ObjectiveTo prepare a recombinant PETase with a dual-catalytic-triad and to evaluate its efficiency in the biodegradation of polyethylene terephthalate （PET）. MethodsBased on the crystal structure of wild-type PETase， point mutations （T88H/L117D） were introduced via site-directed mutagenesis. The recombinant protein was prepared using prokaryotic expression and chromatography purification techniques. The enzymatic hydrolysis of the mutant PETase was assessed by relatively quantifying the products mono （2-hydroxyethyl） terephthalate （MHET） and terephthalic acid （TPA）. ResultsBoth wild-type and mutant PETases accumulated as inclusion bodies， accounting for approximately 20% of the total bacterial protein. After solubilization in urea， the proteins were eluted at 300 mmol/L imidazole during affinity chromatography purification， with concentrations of 1.824 and 1.833 mg/mL and purities of 83.11% and 84.32%， respectively. Subsequent anion-exchange chromatography yielded highly pure enzymes in the 200 mmol/L NaCl fraction： 2.776 mg/mL （96.86% purity） for the wild type and 1.967 mg/mL （95.13% purity） for the mutant. Following refolding， the final concentrations were 0.484 mg/mL for the wild type and 0.991 mg/mL for the mutant. Hydrolysis assays revealed that the mutant released MHET and TPA at （237.67±17.00）% and （197.33±12.01）% of the wild-type levels， respectively. ConclusionThe T88H/L117D dual-catalytic-triad PETase is successfully prepared and it significantly enhanced PET-degrading activity， thus， it′s a promising biocatalyst for PET bioremediation.

Blood cell morphology examination is the foundation and important means of diagnosing blood diseases. Artificial intelligence (AI) assisted blood cell morphology examination compensates for the shortcomings of artificial microscopy in the early detection and diagnosis of blood diseases,improves diagnostic efficiency,accuracy,and sensitivity,greatly reduces labor and time costs,significantly improves medical quality,and promotes personalized health care. Traditional manual microscopy is still the standard method in clinical practice in China. In order to encourage the improvement and development of intelligence-assisted blood cell morphology examination,this article discusses the current situation and characteristics of intelligence-assisted blood cell morphology examination. Considering the standardization,database,and ethical issues of automated blood cell morphology analysis,some challenges and limitations are summarized and analyzed,which can support the diagnosis of blood diseases and assist researchers and clinical doctors in the future.

Extremely unbalanced data refers to datasets with independent or dependent variables showing severe imbalances in proportions, which might lead to deviation of classical test statistics from theoretical distribution and difficulties in controlling type Ⅰ error. The increased availability of genome-wide resources from large population cohorts has highlighted the growing demand for efficient and accurate statistical methods for the process of extremely unbalanced data to improve the development of genetic statistical methods. This paper introduces two widely used correction methods in current genome-wide association study for extremely unbalanced data, i.e. Firth correction and saddle point approximation, describes their effectiveness in controlling type Ⅰ errors confirmed by simulation experiments, finally, and summarizes the commonly used software for extremely unbalanced genomic data to provide theoretical reference and suggestion for its application for the statistical analysis on extremely unbalanced data in future.

Objective:To investigate the application of time series models in forecasting pre-hospital emergency ambulance trips in Handan City and develop the LPro ensemble model for improved prediction accuracy to support emergency resource allocation.Methods:Pre-hospital emergency data from Handan Emergency Medical Command Center (2019-2023) were retrospectively analyzed. From 324 799 original records, 289 949 valid records were included after cleaning. The training set (2019-2022: 215 918 records) included 35 527 records in 2019, 52 015 in 2020, 61 836 in 2021, and 66 540 in 2022. The validation set (2023) contained 74 031 records. ARIMA, linear trend seasonal, exponential smoothing, and Prophet models were fitted to the training set. The LPro ensemble model was constructed using MAPE-based weighting (linear trend seasonal model: 0.38, Prophet: 0.62). Performance metrics included MAPE, RMSE, MAE, and R 2. Results:Data showed annual growth (compound annual growth rate 23.27%) and seasonal patterns (October peaks, February troughs). Ambulance dispatches increased annually with monthly cyclical patterns. For 2023 validation predictions: ARIMA (MAPE 8.76%, RMSE 619, MAE 491, R 2 0.4563), linear trend seasonal (MAPE 9.83%, RMSE 671, MAE 545, R 2 0.3608), Prophet (MAPE 8.43%, RMSE 562, MAE 503, R 2 0.5513), exponential smoothing (MAPE 8.08%, RMSE 643, MAE 410, R 2 0.4124). LPro model showed superior performance (MAPE 7.05%, RMSE 491, MAE 393, R 2 0.6570), with 16.37% lower MAPE, 12.63% lower RMSE, 21.87% lower MAE, and 19.17% higher R 2 versus Prophet. Conclusion:The LPro ensemble model substantially enhances prediction accuracy and reliability, offering scientific support for emergency resource optimization and dispatch scheduling in Handan City.

