BACKGROUND:The mechanism of programmed death receptor-1(PD-1)effect on osteogenic differentiation of bone marrow mesenchymal stem cells in high glucose environment remains unclear. OBJECTIVE:To explore the effect of PD-1 on osteogenic differentiation of rat bone marrow mesenchymal stem cells in high glucose environment and its regulatory mechanism. METHODS:Rat bone marrow mesenchymal stem cells were randomly divided into normal glucose group(5.6 mmol/L),high glucose group(30 mmol/L),PD-1 overexpression group,PD-1 overexpression no-load group,PD-1 knockdown group,PD-1 knockdown no-load group,and PI3K/AKT pathway inhibitor group(PD-1 knockdown+5 μmol/L LY294002).Rat bone marrow mesenchymal stem cells were cultured in high glucose to simulate the diabetic environment in vitro.The mRNA expression of PD-1 and ligand PD-L1 and the mRNA expression of osteogenic markers Runx2 and OSX in rat bone marrow mesenchymal stem cells were detected by qRT-PCR.The osteogenic differentiation ability was observed by alkaline phosphatase staining and alizarin red staining.Cell proliferation was detected by CCK-8 assay.The protein expressions of PD-1,PD-L1,p-PI3K,and p-AKT were detected by western blot assay. RESULTS AND CONCLUSION:(1)The levels of PD-1 and PD-L1 were significantly increased in the high glucose environment in vitro,and the osteogenic differentiation ability of bone marrow mesenchymal stem cells was inhibited in the high glucose environment.(2)Knockdown of PD-1 expression could promote osteogenic differentiation of bone marrow mesenchymal stem cells,increase cell proliferation activity,and activate the PI3K/AKT pathway.(3)After addition of PI3K/AKT pathway inhibitor LY294002,the ability of bone marrow mesenchymal stem cells to differentiate into osteoblasts decreased.The results show that PD-1 is dependent on the PI3K/AKT signaling pathway to inhibit osteogenic differentiation of rat bone marrow mesenchymal stem cells under high glucose environment.

BACKGROUND:Traditional methods of urinary tract reconstruction are limited by donor scarcity,high complication rates,and suboptimal functional recovery.Tissue engineering strategies offer new directions in this field.Since the urinary tract is mainly composed of muscle tissue,the key is to find suitable seed cells and efficiently induce them to differentiate into smooth muscle cells.Comparative studies on the efficacy of different smooth muscle cell induction regimens are still lacking. OBJECTIVE:To isolate,culture,and identify human urine-derived stem cells,and to compare the effects of two different induction protocols. METHODS:Human urine-derived stem cells were isolated from urine samples of 11 healthy adult volunteers by multiple centrifugations.Surface markers were identified by flow cytometry.The multi-directional differentiation potential of human urine-derived stem cells was verified through osteogenic and adipogenic differentiation.Differentiation was induced by transforming growth factor-β1 or transforming growth factor-β1 combined with platelet derived growth factor for 14 days.Immunofluorescence staining and western blot assay were employed to compare the expression differences of smooth muscle-specific proteins(α-SMA and SM22). RESULTS AND CONCLUSION:(1)Urine-derived stem cells were successfully isolated from the eight urine samples of healthy people.These cells exhibit a"rice grain"-like morphology and possess a robust proliferative capacity.(2)Urine-derived stem cells exhibited high expression of mesenchymal stem cell surface markers(CD73,CD90,and CD44)and extremely low expression of hematopoietic stem cell surface markers(CD34 and CD45).These cells did not express CD19,CD105,and HLA-DR.(3)After osteogenic and adipogenic differentiation,the formation of calcium nodules and lipid droplets was observed,with positive staining results from Alizarin Red S and Oil Red O staining.(4)After 14 days of smooth muscle induction culture,immunofluorescence staining revealed that the smooth muscle differentiation rate of urine-derived stem cells treated with a combination of transforming growth factor-β1 and platelet derived growth factor was significantly higher compared to those treated with transforming growth factor-β1 alone(P<0.005).(5)After 14 days of smooth muscle induction culture,western blot assay further demonstrated that the expression levels of α-SMA and SM22 in the transforming growth factor-β1/platelet derived growth factor group were significantly elevated compared to those in the transforming growth factor-β1 only group(P<0.005).These findings confirm that urine-derived stem cells can be non-invasively isolated using multiple rounds of centrifugation.Compared with transforming growth factor-β1 alone,the combination of transforming growth factor-β1 and platelet derived growth factor can improve the efficiency of inducing urine-derived stem cells to differentiate into smooth muscle cells.

