenicity of the blood-stage parasites. ConclusionThis study establishes a role for PbSBP1 in host erythrocyte remodeling and parasite virulence, providing new research strategies for the prevention and treatment of malaria.

Objective This study reports on the obligatory parasitism of water mites Arrenurus sp. on Anopheles messeae at the Manzhouli Port, Inner Mongolia, China. Methods Duing July 2024, a survey on the mosquito diversity was conducted at the Manzhouli Port. Captured mosquitoes and their ectoparasites were identified to species level. Results A total of 1840 adult mosquitoes were collected, representing species from three genera: Culex(Cx. modestus, Cx. pipiens pallens), Aedes(Ae. dorsalis, Ae. flavidorsalis, Ae. flavescens), and Anopheles (An. messeae). Among all the mosqutioes specimens,3 out of 150 captured An. messeae were found to carry ectoparasitic mites, with number of 2,4,27 mites separately. Morphological and molecular identification reached the same result as water mites(Hydrachnidiae, Hydracrina). COI gene sequence showed 94% similarity with the closest species Arrenurus truncatellus. Conlusions Literature review suggests water mites are host-specific parasitism of mosquito species and herein with the first record of Arrenurus sp. parasiting on An. Messeae in the most high-latitude region globally.

Aim To study the role and mechanism of ML210 in glioma.Methods The cell viability was detected by CCK8 assay.The percentage of dead cells was detected by SYTOXstaining.The role of ferroptosis-signaling pathway in gliomas was detected bygenomics.Cell proliferation was observed by EdU staining and clone formation assay.Cell migration ability was detec-ted by scratch healing assay.The apoptosis was detec-ted by flow cytometry.Cell mitochondrial function was assesses by JC-1 staining.The mechanism of action of ML210 was detected by molecular docking coupled with immunoblotting assay(Western blot).The levels of ROS,MDA were observed by ELISA.Results Compared with the control group,ML210 treatment dose-dependently decreased glioma cell viability,in-hibited cell proliferation,migration,and increased cell apoptosis and mitochondrial dysfunction,which were reversed by ferroptosis antagonists.Gene microarray screening showed that 688 genes of the ferroptosissig-naling pathway were aberrant and 10 signaling path-ways were altered in gliomas.Molecular docking re-sults showed that ML210 binding to GPX4 significantly inhibited the protein expression level of GPX4 and pro-moted the elevation of ROS and MDA levels.Conclu-sions ML210 produces anti-glioma cells via GPX4-mediated ferroptosis pathway.

Objective:To analyze the correlation between sex hormone levels and micropenis after mild hypospadias surgery in children.Methods:A case control study was carried out.The clinical data of 71 children aged 1 to 13 years who underwent mild hypospadias surgery at the First Affiliated Hospital of Xinxiang Medical University from April 2022 to April 2024 were analyzed.Preoperatively, the children were divided into a mild hypospadias group (Group A) and a mild hypospadias with micropenis group (Group B) based on the stretched penile length (SPL).Prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone, and testosterone (TES) levels of the 2 groups were measured.Multivariate Logistic regression analysis was used to construct a risk prediction model.The discrimination capability of the model was evaluated using the receiver operating characteristic (ROC) curve.SPL and sex hormone levels were measured again 6 months after surgery.The children were divided into a normal penile group (Group AA) and a micropenis group (Group BB) after mild hypospadias according to SPL.Multivariate Logistic regression analysis was used to construct a risk prediction model, which was evaluated using the ROC curve.Results:The levels of FSH, LH and TES in group A before the operation were 3.28(2.02, 4.46) IU/L, 0.53(0.25, 0.79) IU/L and 25.24(17.94, 36.67) ng/dL, respectively, and those in group B were 1.42(1.10, 1.84) IU/L, 0.14(0.09, 0.23) IU/L and 15.73 (12.92, 17.00) ng/dL, respectively.The difference was all statistically significant (all P<0.05).Multivariate Logistic regression analysis showed statistical significance ( OR=0.515, 95% CI: 0.271-0.977; OR=0.035, 95% CI: 0.002-0.542; OR=0.883, 95% CI: 0.796-0.980).The area under the ROC curve (AUC) of the prediction model was 0.906, with a sensitivity of 75.00% and a specificity of 95.74%.The levels of FSH, LH and TES in the postoperative AA group were 2.97 (1.88, 4.28) IU/L, 0.46 (0.23, 0.78) IU/L and 20.92 (17.34, 33.27) ng/dL, respectively.The median levels of FSH, LH and TES in the BB group were 1.52 (1.27, 1.82) IU/L, 0.17 (0.12, 0.26) IU/L and 15.08(11.68, 16.68) ng/dL, respectively.The difference was all statistically significant (all P<0.05).Multivariate Logistic regression analysis showed statistical significance ( OR=0.484, 95% CI: 0.236-0.992; OR=0.061, 95% CI: 0.004-0.939; OR=0.891, 95% CI: 0.795-0.999).The AUC of the prediction model constructed was 0.877, with a sensitivity of 94.12% and a specificity of 68.52%. Conclusions:Lower FSH, LH and TES levels are risk factors for the micropenis after mild hypospadias surgery, and preoperative hormone levels have higher predictive value.

