Objective:Conventional myeloid cell staining methodologies were employed to stain splenocytes and peripheral blood cells by fixation and permeabilization and non-fixation and permeabilization methods,respectively,and effects of staining were compared following flow cytometry detection.Methods:Peripheral blood and spleen of three 8-week-old C57 male mice were divided into fixation and permeabilization group and non-fixation and permeabilization group for staining,and flow cytometry was used to detect mean fluorescence intensity(MFI)of each fluorescent antibody and proportion of positive cells.Results:In mouse peripheral blood samples,MFI of 7-AAD-PerCP-Cyanine5.5 and CD147-PE were lower in fixation and permeabilization group than non-fixation and permeabilization group,MFI of F4/80-FITC,MHC Class Ⅱ(I-A/I-E)-APC-R700,Ly-6c-APC-Cy7,CD206-BV421,CD45-BV510,CD11b-BV605,CD11c-BV650 and CD86-BV786 were higher than non-fixation and permeabilization group.Proportions of each immune cell in fixation and permeabilization group and non-fixation and permeabilization group were highly similar.In mouse spleen samples,MFI of antibodies in fixation and permeabilization group were higher than those in non-fixation and permeabilization group,with exception of CD147-PE,which had a lower MFI than non-fixation and permeabilization group;proportions of dendritic cells and Mon/Ly-6clow cells were lower than non-fixation and permeabilization group,whereas proportions of rest myeloid subpopula-tions were higher than non-fixation and permeabilization group.Conclusion:Fixation and permeabilization in peripheral blood cells can improve antibody MFI,but it has little effect on proportion of positive cells.Fixation and permeabilization can enhance antibody MFI in splenocytes,while effect on proportion of each positive cell is more significant.It is advised that when designing staining strate-gies,researchers attempt to choose alternative cell surface antigens for assay or optimize protocols through required pre-experiments in order to acquire stable and dependable results.

OBJECTIVE To explore the expression,regulation,and downstream pathways of miR-21-5p in nasal type 2 inflammatory diseases.METHODS The expression levels of miR-21-5p were evaluated in nasal mucosa of patients with chronic rhinosinusitis with nasal polyps(CRSwNP)and healthy controls,as well as in nasal mucosa of CRSwNP and allergic rhinitis(AR)mouse models by real-time PCR.Human primary nasal epithelial cells were cultured and treated with different cytokines.The expression of miR-21-5p were then detected after 48 hours treatment.miR-21-5p lentivirus were used for overexpression in nasal epithelial cells.After 72 hours,cells were collected,and RNA was extracted for transcriptome analysis.Differentially expressed genes were further analyzed for pathway enrichment.RESULTS Compared with healthy controls,the expression of miR-21-5p was significantly increased in the nasal mucosa of CRSwNP patients(P<0.01).The expression of miR-21-5p was also significantly increased in mouse models of CRSwNP and AR respectively,compared to the control groups(P<0.01,respectively).The type 2 inflammation cytokines IL-4 and IL-13 can significantly upregulate miR-21-5p expression in nasal epithelial cells(P<0.01,respectively).Overexpression by lentivirus can significantly increase the expression of miR-21-5p in nasal epithelial cells,and lead to 118 differentially expressed genes.Those genes are significantly enriched in pathways associated with posttranscriptional gene silencing,WNT signaling and MAPKinase signaling.CONCLUSION miR-21-5p is upregulated in nasal type 2 inflammatory diseases partially controlled by IL-4 and IL-13.Overexpression of miR-21-5p lead to the changes of downstream pathways associated with WNT signaling and MAPKinase signaling in nasal epithelial cells.

