OBJECTIVE To investigate the impact of lysosomal associated membrane protein 2b(Lamp2b)modification on the mucosal immune efficacy of engineered exosome-based vaccines.METHODS In vitro experiments:The murine dendritic cell line DC2.4 was transfected with a plasmid encoding the Lamp2b-RBD fusion protein.Real-time quantitative PCR and Western blotting were employed to assess Lamp2b-RBD expressions,flow cytometry was used to evaluate the proportion of Lamp2b-RBD-positive cells,and immunofluorescence staining was performed to determine their membrane localization.Exosomes were isolated via ultracentrifugation,and their morphology and particle size distribution were examined using transmission electron microscopy and nanoparticle tracking analysis.Western blotting was applied to confirm exosomal marker proteins[cluster of differentiation 9(CD9),CD63,ALG-2-interacting protein X(Alix),and Golgi marker GM130]and Lamp2b-RBD expression.In vivo experiments:① Female BALB/c mice were divided into the Lamp2b-RBD-Exo group and the lipid nanoparticle(LNP)group,and administered intratracheally for mucosal immunization.Pulmonary reten-tion was assessed by immunofluorescence staining.② Female BALB/c mice were divided into three groups:placebo group(PBS group),Lamp2b-RBD-Exo intratracheal administration group,and Lamp2b-RBD-Exo intramuscular injection group(im).Immunizations were performed on days 0 and 14,and on days 7 and 21.The titers of RBD-specific immunoglobulin G(IgG)in serum and RBD-specific IgA and IgG antibodies in bronchoalveolar lavage fluid were determined by enzyme-linked immunosor-bent assay(ELISA).RESULTS In vitro experiments:Lamp2b-RBD-positive cells accounted for 71.16％.Lamp2b-RBD mRNA levels were upregulated 1 979-fold compared with controls,with Lamp2b-RBD proteins localized on the cell membrane.Purified engineered exosomes displayed regular morphology,expressed CD9,CD63,and Alix but not GM130,had an average diameter of approximately 124 nm,and carried 3 009 pg of RBD protein per 1×109 exosomes.In vivo experiments:At 4 h after administra-tion,fluorescence signals were observed in the lung tissues of both the Lamp2b-RBD-Exo and LNPs groups.At 24 h,the fluorescence signal in the LNPs group shifted to the liver,while in the Lamp2b-RBD-Exo group,the fluorescence expanded from the trachea to the bronchioles and lung tissue,showing significantly better distribution and retention capacity than the LNPs group.Seven days after immuniza-tion,both the Lamp2b-RBD-Exo and Lamp2b-RBD-Exo(im)groups induced RBD-specific IgG antibody titers.At 21 days after immunization,Lamp2b-RBD-Exo elicited a higher level of RBD-specific immune response,with serum IgG titers reaching 1∶8 100 and bronchoalveolar lavage fluid(BALF)IgA titers reaching 1∶300.No RBD-specific IgA antibody titers were detected in the BALF of the Lamp2b-RBD-Exo(im)group.CONCLUSION Lamp2b-RBD modification enables efficient RBD protein loading and enhances pulmonary retention of engineered exosomes,thereby inducing potent antigen-specific mucosal immune responses.

Astrocytes and microglia engage in extensive and complex communication and mutual effect, which referrs to microglia-astrocyte crosstalk. Recent studies have highlighted that this crosstalk plays a pivotal role in neurodegenerative diseases, exerting either protective or detrimental effects. This review briefly introduces the molecular mechanism of microglia-astrocyte crosstalk and its research progress in Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, and Parkinson's disease, aiming to provide new research directions and therapeutic targets for clinical improvement of neurodegenerative diseases from perspective of microglia-astrocyte crosstalk.