A growing body of research points out that gut microbiota plays a key role in tumor immunotherapy. By optimizing the composition of intestinal microbiota, it is possible to effectively improve immunotherapy resistance and enhance its therapeutic effect. This article comprehensively analyzes the mechanism of intestinal microbiota influencing tumor immunotherapy resistance, expounds the current strategies for targeted regulation of intestinal microbiota, such as traditional Chinese medicine and plant components, fecal microbiota transplantation, probiotics, prebiotics and dietary therapy, and explores the potential mechanisms of these strategies to improve patients' resistance to tumor immunotherapy. At the same time, the article also briefly discusses the prospects and challenges of targeting intestinal microbiota to improve tumor immunotherapy resistance, which provides a reference for related research to help the strategy research of reversing tumor immunotherapy resistance.

Objective To investigate the value of the absolute number of lymphocyte subpopulations as an early warning indicator for children with SMPP(severe mycoplasma pneumonia,SMPP)and to analyze the character-istics observed via bronchoscopy,thereby providing a valuable reference for the early diagnosis of SMPP.Methods This study included 102 children with Mycoplasma pneumoniae pneumonia(MPP),comprising 54 cases of common MPP and 48 cases of SMPP.The lymphocyte subpopulations,clinical characteristics,and laboratory indicators were analyzed.Results There were statistically significant differences between the two groups in the absolute number levels of lymphocyte subpopulations CD3+CD19-T,CD4+T,CD3-CD19+B,CD3-/CD16+CD56+NK cells(P<0.05).The absolute numbers of CD3+CD19-T,CD4+T,CD3-CD19+B,and CD3-/CD16+CD56+NK cells showed negative correlations with serum ferritin,LDH,CRP,and D-D,respectively(P<0.05).Multifactorial logistic regression analysis identified the absolute numbers of CD3+CD19-T,CD4+T,CD3-CD19+B,and CD3-/CD16+CD56+NK cells as independent risk factors for severe Mycoplasma pneumoniae pneumonia(SMPP).ROC analysis demonstrated that the areas under the curve for diagnosing SMPP based on the absolute numbers of CD3+CD19-T,CD4+T,CD3-CD19+B,and CD3-/CD16+CD56+NK cells were 0.711,0.887,0.856,and 0.860,respectively,with sensitivities of 47.4%,80.8%,82.1%,and 92.3%,and specificities of 89.7%,87.2%,75%,and 70.5%,respectively.The combined ROC curve of the four lymphocyte subsets had an area of 0.983,with a sensitivity of 97.4%and specificity of 92.3%.The proportions of bronchoscopy findings and microscopic examination of mucus plugs in the SMPP group were significantly higher than those in the ordinary MPP group(P<0.05).In the mucoid plug subgroup,the absolute numbers of CD3?CD19?T cells and CD4?T cells were significantly lower compared to the non-mucoid plug subgroup,while the percentage of CD8?T cells increased and the CD4?/CD8? ratio decreased(all P<0.05).Conclusions The absolute number of CD3+CD19-T,CD4+T,CD3-CD19+B,and CD3-/CD16+CD56+NK cells in peripheral blood serves as a highly sensitive and specific predictor of small airway mucus plugging phenomenon(SMPP)and can thus be utilized as a potential biomarker for SMPP.Microscopic analysis under SMPP conditions reveals a high prevalence of mucus plugs,necessitating proactive bronchoscopic intervention.Furthermore,the significant imbalance in T cell subpopulations is strongly correlated with the formation of mucus plugs observed during bronchoscopy.

