Objective:To establish a sensing technology of catalytic hairpin self-assembly (CHA) combining with clustered interspaced short palindromic repeats with associated protein 12a (CRISPR-Cas12a) for the detection of exosomal microRNA-21 (miR-21), and to analyze the performance.Methods:Eight patients diagnosed as breast cancer in the First Affiliated Hospital of the Army Military Medical University from September to October 2023 were selected as the breast cancer group; 8 healthy individuals who underwent physical examinations during the same period were selected as the healthy control group. Plasma exosomes and their miR-21 were extracted using the kit. DNA hairpins and CRISPR RNA sequences were designed for miR-21 sequences. The feasibility of detection technology was validated using polyacrylamide gel electrophoresis and fluorescence spectrophotometer. Hairpins concentration, CHA reaction time, Cas12a protein concentration and Cas12a protein reaction time were further optimized. On this basis, miR-21 was detected at different concentrations (0, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0 nmol/L), and fluorescence intensity was collected for unary linear regression analysis to evaluate methodological sensitivity; meanwhile, different types of miRNAs (miR-31, miR-26a, miR-192, miR-25-3p) and blank controls were detected to evaluate methodological specificity. A case-control study was conducted to detect the relative expression level of plasma exosomal miR-21 in breast cancer group and healthy control group using this detection technology and reverse transcription PCR (RT-PCR) to evaluate the detection ability of clinical samples.Results:A detection method for exosomal miR-21 was established using CHA combining with CRISPR-Cas12a. The concentration of miR-21 detected by this method showed a good linear relationship with fluorescence intensity (the linear correlation coefficient 0.966 7), and the linear detection range was 0.1-10.0 nmol/L, and the detection limit was 87.81 pmol/L. The fluorescence intensity of miR-21 was 450.27±23.96 which was higher than that of miR-31, miR-26a, miR-192, miR-25-3p, and the blank group (98.89±7.35, 98.12±2.07, 98.93±2.45, 96.66±2.45, 82.93±3.54, respectively), with statistical significance ( P<0.001). The results of RT-PCR showed that the relative expression levels of plasma exosomal miR-21 in the breast cancer group were higher than that in healthy control group (1.83±0.27 vs 0.93±0.12, P<0.001); CHA combining with CRISPR-Cas12a detection technology showed that the relative expression levels of plasma exosomal miR-21 in breast cancer group were higher than that in healthy control group (1.94±0.21 vs 0.98±0.08, P<0.001); There was no significant difference in the relative expression levels of plasma exosomal miR-21 between CHA combining with CRISPR-Cas12a detection technology and reverse transcription PCR in breast cancer group and healthy control group ( P>0.05). Conclusion:In this study, a highly sensitive and specific sensing technology of CHA combining with CRISPR-Cas12a for exosomal miR-21 was established. The results of detecting plasma exosomal miR-21 were consistent with the results of reverse transcription PCR, which can be used for screening of breast cancer patients.

Objective:To explore the produced-radiation brain damage in simulated solar particle events and to provide evidence for health risk assessment of radiation from manned deep space exploration.Methods:According to the main characteristics of solar particle events, mice were treated with total body irradiation (TBI) with 90 MeV protons in a dose range from 0.1 to 2 Gy, with irradiation dose of 0, 0.1, 0.3, 0.5, 1, 2 Gy, respectively. At 3 and 7 d after irradiation, the behavior of mice was examined using balance beam tests, rotarod tests, and new object recognition tests. Then, the density of dendritic spines and the number of Nissl bodies in the hippocampus were measured using Golgi and Nissl staining. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and neurotransmitter content in brain tissue were detected using the WST-8 method, TBA method, and high pressure liquid chromatography (HPLC), respectively. Besides, cell apoptosis was determined using the TUNEL method, and the dose-response relationship, a function of dose change with damage index, was analyzed using linear and linear square fitting method. Finally, the minimum radiation dose causing a significant change in all indicators of brain damage was determined as the brain damage threshold.Results:Compared to the control group, 1 Gy proton irradiation result ed in a significant decrease in the density of filopod dendritic spines ( t = 1.82, 2.30, P < 0.05) and a significant increase in abnormal Nissl bodies in the CA1 region ( t = 2.44, 3.77, P < 0.05). At 3 and 7 d after irradiation, as well as a significant increase in the DA ( t = 2.52, P<0.05) and Glu contents ( t = 4.04, P < 0.05) on day 7. In contrast, 2 Gy proton irradiation result ed in a decrease in SOD activity on day 3 ( t = 3.44, P < 0.05), and an increase in the MDA content ( t = 1.90, 2.14, P < 0.05), hippocampal cell apoptosis (t = 3.91, 3.54, P < 0.05), and 5-HT levels ( t = 2.81, 2.69, P < 0.05), together with a decrease in climbing time in the rotarod tests ( t = 2.85, 2.64, P<0.05) and propensity to recognize new objects ( t = 2.87, 2.84, P < 0.05) on days 3 and 7. Furthermore, a dose-response relationship was observed in the dose range from 0.1 to 2 Gy ( R2=0.74-0.99). Conclusions:The dose threshold of 90 MeV protons inducing brain damage in mice is inferred to be 1 Gy, and 14 dose-response models are developed, providing a biological basis for organ dose capping and risk assessment of crew experiencing short-term deep space flights.