Objective:An in vivo mammalian hepatocyte Unscheduled DNA Synthesis(UDS)test was used to evaluate the genotoxicity of Cross-linked Sodium Hyaluronate Gel and Bone Repair Materials,providing experimental evidence for establishing a UDS testing method for medical devices and materials.Methods:0.9％sodium chloride injection and cottonseed oil were used as the solvent for test materials and negative control,respectively.N-dimethylnitrosamine(NDMA)was used as the positive control for the early sampling times,and 2-acetylaminofluorene(2-AAF)was used as the positive control for the late sampling times.SD rats were administered a single dose for toxic exposure,and liver tissues were collected at 4 h and 16 h,respectively.Hepatocytes were isolated using collagenase perfusion.After labeling with 5-ethynyl-2'-deoxyuridine(EdU),and the net average fluorescence intensity(NAFI)of cell nuclei and nucleoplasm was measured by fluorescence microscope.Data from 50 cells were used to analyze the DNA repair level.Results:Compared with the negative control groups,the positive control groups(NDMA and 2-AAF)showed highly statistically significant differences in NAFI(P＜0.01),indicating successful induction of DNA damage.There was no statistically significant differences between the cross-linked sodium hyaluronate gel groups,bone repair material groups and the negative control group(P＞0.05),suggesting that these materials did not significantly induce DNA damage under the experimental conditions.Conclusion:This study first applied EdU labeling technology to the in vivo hepatic UDS assay,achieving non-radioactive labeling through click chemistry reactions.Under the conditions of this study,cross-linked sodium hyaluronate gel and bone repair materials did not exhibit genotoxicity.In the follow-up,the sample range can be expanded and the observation period can be prolonged to further improve the genotoxicity evaluation system of medical devices.

AIM:Flow cytometry was used to evaluate the effects of short-term storage conditions(fresh,frozen at-80℃for 7 d,and stored at 4℃for 7 d)on the median fluorescence intensity(MFI)of antibodies and the percentage of immune cell subsets in mouse peripheral blood mononuclear cells(PBMCs)and splenocytes.METHODS:The PBMC and splenocyte suspensions from six male Kunming mice were collected and analyzed under three different processing con-ditions to compare differences in the antibody MFI and percentages of monocyte subsets(Ly-6clow/Ly-6cmedium/Ly-6chigh),macrophages(M1/M2),and dendritic cells.RESULTS:Both tissue and antibody specificity were demonstrated by changes in the antibody MFI values.Following storage at-80℃,the MFIs of certain antibodies(such as CD45 and F4/80 in PBMCs,and CD115,Ly-6c,F4/80,CD80 and MHC-II in the spleen)were similar to those of the fresh groups,where-as after storage at 4℃,the MFIs of other antibodies(such as 7-AAD,CD115,Ly-6c and MHC-II in PBMCs,and CD11b,CD206 and CD11c in the spleen)were closer to those of the fresh groups.The MFI of most of the examined anti-bodies varied significantly following storage.Both storage conditions significantly reduced the viability of PBMCs and sple-nocytes.In PBMCs stored at 4℃,the percentages of total monocytes,Ly-6cmedium/Ly-6chigh monocytes,total macrophages,and dendritic cells were similar to those in the fresh group.Compared with the fresh group,both storage groups presented significantly lower percentages of M1 macrophages and dendritic cells(P<0.05).There were no statistically significant differences in the percentages of total monocytes,Ly-6cmedium monocytes,Ly-6chigh monocytes,total macrophages,M1 and M2 macrophages,or dendritic cells in the spleen among the three groups(P>0.05).The percentage of Ly-6clow monocytes did not differ substantially(P>0.05)between the fresh and-80℃frozen groups but was significantly lower in the 4℃storage group than in the fresh group(P<0.05).CONCLUSION:The storage conditions of the samples had a substantial effect on the flow cytometry results(antibody MFI and cell subset percentages)of the PBMCs and splenic cells,with tissue specificity.If the percentage of immune cell subgroups(particularly monocytes/macrophages/dendritic cells)in PBMCs is highly important,storage at 4℃for 7 d is preferable.If the MFI values of specific antibodies(such as CD45 and F4/80)are important,freezing at-80℃may be more appropriate.If the MFI values of most antibodies or the percentages of criti-cal subgroups(such as total monocytes/Ly-6chigh/total macrophages/dendritic cells)in splenic cells need to be close to those of fresh samples,4 ℃ storage for 7 d is more effective.Freezing at-80℃is preferable if the MFI values of particular anti-bodies(such as CD115 and Ly-6c)need to be determined.