Objective:To quantitatively evaluate the level of the three medical linkage in China from 2009 to 2022,explore the influencing factors and driving paths of the three medical linkage in China,and provide a new perspective for promoting the development of the three medical linkage.Methods:An optimized coupling coordination degree model was used to calculate the coupling coordination degree between the trinity healthcare systems and different binary systems within the systems in 31 provinces of China(excluding Hong Kong,Macao and Taiwan),and the Fuzzy-set Qualitative Comparative Analysis method was used to explore the condition configurations of multi-factor-driven three medical linkage.Results:From 2009 to 2022,the coupling coordination degree between the trinity healthcare systems in each province of China generally showed an increasing trend year by year.Among the binary systems,the overall coordinated development situation between the medical and medical insurance systems was the best and the regional development was the most balanced.The coupling coordination degree gap between the trinity healthcare system and the internal binary systems among provinces gradually widened,and the multi-polarization trend intensified.The paths to promote high-level three medical linkage can be summarized into two types:internal and external balanced development type(H1)and government-led type(H2,H3),among which the H1 path with per capita GDP and health expenditure as core conditions was the most common.Conclusion:It is suggested to enhance institutional and technological innovation,and integrate resources through a cross-departmental collaboration mechanism and digital technology.Provinces should select high-level optimization paths by leveraging regional endowments to narrow the regional development gap.Meanwhile,under the impetus of high-level policies,the protection and supervision system continues to improve,thereby promoting the three medical linkage.

Aim To explore the mechanism of baicalin combined with heat stimulation in treating acute lym-phoblastic leukemia(ALL)based on network pharma-cology and in vitro experiments.Methods The CCK-8 assay was used to screen the suitable conditions for heat stimulation to interfere ALL cell lines Jurkat,CCRF-CEM,Hut-78 and a normal lymphocyte HMy2.CIR,and the effects of baicalin combined with heat stimulation on the proliferation of three ALL cell lines and a normal lymphocyte were tested.The key targets of baicalin combined with fever stimulation for the treatment of ALL were obtained based on network phar-macological analysis,and the potential mechanisms were predicted by gene ontology(GO)annotation and kyoto encyclopedia of genes and genomes(KEGG)en-richment.The expression levels of TNF-α,AKT1,TYMS and CASP3 mRNA in ALL cell lines Jurkat and CCRF-CEM were examined by RT-qPCR with baicalin alone and baicalin combined with heat stimulation.Results The optimal conditions for heat stimulation to intervene ALL cells were 41 ℃ for 24 h,and heat stimulation combined with baicalin synergistically inhibited the growth of ALL cell lines and effectively reduced the cy-totoxicity of baicalin.Based on the network pharmaco-logical analysis,55 intersecting targets of baicalin with ALL diseases and 77 intersecting targets of baicalin with fever were obtained.The results of GO annotation and KEGG enrichment suggested that baicalin com-bined with fever stimulation to intervene ALL might be associated with influencing intracellular reactive oxygen species metabolism,DNA transcription and apoptotic processes involved in cysteine enzymes.Apoptosis,TNF and IL-17 signaling pathways were the key pathways for baicalin combined with heat stimulation in treating ALL.Under heat stimulation at 41 ℃ using SDHA gene as housekeeping gene,in vitro experiments showed that baicalin significantly up-regulated the expression of TNF-α and CASP3,and down-regulated the expression of TYMS in ALL cells.Conclusions Based on net-work pharmacologic analyses and in vitro experiments,baicalin combined with heat stimulation can regulate TNF-α and CASP3 gene levels in ALL cells and de-stroy cellular structure to promote cell apoptosis,thus synergistically treating ALL.

Objective:To apply mass spectrometry as well as bioinformatics techniques to analyze HBV peptides derived from Pol and X proteins presented by human immortalized B lymphocytes(BLCLs),and to screen for effective T-cell epitopes,which lays the foundation for the development of therapeutic vaccines.Methods:The group has constructed a genome-wide expression plasmid containing 1.2-fold HBVC2 isoforms and transfected it into BLCLs by electro-transfection,isolated and identified HBV peptides by LC-MS/MS,bioinformatically predicted peptide sequences in terms of sensitization,antigenicity,toxicity,HLA molecular affinity,and the ability of peptide HLA-class Ⅰ complex to bind to specific T cells,and retrieved reported sequences.Results:HBV peptides from lysates of five immortalized B cells(BLCLs-1 to BLCLs-5)carrying HBVC2 subtype-expressing recombinants were analyzed,and 141 peptides with sequence lengths of no less than 8 amino acid residues(≥8 aa)were successfully isolated and identified,of which 133 were derived from Pol,and 8 from X proteins.The 141 HBV peptides were analyzed for sensitization,antigenicity and toxicity,and 50 antigenic,non-toxic and sensitizing HBV peptides(47 from Pol and 3 from X protein)were screened for affinity analysis with HLA-class Ⅰ and Ⅱ molecules and for prediction of the binding ability of the peptide HLA-class Ⅰ complexes to specific T cells.Among these peptides,37 had affinity to the corresponding genotypes of HLA-class Ⅰ and Ⅱ molecules.Finally,we obtained 37 peptides with antigenic,nontoxic,and sensitizing properties with IC50<500 nmol/L to HLA-class Ⅰ and Ⅱ molecules,which have affinitied to the corresponding genotypes but have not been reported,and can be continued to be explored as potential epitopes.Finally,15 HBV peptide hotspot core regions were formed by intra-and inter-strain common,contained,overlapping or neighboring sequence analysis of 141 peptides,with 7 core region sequences with antigenic,nontoxic,and sensitizing properties restricted to the corresponding geno-types.Peptides with affinity IC50<500 nmol/L had an affinity core sequence overlap of no less than 8 amino acids,and can be priori-tized for candidate T cell epitopes for subsequent studies.Conclusion:Peptides derived from Pol and X proteins have been successfully isolated and identified,and potential T cell epitopes have been screened.