Objective:To analyze the safety and short-term efficacy of thoracic radiotherapy combined with anlotinib in the treatment of inoperable non-small cell lung cancer (NSCLC).Methods:A prospective study was conducted on patients with unresectable locally advanced NSCLC who were intolerant to concurrent chemoradiotherapy and treated at the Department of Radiation Oncology, Cancer Hospital, Chinese Academy of Medical Sciences, from October 2020 to September 2023. Anlotinib was administered orally concurrently with radiotherapy (days 1-14, 21 days per cycle, for 3 cycles). Adverse effects and short-term tumor recurrence were observed from the beginning of radiotherapy to the 3-month post-radiotherapy. Kaplan-Meier method was used to calculate progression-free survival (PFS) and overall survival (OS) rates from the date of initial treatment (induction therapy), and intergroup comparisons were performed using the log-rank test.Results:The median age was 62 years (range:42-76 years), with a male predominance ( n=36, 88%) of the included 41 patients. The incidence of grade 3-4 acute hematologic adverse events was 20% (8 cases); the incidence of grade 3 hemoptysis was 2% (1 case), with no grade 4 hemoptysis; the incidence of grade 3-4 radiation pneumonitis was 10% (4 cases). No grade 5 adverse events were observed in the entire cohort. With a median follow-up of 19.7 months (range: 7.1-50.1 months), 19 patients (46%) experienced recurrence, including 4 patients (10%) with local recurrence, 6 patients (15%) with regional lymph node recurrence, and 11 patients (27%) with distant metastases. The 1-year PFS rate was 78.3%. 8 patients (20%) died, including 3 patients died from COVID-19 infection during the follow-up period, 1 patient who died from hypostatic pneumonia due to prolonged bed rest after cerebral infarction, and 4 patients died from tumor-related causes. The 1-year OS rate was 78.0%. Conclusions:Thoracic radiotherapy combined with anlotinib demonstrates good safety, manageable adverse events, and favorable short-term efficacy in NSCNC patients intolerant to concurrent chemoradiotherapy.

Objective:To evaluate the role of a single PET-CT scan in predicting survival and prognosis in patients with non-small cell lung cancer (NSCLC) who did not undergo surgery but received radiotherapy after neoadjuvant chemotherapy combined with immunotherapy.Methods:A retrospective analysis was conducted on the data of 23 NSCLC patients treated at the Cancer Hospital of the Chinese Academy of Medical Sciences from May 2022 to June 2024. All patients were pathologically confirmed, received neoadjuvant chemotherapy combined with immunotherapy, did not undergo surgery for various reasons, and instead received radiotherapy. Each patient underwent only one PET-CT scan after neoadjuvant chemotherapy combined with immunotherapy and before radiotherapy. According to the maximum standardized uptake value (SUV max) on PET-CT, patients were divided into the low-uptake group (SUV max < 8, n=12) and high-uptake group (SUV max ≥ 8, n=11). Survival analysis was performed using the Kaplan-Meier method with survival curves plotted. Univariate analysis of influencing factors of survival was conducted using the Cox proportional hazards regression model. Clinical characteristics and survival outcomes of the two groups were compared, including progression-free survival (PFS) and overall survival (OS). Results:The 1-year PFS rates were 100% in the low-uptake group, 54.5% in the high-uptake group. This difference was statistically significant ( P=0.007). The 1-year and 2-year OS rates were both 100% in the low-uptake group, the 1-year and 2-year OS rates were both 90.9% in the high-uptake group, with no statistically significant difference ( P=0.394). Univariate Cox analysis identified age as an independent factor affecting PFS. Conclusions:For NSCLC patients who did not undergo surgical resection but received radiotherapy after neoadjuvant chemotherapy combined with immunotherapy, a single PET-CT scan before radiotherapy has potential value in predicting PFS. However, clinical studies with larger sample size and longer follow-up are required to evaluate its predictive value for OS.