Objective:To investigate the association between photodynamic diagnosis(PDD)and treatment efficacy of 5-aminolevu-linic acid(ALA)photodynamic therapy(PDT)in patients with cutaneous squamous cell carcinoma in situ(cSCCis).Methods:A pro-spective cohort study was conducted among the patients with cSCCis who underwent ALA-PDT in Department of Dermatology,Huashan Hospital,Fudan University,from January 2020 to November 2024.All patients were diagnosed with cSCCis based on biopsy,and invasive squamous cell carcinoma was excluded.Clinical information was collected and PDD was performed before treatment,and the patients were divided into moderate fluorescence group and strong fluorescence groups based on fluorescence intensity.All patients received six sessions of PDT treatment at an interval of 1-2 weeks.The primary endpoint was the initial complete clearance rate at 3 months after the last treatment session,and secondary endpoints were the sustained complete clearance rate at 12 months after the last treatment session and treatment failure rate.The multivariate regression analysis,the survival curves,and the Cox regression analysis were used to investigate the association between PDD fluorescence intensity and treatment efficacy,as well as other influencing factors for treatment efficacy.Results:Compared with the strong fluorescence group,the moderate fluorescence group had significantly lower initial complete clearance rate[57.14%(8/14)vs.93.33%(28/30),odds ratio OR=0.100,P=0.010]and sustained complete clearance rate[42.86%(6/14)vs.76.67%(23/30),OR=0.230,P=0.030)].The multivariate regression analysis showed that moderate fluores-cence was an independent risk factor for initial complete clearance(OR=0.030,P=0.030).The moderate fluorescence group had a significantly higher treatment failure rate than the strong fluorescence group[57.14%(8/14)vs.23.33%(7/30),P=0.030].The survival analysis and the Cox regression analysis showed that moderate fluo-rescence was an independent risk factor for PDT treatment failure(hazard ratio=3.040,P=0.048).There were no significant differences in adverse reactions between the two groups.Conclusion:PDD fluorescence intensity can predict the efficacy of ALA-PDT in patients with cSCCis.Moderate fluorescence indicates a higher risk of treatment failure,which can help to guide clinicians in develop-ing individualized treatment strategies.

OBJECTIVE: To analyze the efficacy and safety of decitabine-based myeloablative preconditioning regimen for allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with acute myeloid leukemia (AML). METHODS: The clinical characteristics and efficacy of 115 AML patients who underwent allo-HSCT at the First Medical Center of Chinese PLA General Hospital from August 2018 to August 2022 were retrospectively analyzed, including 37 patients treated with decitabine conditioning regimen (decitabine group) and 78 patients without decitabine conditioning regimen (non-decitabine group). The cumulative incidence of relapse (CIR), overall survival (OS), leukemia-free survival (LFS), non-relapse mortality (NRM) and graft versus host disease (GVHD) were analyzed. RESULTS: For the patients in first complete remission (CR1) state before allo-HSCT, the 1-year relapse rates of decitabine group(22 cases) and non-decitabine group(69 cases) were 9.1% and 29.6%, respectively, the difference was statistically significant(P =0.042). The 1-year cumulative incidence of acute graft-versus-host disease (aGVHD) in decitabine group and non-decitabine group was 62.2% and 70.5%, respectively, and the 1-year cumulative incidence of chronic inhibitor-versus-host disease (cGVHD) was 18.9% and 14.1%, respectively, there were no significant differences in the incidence of aGVHD and cGVHD between the two groups (P >0.05). Of the 115 patients, there were no significantly differences in the 1-year CIR(21.7% vs 28.8%, P =0.866), NRM(10.9% vs 3.9%, P =0.203), OS(75.2% vs 83.8%, P =0.131) and LFS(74.6% vs 69.1%, P =0.912) between the decitabine group(37 cases) and the non-decitabine group(78 cases). CONCLUSION Decitabine-based conditioning regimen could reduce the relapse rate of AML CR1 patients with good safety.
Humans ; Leukemia, Myeloid, Acute/therapy* ; Hematopoietic Stem Cell Transplantation/methods* ; Decitabine/therapeutic use* ; Transplantation Conditioning/methods* ; Retrospective Studies ; Graft vs Host Disease ; Transplantation, Homologous ; Male ; Female ; Adult ; Middle Aged ; Adolescent ; Young Adult