Objective To construct a risk prediction model for healthcare-associated infection(HAI)in stroke pa-tients,accurately and effectively screen out potential high-risk groups,and formulate targeted preventive interven-tions to reduce the occurrence of infection.Methods Stroke patients in the"Henan Stroke Cohort"in 2019-2021 were selected as the study objects,and relevant clinical data were collected as the main analysis data for model con-struction and internal validation.The relevant data of stroke patients in three hospitals that had never participated in the cohort construction from January to September 2022 were randomly selected as a test set for external validation of the risk prediction model.The main analysis data were randomly divided into a training set and a test set,and a risk prediction model was constructed based on logistic regression,artificial neural network(ANN)algorithm,extreme gradient boosting algorithm and random forest algorithm,respectively.Multiple indicators were used to evaluate the prediction performance of the model,and the optimal model was externally validated based on the test set data.Results The infection rate of stroke patients was 20.6％in the main analysis data and 56.4％in the test set data.The accuracy of the risk prediction model based on logistic regression was 91.2％,the area under the re-ceiver operating characteristic(ROC)curve(AUC)was 0.938,the precision rate,recall rate,specificity,and the F1 score were 0.851,0.695,0.968,and 0.765,respectively.The accuracy rate,precision rate,specificity and AUC of the logistic risk prediction model and the ANN risk prediction model were all significantly better than other models,while the recall rate and F1 score of the logistic risk prediction model were slightly better than the ANN risk prediction model.The logistic risk prediction model had excellent prediction performance in external validation.Conclusion HAI risk prediction model of stroke patients based on logistic regression can better screen out high-risk stroke patients with infection risk,and can contribute to formulate targeted preventive interventions to reduce the occurrence of infection.

Objective To understand the whole genome sequence characteristics of a Coxsackievirus A4(CVA4)isolated from Yunnan,China in 2022,and explore the phylogenetic characteristics of CVA4.Methods The whole genome sequence of CVA4 isolate 194R3/YN/CHN/2022 was amplified and sequenced,and the phylogenetic tree of CVA4 isolate was constructed by using Mega 7.0,Geneious 9.1.4 and Simplot 3.5.1 softwares.The whole ge-nome sequence characteristics were analyzed.Results The 194R3/YN/CHN/2022 isolate was identified as CVA4,belonging to the C2 gene subtype,which was consistent with the dominant gene subtype in recent years in China.Recombination analysis showed that recombination of CVA4 virus isolate with EVA114 prototype(V13-0285),CVA16 prototype(G-10),and CVA14 prototype(G-14)at the non-structural coding regions of P2 and P3 may have occurred.Conclusion The 194R3/YN/CHN/2022 isolated from Yunnan belongs to the C2 gene subtype,which is the prevalent CVA4 in China,but with certain mutations.

Following the principles of evidence-based medicine,in accordance with the structure and drafting rules of standardized documents,based on literature research,according to the characteristics of chronic cough in children and issues that need to form a consensus,the TCM Guidelines for Diagnosis and Treatment of Chronic Cough in Children was formulated based on the Delphi method,expert discussion meetings,and public solicitation of opinions.The guideline includes scope of application,terms and definitions,eti-ology and diagnosis,auxiliary examination,treatment,prevention and care.The aim is to clarify the optimal treatment plan of Chinese medicine in the diagnosis and treatment of this disease,and to provide guidance for improving the clinical diagnosis and treatment of chronic cough in children with Chinese medicine.