Purpose To explore the expression level of let-7b-5p in glioma and its effects and potential mecha-nisms on U251 cell growth.Methods The expression of let-7b-5p in glioma was detected using qRT-PCR.Data from the CGGA database were analyzed to examine the relationship between the let-7b-5p expression levels,WHO grade and overall survival rates of glioma patients.Transient transfection was used to downregulate the expression of let-7b-5p and IGF1R in U251 cells.The role and potential mechanism of let-7b-5p in the U251 cell were evaluated using qRT-PCR,CCK8 assays,clone formation assays,Western blotting,and double luciferase reporter assays.Results The expres-sion of let-7b-5p in glioma cells(A172:3.64±0.64,V251:4.56±0.52,U87-MG:3.31±0.50)and tissues(2.18±0.22)was significantly higher than that in astrocytes(HMC3:1.00±0.21,P＜0.05 or P＜0.01)and nor-mal brain tissues(1.01±0.19,P＜0.05).Let-7b-5p expression was negatively correlated with WHO grades but pos-itively correlated with survival rates in primary and recurrent glioma patients(P＜0.000 1 and P=0.028,respective-ly).Knockdown of let-7b-5p in U251 cells significantly promoted the growth of glioma cells(CCK8:knockdown group 126.00±12.09 vs miR-NC group 90.93±5.13,P＜0.05)and activated PI3K/AKT signal pathway.Suppressing IGF1R expression in U251 cells reversed the effects of let-7b-5p knockdown on glioma cell growth[CCK8:let-7b-5p knockdown+IGF1R knockdown group(92.08±6.14)vs let-7b-5p knockdown+sh-NC group(116.67.08±8.50)]and PI3K/AKT signal pathway activation.Conclusion Let-7b-5p functions as a tumor suppressor gene in glioma.It may regulate glioma cell growth by targeting IGF1R and modulating PI3K/AKT signal pathway.

OBJECTIVE To investigate the impact of lysosomal associated membrane protein 2b(Lamp2b)modification on the mucosal immune efficacy of engineered exosome-based vaccines.METHODS In vitro experiments:The murine dendritic cell line DC2.4 was transfected with a plasmid encoding the Lamp2b-RBD fusion protein.Real-time quantitative PCR and Western blotting were employed to assess Lamp2b-RBD expressions,flow cytometry was used to evaluate the proportion of Lamp2b-RBD-positive cells,and immunofluorescence staining was performed to determine their membrane localization.Exosomes were isolated via ultracentrifugation,and their morphology and particle size distribution were examined using transmission electron microscopy and nanoparticle tracking analysis.Western blotting was applied to confirm exosomal marker proteins[cluster of differentiation 9(CD9),CD63,ALG-2-interacting protein X(Alix),and Golgi marker GM130]and Lamp2b-RBD expression.In vivo experiments:① Female BALB/c mice were divided into the Lamp2b-RBD-Exo group and the lipid nanoparticle(LNP)group,and administered intratracheally for mucosal immunization.Pulmonary reten-tion was assessed by immunofluorescence staining.② Female BALB/c mice were divided into three groups:placebo group(PBS group),Lamp2b-RBD-Exo intratracheal administration group,and Lamp2b-RBD-Exo intramuscular injection group(im).Immunizations were performed on days 0 and 14,and on days 7 and 21.The titers of RBD-specific immunoglobulin G(IgG)in serum and RBD-specific IgA and IgG antibodies in bronchoalveolar lavage fluid were determined by enzyme-linked immunosor-bent assay(ELISA).RESULTS In vitro experiments:Lamp2b-RBD-positive cells accounted for 71.16％.Lamp2b-RBD mRNA levels were upregulated 1 979-fold compared with controls,with Lamp2b-RBD proteins localized on the cell membrane.Purified engineered exosomes displayed regular morphology,expressed CD9,CD63,and Alix but not GM130,had an average diameter of approximately 124 nm,and carried 3 009 pg of RBD protein per 1×109 exosomes.In vivo experiments:At 4 h after administra-tion,fluorescence signals were observed in the lung tissues of both the Lamp2b-RBD-Exo and LNPs groups.At 24 h,the fluorescence signal in the LNPs group shifted to the liver,while in the Lamp2b-RBD-Exo group,the fluorescence expanded from the trachea to the bronchioles and lung tissue,showing significantly better distribution and retention capacity than the LNPs group.Seven days after immuniza-tion,both the Lamp2b-RBD-Exo and Lamp2b-RBD-Exo(im)groups induced RBD-specific IgG antibody titers.At 21 days after immunization,Lamp2b-RBD-Exo elicited a higher level of RBD-specific immune response,with serum IgG titers reaching 1∶8 100 and bronchoalveolar lavage fluid(BALF)IgA titers reaching 1∶300.No RBD-specific IgA antibody titers were detected in the BALF of the Lamp2b-RBD-Exo(im)group.CONCLUSION Lamp2b-RBD modification enables efficient RBD protein loading and enhances pulmonary retention of engineered exosomes,thereby inducing potent antigen-specific mucosal immune responses.