Objective To explore the accuracy of machine learning algorithms based on SHOX2 and RASSF1A methylation levels in predicting early-stage lung adenocarcinoma pathological types. Methods A retrospective analysis was conducted on formalin-fixed paraffin-embedded (FFPE) specimens from patients who underwent lung tumor resection surgery at Affiliated Hospital of Nantong University from January 2021 to January 2023. Based on the pathological classification of the tumors, patients were divided into three groups: a benign tumor/adenocarcinoma in situ (BT/AIS) group, a minimally invasive adenocarcinoma (MIA) group, and an invasive adenocarcinoma (IA) group. The methylation levels of SHOX2 and RASSF1A in FFPE specimens were measured using the LungMe kit through methylation-specific PCR (MS-PCR). Using the methylation levels of SHOX2 and RASSF1A as predictive variables, various machine learning algorithms (including logistic regression, XGBoost, random forest, and naive Bayes) were employed to predict different lung adenocarcinoma pathological types. Results A total of 272 patients were included. The average ages of patients in the BT/AIS, MIA, and IA groups were 57.97, 61.31, and 63.84 years, respectively. The proportions of female patients were 55.38%, 61.11%, and 61.36%, respectively. In the early-stage lung adenocarcinoma prediction model established based on SHOX2 and RASSF1A methylation levels, the random forest and XGBoost models performed well in predicting each pathological type. The C-statistics of the random forest model for the BT/AIS, MIA, and IA groups were 0.71, 0.72, and 0.78, respectively. The C-statistics of the XGBoost model for the BT/AIS, MIA, and IA groups were 0.70, 0.75, and 0.77, respectively. The naive Bayes model only showed robust performance in the IA group, with a C-statistic of 0.73, indicating some predictive ability. The logistic regression model performed the worst among all groups, showing no predictive ability for any group. Through decision curve analysis, the random forest model demonstrated higher net benefit in predicting BT/AIS and MIA pathological types, indicating its potential value in clinical application. Conclusion Machine learning algorithms based on SHOX2 and RASSF1A methylation levels have high accuracy in predicting early-stage lung adenocarcinoma pathological types.

Background The decline of physical activity in the elderly due to aging may increase the risk of sarcopenia. Currently, there is a lack of evidence from large natural populations on the relationship between PA and sarcopenia. Objective To explore the relationship between PA and sarcopenia in the elderly aged 60 years and above in 10 provinces (autonomous regions) of China. Methods Data were retrieved from the 2022—2023 round of the China Development and Nutrition Health Impact Cohort. Personal basic information and PA data were collected by questionnaire survey. Skeletal muscle mass was measured by bio-electrical impedance analysis, muscle strength was measured using a grip dynamometer, and physical performance was reflected by 6-meter walk speed. The Asian Working Group for Sarcopenia (AWGS) 2019 criteria were used to diagnose sarcopenia. Light physical activity (LPA) duration, moderate-to-vigorous physical activity (MVPA) duration, and total physical activity volume were calculated. A total of 3326 residents aged ≥60 years with complete data were selected as the survey participants. Multiple logistic regression models and restricted cubic splines (RCS) were used to assess the association between PA duration/volume and sarcopenia. Results In 10 provinces (autonomous regions) of China, the prevalence of sarcopenia in the elderly aged 60 years and above was 5.5% (95%CI: 4.7%, 6.2%). The logistic regression analysis showed that compared with the elderly reporting 0-7.0 h·week⁻¹ in LPA duration, the risk of sarcopenia in the elderly with LPA duration of >14.0-21.0 h·week⁻¹ was reduced by 51% (OR=0.49, 95%CI: 0.26, 0.96). Compared with the elderly with total physical activity ≥15.0 metabolic equivalent of task (MET)-h·week⁻¹, those with <7.5 MET-h·week⁻¹ had a 2.24-fold increased risk of sarcopenia (OR=2.24, 95%CI: 1.17, 4.29). The RCS results found that LPA duration and total physical activity volume each showed a nonlinear dose-response relationship with the risk of sarcopenia (P_overall<0.05, P_non-linear<0.05). Compared with the elderly with an LPA duration of 0 h· week⁻¹, the risk of sarcopenia in the elderly with an LPA duration of 12.6 h· week⁻¹ was the lowest (OR=0.42, 95%CI: 0.21, 0.83). Compared with the elderly with a total physical activity volume of 15.0 MET-h· week⁻¹, the elderly with a total physical activity volume of 46.0 MET-h· week⁻¹ had the lowest risk of sarcopenia (OR=0.57, 95%CI: 0.38, 0.84). There was no statistically significant association between weekly MVPA duration and the risk of sarcopenia (P>0.05). Conclusion Increasing weekly LPA duration and total physical activity volume would help to reduce the risk of sarcopenia.