This study aims to investigate the effects of Pulsatilla powder(PP)on colonic intestinal flora and tryptophan metabolism in piglets with diarrhea.Twenty-four weaned piglets were ran-domly divided into the normal control group,model group,self-healing group,and PP group.The model of piglet diarrhea was established by the compound factor of external evil+internal injury+epi-demic virus,and treated with PP.During the modeling period,the body weight,mental status and fecal patterns of piglets were monitored daily.After the experiment,the colon tissues were collect-ed for histopathological observation,and the inflammatory factors IL-1β and IL-6 in the colon tis-sue was detected by ELISA,16S rRNA was used to analyze colonic intestinal flora and RT-qPCR was used to determine the expression of genes related to tryptophan metabolism in the colon.The results showed that,compared with the model group,PP significantly improved the pathological status of the colon in piglets with diarrhea,with elevated body mass and a significant increase in goblet cells(P＜0.05);and significantly reduced the levels of IL-1β and IL-6 in the colon of diar-rhea piglets(P＜0.05).The results of intestinal flora showed that PP increased the abundance of Firmicutes(P＜0.01),Spirochaeota(P＜0.01)and Lactobacillus(P＜0.05),reduced the abun-dance of Bacteroidota(P＜0.05)、Prevotella_NK3B31_group(P＜0.05)and Clostridium_sensu_stricto 1(P＜0.01).Meanwhile,PP significantly reduced the expression level of TPH1 mRNA(P＜0.05),increased the expression levels of AhR(P＜0.05)and IL-22 mRNA in the co-lon of diarrhea piglets.These results indicates that PP may alleviate diarrhea in piglets by regula-ting colonic intestinal flora and genes related to intestinal tryptophan metabolism.This study pro-vides data support for the subsequent investigation of piglet diarrhea prevention and treatment.

Objective To investigate the value of the absolute number of lymphocyte subpopulations as an early warning indicator for children with SMPP(severe mycoplasma pneumonia,SMPP)and to analyze the character-istics observed via bronchoscopy,thereby providing a valuable reference for the early diagnosis of SMPP.Methods This study included 102 children with Mycoplasma pneumoniae pneumonia(MPP),comprising 54 cases of common MPP and 48 cases of SMPP.The lymphocyte subpopulations,clinical characteristics,and laboratory indicators were analyzed.Results There were statistically significant differences between the two groups in the absolute number levels of lymphocyte subpopulations CD3+CD19-T,CD4+T,CD3-CD19+B,CD3-/CD16+CD56+NK cells(P<0.05).The absolute numbers of CD3+CD19-T,CD4+T,CD3-CD19+B,and CD3-/CD16+CD56+NK cells showed negative correlations with serum ferritin,LDH,CRP,and D-D,respectively(P<0.05).Multifactorial logistic regression analysis identified the absolute numbers of CD3+CD19-T,CD4+T,CD3-CD19+B,and CD3-/CD16+CD56+NK cells as independent risk factors for severe Mycoplasma pneumoniae pneumonia(SMPP).ROC analysis demonstrated that the areas under the curve for diagnosing SMPP based on the absolute numbers of CD3+CD19-T,CD4+T,CD3-CD19+B,and CD3-/CD16+CD56+NK cells were 0.711,0.887,0.856,and 0.860,respectively,with sensitivities of 47.4%,80.8%,82.1%,and 92.3%,and specificities of 89.7%,87.2%,75%,and 70.5%,respectively.The combined ROC curve of the four lymphocyte subsets had an area of 0.983,with a sensitivity of 97.4%and specificity of 92.3%.The proportions of bronchoscopy findings and microscopic examination of mucus plugs in the SMPP group were significantly higher than those in the ordinary MPP group(P<0.05).In the mucoid plug subgroup,the absolute numbers of CD3?CD19?T cells and CD4?T cells were significantly lower compared to the non-mucoid plug subgroup,while the percentage of CD8?T cells increased and the CD4?/CD8? ratio decreased(all P<0.05).Conclusions The absolute number of CD3+CD19-T,CD4+T,CD3-CD19+B,and CD3-/CD16+CD56+NK cells in peripheral blood serves as a highly sensitive and specific predictor of small airway mucus plugging phenomenon(SMPP)and can thus be utilized as a potential biomarker for SMPP.Microscopic analysis under SMPP conditions reveals a high prevalence of mucus plugs,necessitating proactive bronchoscopic intervention.Furthermore,the significant imbalance in T cell subpopulations is strongly correlated with the formation of mucus plugs observed during bronchoscopy.