Purpose To explore the expression level of let-7b-5p in glioma and its effects and potential mecha-nisms on U251 cell growth.Methods The expression of let-7b-5p in glioma was detected using qRT-PCR.Data from the CGGA database were analyzed to examine the relationship between the let-7b-5p expression levels,WHO grade and overall survival rates of glioma patients.Transient transfection was used to downregulate the expression of let-7b-5p and IGF1R in U251 cells.The role and potential mechanism of let-7b-5p in the U251 cell were evaluated using qRT-PCR,CCK8 assays,clone formation assays,Western blotting,and double luciferase reporter assays.Results The expres-sion of let-7b-5p in glioma cells(A172:3.64±0.64,V251:4.56±0.52,U87-MG:3.31±0.50)and tissues(2.18±0.22)was significantly higher than that in astrocytes(HMC3:1.00±0.21,P＜0.05 or P＜0.01)and nor-mal brain tissues(1.01±0.19,P＜0.05).Let-7b-5p expression was negatively correlated with WHO grades but pos-itively correlated with survival rates in primary and recurrent glioma patients(P＜0.000 1 and P=0.028,respective-ly).Knockdown of let-7b-5p in U251 cells significantly promoted the growth of glioma cells(CCK8:knockdown group 126.00±12.09 vs miR-NC group 90.93±5.13,P＜0.05)and activated PI3K/AKT signal pathway.Suppressing IGF1R expression in U251 cells reversed the effects of let-7b-5p knockdown on glioma cell growth[CCK8:let-7b-5p knockdown+IGF1R knockdown group(92.08±6.14)vs let-7b-5p knockdown+sh-NC group(116.67.08±8.50)]and PI3K/AKT signal pathway activation.Conclusion Let-7b-5p functions as a tumor suppressor gene in glioma.It may regulate glioma cell growth by targeting IGF1R and modulating PI3K/AKT signal pathway.

Astrocytes and microglia engage in extensive and complex communication and mutual effect, which referrs to microglia-astrocyte crosstalk. Recent studies have highlighted that this crosstalk plays a pivotal role in neurodegenerative diseases, exerting either protective or detrimental effects. This review briefly introduces the molecular mechanism of microglia-astrocyte crosstalk and its research progress in Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, and Parkinson's disease, aiming to provide new research directions and therapeutic targets for clinical improvement of neurodegenerative diseases from perspective of microglia-astrocyte crosstalk.

It is difficult to accurately distinguish pharyngitis caused by Group A Streptococcus(GAS) from other pathogens according to the clinical presentation alone, which cannot effectively guide the rational use of antimicrobials.The pharyngeal swab culture, rapid antigen detection test, nucleic acid test, and blood test can help definitively diagnose GAS pharyngitis.However, there are differences in different guidelines on who the laboratory test methods are intended for, interpretation of laboratory test results and so on.This article summarizes and analyses the laboratory diagnostic modalities and their characteristics, as well as recommendations for GAS pharyngitis in different guidelines to provide references for the clinical diagnosis, antimicrobial treatment, and further study of GAS pharyngitis.