Objective:To investigate the impact of donor human leukocyte antigen (HLA) -Bw4 expression on natural killer (NK) cell reconstitution and transplant outcomes in recipients undergoing haploidentical hematopoietic stem cell transplantation (HSCT) from maternal or related donors without ex vivo T-cell depletion.Methods:This study prospectively enrolled 32 patients who received T-replete haploidentical HSCT from maternal or collateral donors (cohort 1) to evaluate the facilitating effect of donor HLA-Bw4 expression on NK cell reconstitution. Furthermore, a retrospective analysis was conducted on 278 patients who underwent T-replete haploidentical HSCT from maternal or collateral donors (cohort 2) to analyze the impact of donor HLA-Bw4 expression on HSCT outcomes. Thus, a comparison was made between the effects of donor HLA-Bw4 expression on HSCT outcomes in patients receiving or not receiving post-transplant cyclophosphamide (PT-Cy) conditioning.Results:Donors expressing HLA-Bw4 alleles facilitated NK cell reconstitution and functional recovery, which remained unaffected by PT-Cy. Donors with HLA-Bw4 expression were associated with reduced transplant-related mortality (TRM), particularly mortality related to infections. The use of PT-Cy did not impact the ability of donor HLA-Bw4 to decrease TRM.Conclusion:In haploidentical HSCT from maternal or related donors without ex vivo T-cell depletion, the presence of donor HLA-Bw4 expression promotes rapid NK cell reconstitution and functional recovery and is significantly associated with lower TRM, especially infection-related mortality. These findings underscore the clinical significance of donor HLA-Bw4 expression in patients who underwent HSCT. Hence, the consideration of donor HLA-Bw4 in recipient selection and HSCT strategies holds important clinical implications.

Activation of nuclear factor erythroid 2-related factor 2(Nrf2)by Kelch-like ECH-associated protein 1(Keap1)alkylation plays a central role in anti-inflammatory therapy.However,activators of Nrf2 through alkylation of Keap1-Kelch domain have not been identified.Deoxynyboquinone(DNQ)is a natural small molecule discovered from marine actinomycetes.The current study was designed to investigate the anti-inflammatory effects and molecular mechanisms of DNQ via alkylation of Keap1.DNQ exhibited signif-icant anti-inflammatory properties both in vitro and in vivo.The pharmacophore responsible for the anti-inflammatory properties of DNQ was determined to be the α,β-unsaturated amides moieties by a chemical reaction between DNQ and N-acetylcysteine.DNQ exerted anti-inflammatory effects through activation of Nrf2/ARE pathway.Keap1 was demonstrated to be the direct target of DNQ and bound with DNQ through conjugate addition reaction involving alkylation.The specific alkylation site of DNQ on Keap1 for Nrf2 activation was elucidated with a synthesized probe in conjunction with liquid chromatography-tandem mass spectrometry.DNQ triggered the ubiquitination and subsequent degra-dation of Keap1 by alkylation of the cysteine residue 489(Cys489)on Keap1-Kelch domain,ultimately enabling the activation of Nrf2.Our findings revealed that DNQ exhibited potent anti-inflammatory capacity through α,β-unsaturated amides moieties active group which specifically activated Nrf2 signal pathway via alkylation/ubiquitination of Keap1-Kelch domain,suggesting the potential values of targeting Cys489 on Keap1-Kelch domain by DNQ-like small molecules in inflammatory therapies.

Background: The incidence density of metabolic dysfunction-associated fatty liver disease (MAFLD) and the effect of a healthy lifestyle on the risk of MAFLD remain unknown. We evaluated the prevalence and incidence density of MAFLD and investigated the association between healthy lifestyle and the risk of MAFLD. Methods: A cross-sectional analysis was conducted on 37,422 participants to explore the prevalence of MAFLD. A cohort analysis of 18,964 individuals was conducted to identify the incidence of MAFLD, as well as the association between healthy lifestyle and MAFLD. Cox proportional hazards regression was used to calculate the hazard ratio (HR) and 95% confidence interval (CI) with adjustments for confounding factors. Results: The prevalence of MAFLD, non-alcoholic fatty liver disease, and their comorbidities were 30.38%, 28.09%, and 26.13%, respectively. After approximately 70 thousand person-years of follow-up, the incidence densities of the three conditions were 61.03, 55.49, and 51.64 per 1,000 person-years, respectively. Adherence to an overall healthy lifestyle was associated with a 19% decreased risk of MAFLD (HR, 0.81; 95% CI, 0.72 to 0.92), and the effects were modified by baseline age, sex, and body mass index (BMI). Subgroup analyses revealed that younger participants, men, and those with a lower BMI experienced more significant beneficial effects from healthy lifestyle. Conclusion Our results highlight the beneficial effect of adherence to a healthy lifestyle on the prevention of MAFLD. Health management for improving dietary intake, physical activity, and smoking and drinking habits are critical to improving MAFLD.