Objective To observe the value of vector flow mapping(VFM)technique for assessing changes of left ventricular diastolic function in ovarian cancer(OC)patients who underwent postoperative chemotherapy.Methods Totally 37 OC patients who received postoperative chemotherapy were prospectively enrolled in chemotherapy group,while 40 healthy adults were taken as controls(control group).Routine echocardiography and VFM were performed for chemotherapy group before chemotherapy,after 3 and 6 cycles of chemotherapy,also for controls at enrollment,and comparison was performed between groups before chemotherapy,as well as among different time points within chemotherapy group,and the correlations of VFM results with hemoglobin and routine echocardiographic results in chemotherapy group were analyzed.Results No significant difference of age,body mass,body surface area(BSA),nor hemoglobin level,routine echocardiographic and VFM results before chemotherapy was found between groups(all P＞0.05).With the process of chemotherapy,hemoglobin level gradually decreased,the isovolumic relaxation period(IR),atrial systole period(AS)intraventricular pressure difference(IVPD)and intraventricular pressure gradient(IVPG)of the left ventricle gradually increased(adjusted P＜0.05),whereas routine echocardiography only showed that the left atrial volume index(LAVI)and the ratio of early mitral inflow velocity and the mean mitral annular early diastolic velocity(E/e')increased after 6 cycles of chemotherapy compared with those pre-chemotherapy(adjusted P＜0.05).In chemotherapy group,VFM results in all diastolic subphases were strongly correlated with hemoglobin levels(|r|=0.718 to 0.836,all P＜0.05),weakly to moderately correlated with LAVI(|r|=0.375 to 0.525,all P＜0.05)and moderately correlated with E/e'(|r|=0.424 to 0.537,all P＜0.05).Conclusion The diastolic function of left ventricle was probably damaged in early stage after postoperative chemotherapy in OC patients.VFM might detect slight changes of early diastolic function of left ventricle more sensitively than routine echocardiography.

Objective:To analyze the genetic evolution and molecular characteristics of H7N9 avian influenza virus (AIV) isolated in a live poultry market.Methods:Samples such as poultry feces, sewage, and hair removal machine and chopping board swabs were collected. Real-time fluorescent quantitative PCR was used to detect influenza A virus and H7N9 AIV in the samples. The whole genome of H7N9 AIV was amplified with influenza A virus universal primers and sequenced. BLAST and MEGA X were used for sequence alignment, phylogenetic analysis and molecular characterization.Results:Seven poultry-related environment samples were collected in the live poultry market in Xuchang city in February 2023, and four were positive for H7N9 AIV. The whole genome sequences of three H7N9 AIV isolates were successfully obtained, and the isolates shared high nucleotide identity in different genes (98.37%-100.00%). BLAST analysis showed they were highly identical to H7N9 strains isolated from domestic poultry in China from 2020 to 2021. Genetic evolution analysis showed that the three isolates clustered in the same branch and were closer to the recent environmental isolates than to the recent strains isolated from human or avian. Through comparison with the sequences of the representative strains in different periods, it was found that the isolated strains in this study showed high avian pathogenicity with four amino acids KRAA inserted at the cleavage site; the hemagglutinin receptor-binding site was QSG, which was an avian binding receptor; there was a G186I mutation in hemagglutinin. Mammalian-adaptive mutation E627K was not detected in polymerase basic protein 2. Mutations (R292K and I38T) associated with drug resistance to neuraminidase inhibitor (oseltamivir) and polymerase acidic protein inhibitor (baloshavir) were not detected, suggesting that these isolates remained susceptible to these drugs. A S31N mutation was found in M2 protein, indicating they were resistant to alkamines.Conclusions:The three H7N9 AIV strains isolated in the live poultry market have high avian pathogenicity, but there are no significant increase in mutations related to the binding ability to human receptors, mammalian pathogenicity, viral transmissibility, or drug resistance as compared with previous representative strains causing human or avian infection.