Objective:To explore the characteristics of anorectal motility and sensation in patients with functional anorectal pain (FAP) by high-resolution anorectal manometry (HR-ARM) .Methods:The clinical data of 81 FAP patients (FAP group) who underwent HR-ARM in Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine from January 1, 2020 to January 31, 2022 were retrospectively collected, and 80 healthy volunteers were recruited as healthy control group during the same period. The HR-ARM characteristics were compared between FAP group and the healthy control group, between the patients with different genders in the FAP group, the patients with different subtypes (proctalgia fugax, levator syndrome, and non-specific FAP) in the FAP group, which included anal resting pressure, anal squeeze pressure, rectal pressure during simulated defecation, anal residual pressure during simulated defecation, paradoxical contractions, initial sensation threshold, defecation threshold, defecation urgency threshold, and tolerance threshold. Visual analogue scale (VAS) was used to assess the pain level of the patients in the FAP group, and Spearman correlation analysis was used to analyze the correlation between VAS and HR-ARM characteristics. Independent sample t-test, least significant difference test, Tamhane′s T2 test, and Mann-Whitney U test were used for statistical analysis. Results:The anal resting pressure, anal squeeze pressure, anal residual pressure during simulated defecation, defecation urgency threshold, and tolerance threshold of the FAP group were all lower than those of the healthy control group ((59.56±24.71) mmHg (1 mmHg=0.133 kPa) vs. (81.94±15.87) mmHg, (119.04±46.94) mmHg vs.(154.62±37.95) mmHg, 59.00(40.75, 80.95) mmHg vs. 83.10(61.78, 94.30) mmHg, 70.00(55.00, 90.00) mL vs. 85.00(60.00, 110.00) mL, 105.00(87.50, 150.00) mL vs. 140.00(100.00, 180.00) mL), and the differences were all statistically significant ( t=-6.83 and -5.29, Z=-4.12, -3.12 and -2.82; all P<0.01).The rectal pressure during simulated defecation of male patients in the FAP group was higher than that of males in the healthy control group, and the defecation urgency threshold was lower than that of males in the healthy control group (42.40(29.60, 57.95) mmHg vs. 31.10(25.85, 36.80) mmHg, 80.00(62.50, 107.50) mL vs. 92.00(81.00, 140.00) mL), and the differences were statistically significant ( Z=-1.99 and -2.53, both P<0.05). The anal resting pressure, anal squeeze pressure, anal residual pressure during simulated defecation, defecation urgency threshold, and tolerance threshold of female patients in FAP group were all lower than those of female in the healthy control group ((55.67±21.61) mmHg vs. (87.04±15.54) mmHg, (102.70±37.09) mmHg vs. (155.98±31.44) mmHg, 52.55(40.53, 67.48) mmHg vs. 83.10(61.10, 94.50) mmHg, 60.00(52.50, 81.50) mL vs. 80.00(60.00, 100.00) mL, 101.00(80.00, 128.75) mL vs. 120.00(94.00, 155.00) mL), and the differences were statistically significant ( t=-8.77 and -8.16, Z=-4.57, -2.24 and -2.14; all P<0.05). The anal resting pressure, anal squeeze pressure, anal residual pressure during simulated defecation, incidence rate of paradoxical contractions, defecation urgency threshold, and tolerance threshold of female patients in FAP group were all lower than those of male patients in FAP group ((55.67±21.61) mmHg vs. (68.28±29.16) mmHg, (102.70±37.09) mmHg vs. (155.62±46.66) mmHg, 52.55(40.53, 67.48) mmHg vs. 79.00(59.55, 99.25) mmHg, 28.6%(16/56) vs. 68.0%(17/25), 44.00(35.00, 60.00) mL vs. 60.00(45.00, 70.00) mL, 60.00(52.50, 81.50) mL vs. 80.00(62.50, 107.50) mL), and the differences were statistically significant( t=2.17 and 5.47, Z=-2.96, χ2=11.10, Z=-2.93 and -2.34; all P<0.05). The anal squeeze pressure of patients with proctalgia fugax subtype was higher than that of patients with levator syndrome subtype ((140.19±56.51) mmHg vs. (80.56±30.79) mmHg), and the tolerance threshold was lower than that of patients with non-specific FAP subtype ((87.86±17.80) mL vs. (125.14±48.31) mL), and the differences were statistically significant ( t=2.35 and 2.02, both P<0.05). The results of Spearman correlation analysis showed that VAS was negatively correlated with anal resting pressure, anal squeeze pressure, and defecation urgency threshold in the patients of the FAP group ( r= -0.28, -0.23, and -0.24; all P< 0.05). Conclusion:The presence of anorectal dismotility and sensory dysfunction in FAP may be related to pelvic floor muscle abnormalities, muscle coordination disorders during defecation, and rectal hypersensitivity.

Astrocytes(AS)are the most abundant glial cells in the central nervous system and are involved in many physiological and pathological processes in the nervous system.Alterations in their phenotype are particularly important for the health of the CNS.Epigenetic mechanisms,including DNA methylation,histone modification,non-coding RNA regulation,and chromatin remodeling,are closely linked to alterations in AS proliferation,differentiation,inflammation,and other phenotypic features,but how these mechanisms function needs to be explored and summarized.By reviewing the recent advances in the role of epigenetic mechanisms in AS under various physiological and pathological states,we aim to provide new ideas for the understanding and treatment of related diseases.

Mori Cortex is sweet and pungent in taste， cold in nature， and has the tropism to the lung meridian. It has the functions of purging the lung and relieving asthma and can treat oliguria and edema， being one of the commonly used herbal medicines in clinical practice. The prescriptions with Mori Cortex， such as Sangbaipi Tang， Qingjin Huatanfang， and Qingfei Huatantang， are widely used in clinical practice. The main active components in Mori Cortex are the material basis for its efficacy. Owing to the mature methods for the identification of pharmacodynamic substances in Chinese herbal medicines， the research on the chemical components of Mori Cortex has been in-depth and systematic. This article reviews the recent studies about the chemical components and pharmacological effects of Mori Cortex， as well as the treatment of respiratory diseases by the prescriptions with Mori Cortex. On this basis， the effect and mechanism of Mori Cortex and related prescriptions in the treatment of respiratory diseases are summarized. Furthermore， this article analyzes the formulation compatibility and commonly used dosages of Mori Cortex-related prescriptions in clinical practice. It provides reference for the clinical application of Mori Cortex and related prescriptions in the treatment of respiratory diseases.