Objective To investigate the impact of overweight on the prognosis of patients with acute mild ischemic stroke or moderate-high risk transient ischemic attack (TIA). Methods A total of 366 patients with acute mild ischemic stroke or moderate-high risk TIA who carried CYP2C19 loss-of-function alleles were included in the study. Baseline data were collected at admission, and clinical outcomes were collected within 3 months post-onset (primary outcomes: stroke recurrences within 3 months post-onset; secondary outcomes: composite outcome of stroke recurrence or death within 3 months post-onset, vascular events, and quality of life within 3 months). Kaplan-Meier method was used to plot the cumulative incidence curve of outcomes. Multivariate Cox proportional hazards model and multivariate Logistic regression model were used to evaluate the relationship between overweight and clinical outcomes within 3 months post-onset in patientswith ischemic stroke or TIA. The integrated discrimination improvement index (IDI) and net reclassification improvement index (NRI) were calculated to assess the predictive value of adding body mass index (BMI, whether overweight or not) based on traditional models for predicting the prognosis of acute ischemic stroke or TIA. Results After 3 months of follow-up, 28 patients had stroke recurrence, 1 patient died, and 31 patients had vascular events. The Kaplan-Meier cumulative incidence curve showed that the cumulative incidences of stroke recurrence, stroke recurrence or death, and vascular events after 3 months of onset were lower in overweight patients compared with non-overweight patients (Log-rank P < 0.05). Multivariate analysis showed that compared with non-overweight patients, overweight patients had significantly reduced risks of stroke recurrence within 3 months (HR=0.24; 95%CI, 0.08 to 0.71), composite outcome of stroke recurrence or death (HR=0.24; 95%CI, 0.08 to 0.69), and vascular events (HR=0.22; 95%CI, 0.07 to 0.63), and significantly improved quality of life within 3 months (OR=0.39; 95%CI, 0.20 to 0.76). The IDI and NRI calculations showed that compared with traditional models, the new model adding BMI (whether overweight) had significantly improved predictive ability for the prognosis of acute ischemic stroke or TIA. Conclusion Overweight may be a protective factor for the prognosis of TIA patients with acute mild ischemic stroke or moderate-high risk who carry CYP2C19 loss-of-function alleles. Compared with non-overweight patients, overweight patients have reduced risks of stroke recurrence, composite outcome of stroke recurrence or death, and vascular events within 3 months post-onset, and improved quality of life within 3 months post-onset.

The major vascular complications associated with diabetes make the management of diabetic mellitus erectile dysfunction (DMED) a challenging endeavor. Notable factors contributing to DMED include oxidative stress, nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway activation, and apoptosis, while nitro-oleic acid (NO2-OA) has been shown to be beneficial in treating these aspects of this condition. We, herein, investigated the effects and possible mechanisms of NO2-OA on erectile function as assessed in a streptozotocin-induced rat model of diabetes. Our results revealed that the erectile function of DMED rats was significantly impaired compared with that of the control group. However, in response to 4 weeks of NO2-OA treatment, there was an improvement in erectile function. The expression of oxidative stress-related indicators was significantly increased and the NO/cGMP pathway was impaired in the DMED group. The expression of proapoptotic factors was increased, while that of antiapoptotic factors was decreased in the DMED group. Moreover, the cell morphology in the cavernous tissue of the DMED group also changed adversely. NO2-OA treatment significantly reversed all these changes observed in the DMED group. In conclusion, NO2-OA treatment partially improved erectile function in DMED rats through mechanisms that included inhibition of oxidative stress, activation of the NO/cGMP pathway, and a reduction in apoptosis.