Objectives:To investigate the value of metagenomic next-generation sequencing (mNGS) in the diagnosis of Pneumocystis jirovecii pneumonia (PJP) in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) .Methods:The data of 98 patients with suspected pulmonary infection after allo-HSCT who underwent pathogen detection from bronchoalveolar lavage fluid between June 2016 and August 2023 at Nanfang Hospital were analyzed. The diagnostic performance of mNGS, conventional methods, and real-time quantitative polymerase chain reaction (qPCR) for PJP were compared.Results:A total of 12 patients were diagnosed with PJP, including 11 with a proven diagnosis and 1 with a probable diagnosis. Among the patients with a proven diagnosis, 1 was positive by both conventional methods and qPCR, and 10 were positive by qPCR only. Pneumocystis jirovecii was detected by mNGS in all 12 patients. The diagnostic sensitivity of mNGS for PJP was 100%, which was greater than that of conventional methods (8.3%, P=0.001) and similar to that of qPCR (91.6%, P=1.000) . A total of 75% of the patients developed mixed pulmonary infections, and cytomegalovirus and Epstein-Barr virus were the most common pathogens. Mixed infection was detected in eight patients by mNGS and in five patients by qPCR, but not by conventional methods ( P=0.008) . Conclusions:mNGS had good sensitivity for diagnosing PJP after allo-HSCT and was advantageous for detecting mixed infectious pathogens; therefore, mNGS might be an effective supplement to regular detection methods and qPCR.

Epstein-Barr virus (EBV) associated post-transplant lymphoproliferative disorders (PTLD) are one of the most severe complications after hematopoietic stem cell transplantation (HSCT). This study includes 31 cases of aplastic anemia (AA) patients who developed PTLD after haploidentical transplantation, summarizing their clinical characteristics and categorizing them into either rituximab monotherapy group or combination therapy group based on whether their condition improved by 1 log after a single dose of rituximab. The incidence of PTLD after HSCT in children with AA was 10.16%, and the incidence of PTLD in patients with age >10 years was significantly increased ( χ2=11.336, P=0.010). Of the 31 patients, 27 were clinically diagnosed and 4 were pathologically confirmed. Finally, 15 patients were classified into the rituximab treatment group and 15 patients into the combination treatment groups. Finally three patients died, and the 2-year overall survival rate was (89.7±5.6) %. Standard pre-treatment protocols and EBV reactivation are risk factors affecting the prognosis of PTLD. There was no statistically significant difference in the impact of the two treatment schemes on prognosis.

Objective:To investigate the efficacy and safety of venetoclax combined with the decitabine, cytarabine, and homoharringtonine (HHT) regimen and donor lymphocyte infusion (DLI) for the preventive and salvage therapy of pediatric acute myeloid leukemia (AML) /myelodysplastic syndrome (MDS) after allogeneic hematopoietic stem cell transplantation (HSCT) .Methods:A total of 29 relapsed pediatric/minimal residual disease-positive AML after HSCT were recruited at the Peking University Institute of Hematology from January 1, 2021, to June 1, 2023. They were treated with the above combination regimen and administered with DLI after 24-48 hours at the end of chemotherapy, and the treatment response and adverse reactions were regularly assessed.Results:The overall response rate (ORR) was 75.8%, CR rate was 88.9% (8/9) in the hematologic relapse group, and MRD negativity rate was 61.1% (11/18) in the MRD-positive group. The incidence of agranulocytosis, anemia, and thrombocytopenia with a classification above grade 3 were 100%, 82.7%, and 100%, respectively. The median time of the granulocyte deficiency period was 15 days. Acute graft-versus-host diseases (aGVHD) with a classification of grades Ⅲ-Ⅳ occurred in 11.1% of the patients after DLI, while moderate or severe cGVHD occurred in 7.4% of the patients. The single risk factor for ORR was MNC counts of less than 10×10 8/kg, and the relapse occurred within 100 days. At a median follow-up of 406 days, the 1-year OS was 65%, and the 1-year OS was 57% in the group with no reaction ( P=0.164) compared with 71% in the group who had an overall reaction. Conclusion:The combined regimen based on the DAC, VEN, and modified HA regimen showed a high response rate in the salvage therapy for pediatric AML after the relapse of HSCT. However, bridging to transplantation should be performed immediately after remission to result in a long survival rate.

In recent years, with the wide application of immune checkpoint inhibitors, the landscape of non small cell lung cancer has changed dramatically. Immune checkpoints like programmed death protein/ligand 1(PD-1/PD-L1), cytotoxic T lymphocyte associated antigen-4(CTLA-4) are negative regulators of T-cell immune response. Inhibition of these immune checkpoints can further activate the immune system and promote anti-tumor response. Compared to the limited efficacy of monotherapy, reasonable dual immunotherapy shows great benefit at different stages of anti-tumour immunity. This article aims to comprehensively review the mechanism, treatment regimen and the latest clinical progress of dual immunotherapy so as to provide more precise and individualised immunotherapy for NSCLC patients.