AIM To prepare the sustained-release microspheres of ginsenosides.METHODS The sustained-release microspheres were prepared by SPG membrane emulsification technology with poly(lactic-co-glycolic acid)(PLGA)as a shell carrier.With PLGA concentration,feed rate and Span 60 concentration as influencing factors,comprehensive score for appearance,drug loading and encapsulation efficiency as an evaluation indice,the preparation process was optimized by response surface method.The morphology of sustained-release microspheres was observed,after which the particle size,drug loading and encapsulation efficiency were determined,and the in vitro drug release was investigated.RESULTS The optimal conditons were determined to be 45 s for agitation time of primary emulsion,74.68 mg/mL for PLGA concentration,11％for feed rate,and 4.18 mg/mL for Span 60 concentration,the comprehensive score was 74.98.The round sustained-release microspheres demonstrated the average particle size of 4.33 μm,drug loading of(8.24±0.13)％,and encapsulation efficiency of(74.94±1.17)％,respectively.At 336 h,ginsenosides Rg1,Rb1,Rb2 displayed the accumulative release rates of 84.12％,78.04％,65.88％,respectively.CONCLUSION This reasonable and feasible method can be used for the preparation of sustained-release microspheres of ginsenosides with good appearance and high drug loading,which can provide references for the preparation of other water-soluble drug microspheres and solution of microsphere collapse problem.

Objective:To investigate the significance of serum β2-microglobulin(β2-MG)for survival and relapse of patients with diffuse large B-cell lymphoma(DLBCL)in the rituximab era.Methods:Clinical data of 92 patients with DLBCL admitted from December 2003 to July 2015 were retrospectively analyzed.The optimal cutoff value of β2-MG levels for predicting prognosis of the DLBCL patients was determined using receiver operating characteristic(ROC)curve.Kaplan-Meier analysis was used to estimate progression-free survival(PFS)and overall survival(OS).Cox logistic regression analysis was used to explore potential prognostic factors associated with survival.Binary logistic regression analysis was used to analyze the relationship between various factors and relapse.Results:The most discriminative cutoff value for β2-MG level was determined to be 2.25 mg/L by the ROC curve.Subgroup analysis showed that patients in the elevated β2-MG(＞2.25 mg/L)group had significantly worse PFS(P=0.006)and a trend toward worse OS compared with those in the low β2-MG(≤2.25 mg/L)group(P=0.053).Univariate analysis showed that elevated β2-MG,age＞60 years,Ann Arbor stage Ⅲ-Ⅳ,as well as IPI score ≥ 3 were associated with worse PFS.Binary logistic regression analysis showed that age＞60 years and β2-M G＞2.25 mg/L were potential influencing factors for relapse of DLBCL patients.Conclusion:Serum β2-MG might be an important predictor for the survival and relapse of DLBCL patients in the rituximab era.

AIM To prepare the sustained-release microspheres of ginsenosides.METHODS The sustained-release microspheres were prepared by SPG membrane emulsification technology with poly(lactic-co-glycolic acid)(PLGA)as a shell carrier.With PLGA concentration,feed rate and Span 60 concentration as influencing factors,comprehensive score for appearance,drug loading and encapsulation efficiency as an evaluation indice,the preparation process was optimized by response surface method.The morphology of sustained-release microspheres was observed,after which the particle size,drug loading and encapsulation efficiency were determined,and the in vitro drug release was investigated.RESULTS The optimal conditons were determined to be 45 s for agitation time of primary emulsion,74.68 mg/mL for PLGA concentration,11％for feed rate,and 4.18 mg/mL for Span 60 concentration,the comprehensive score was 74.98.The round sustained-release microspheres demonstrated the average particle size of 4.33 μm,drug loading of(8.24±0.13)％,and encapsulation efficiency of(74.94±1.17)％,respectively.At 336 h,ginsenosides Rg1,Rb1,Rb2 displayed the accumulative release rates of 84.12％,78.04％,65.88％,respectively.CONCLUSION This reasonable and feasible method can be used for the preparation of sustained-release microspheres of ginsenosides with good appearance and high drug loading,which can provide references for the preparation of other water-soluble drug microspheres and solution of microsphere collapse problem.