Tubulointerstitial fibrosis (TIF) is one of the key indicators in evaluating the renal function of patients. Mild TIF can cause a vicious cycle of renal tubular glomerular injury and aggravate renal disease. Therefore, studying the mechanisms underlying TIF is essential to identify therapeutic targets, thereby protecting the renal function of patients with timely intervention. Astragaloside IV (AS-IV) is a Chinese medicine component that has been shown to inhibit the occurrence and progression of TIF via multiple pathways. Previous studies have reported that AS-IV protected against TIF by inhibiting inflammation, autophagy, endoplasmic reticulum stress, macrophages, and transforming growth factor-β1, which laid the foundation for the development of a new preventive and therapeutic option for TIF.
Saponins/pharmacology* ; Triterpenes/pharmacology* ; Humans ; Fibrosis ; Animals ; Kidney Tubules/drug effects* ; Kidney Diseases/pathology*

Traditional Chinese medicine formula (TCMF) represents a fundamental component of Chinese medical practice, incorporating medical knowledge and practices from both Han Chinese and various ethnic minorities, while providing comprehensive insights into health and disease. The foundation of TCMF lies in its holistic approach, manifested through herbal compatibility theory, which has emerged from extensive clinical experience and evolved into a highly refined knowledge system. Within this framework, Chinese herbal medicines exhibit intricated characteristics, including multi-component interactions, diverse target sites, and varied biological pathways. These complexities pose significant challenges for understanding their molecular mechanisms. Contemporary advances in artificial intelligence (AI) are reshaping research in traditional Chinese medicine (TCM), offering immense potential to transform our understanding of the molecular mechanisms underlying TCMFs. This review explores the application of AI in uncovering these mechanisms, highlighting its role in compound absorption, distribution, metabolism, and excretion (ADME) prediction, molecular target identification, compound and target synergy recognition, pharmacological mechanisms exploration, and herbal formula optimization. Furthermore, the review discusses the challenges and opportunities in AI-assisted research on TCMF molecular mechanisms, promoting the modernization and globalization of TCM.
Artificial Intelligence ; Drugs, Chinese Herbal/pharmacokinetics* ; Humans ; Medicine, Chinese Traditional ; Animals

Direct electrical stimulation of the human cortex can produce subjective visual sensations, yet these sensations are unstable. The underlying mechanisms may stem from differences in electrophysiological activity within the distributed network outside the stimulated site. To address this problem, we recruited 69 patients who experienced visual sensations during invasive electrical stimulation while intracranial electroencephalography (iEEG) data were recorded. We found significantly flattened power spectral slopes in distributed regions involving different brain networks and decreased integrated information during elicited visual sensations compared with the non-sensation condition. Further analysis based on minimum information partitions revealed that the reconfigured network interactions primarily involved the inferior frontal cortex, posterior superior temporal sulcus, and temporoparietal junction. The flattened power spectral slope in the inferior frontal gyrus was also correlated with integrated information. Taken together, this study indicates that the altered electrophysiological signatures provide insights into the neural mechanisms underlying subjective visual sensations.
Humans ; Male ; Female ; Adult ; Visual Perception/physiology* ; Electric Stimulation ; Middle Aged ; Young Adult ; Electrocorticography ; Electroencephalography ; Brain Mapping

Cemental tear is a rare and indetectable condition unless obvious clinical signs present with the involvement of surrounding periodontal and periapical tissues. Due to its clinical manifestations similar to common dental issues, such as vertical root fracture, primary endodontic diseases, and periodontal diseases, as well as the low awareness of cemental tear for clinicians, misdiagnosis often occurs. The critical principle for cemental tear treatment is to remove torn fragments, and overlooking fragments leads to futile therapy, which could deteriorate the conditions of the affected teeth. Therefore, accurate diagnosis and subsequent appropriate interventions are vital for managing cemental tear. Novel diagnostic tools, including cone-beam computed tomography (CBCT), microscopes, and enamel matrix derivatives, have improved early detection and management, enhancing tooth retention. The implementation of standardized diagnostic criteria and treatment protocols, combined with improved clinical awareness among dental professionals, serves to mitigate risks of diagnostic errors and suboptimal therapeutic interventions. This expert consensus reviewed the epidemiology, pathogenesis, potential predisposing factors, clinical manifestations, diagnosis, differential diagnosis, treatment, and prognosis of cemental tear, aiming to provide a clinical guideline and facilitate clinicians to have a better understanding of cemental tear.
Humans ; Dental Cementum/injuries* ; Consensus ; Diagnosis, Differential ; Cone-Beam Computed Tomography ; Tooth Fractures/therapy*