Background/Aims: The pathophysiology of refractory gastroesophageal reflux disease (RGERD), which differs from proton pump inhibitor dependent gastroesophageal reflux disease (DGERD), remains incompletely elucidated. This study aims to compare esophageal motility patterns, transdiaphragmatic pressure gradients (TPG), and reflux profiles between RGERD and DGERD patients, and to delineate dynamic pressure gradient-esophagogastric junction (EGJ) interactions in these patients. Methods: In this retrospective study, 274 patients who underwent 24-hour impedance-pH monitoring and high-resolution manometry, along with an assessment of proton pump inhibitor responsiveness, were classified as RGERD (32.5%), DGERD (54.4%), or non-GERD (13.1%). Clinical characteristics, TPG, esophageal motility, and reflux metrics were compared between RGERD and DGERD patients. Subgroup analysis excluding hiatal hernia (HH) was conducted to investigate the pathophysiology of RGERD. Results: The RGERD group exhibited a significantly higher proportion of chest pain compared to the DGERD group. Regarding reflux profiles, RGERD patients without HH (RGERDHH- group) experienced increased weakly acidic reflux (P < 0.001) and prolonged bolus exposure (P = 0.006) compared to their counterparts (DGERDHH- group). Mechanistically, the RGERDHH- group showed reduced lower esophageal sphincter basal pressure (P = 0.010) and EGJ contractile integral (P = 0.005). Notably, following a wet-swallow, the RGERDHH- group experienced the significant elevation in gastric pressure and TPG. Correlation analyses revealed weakly acidic reflux and bolus exposure were positively correlated with gastric pressure variation, and inversely correlated with lower esophageal sphincter basal pressure. Conclusions Transient gastric pressure elevation and compromised EGJ barrier function drive weakly acidic reflux and esophageal bolus exposure. This pressure gradient-barrier mismatch underpins the refractoriness of RGERD.

ObjectiveTo investigate the association between sarcopenia and short-term prognosis in patients with acute-on-chronic liver failure （ACLF）， to assess the value of Model for End-Stage Liver Disease （MELD）， MELD combined with serum sodium concentration （MELD-Na）， MELD 3.0， and Chinese Group on the Study of Severe Hepatitis B （COSSH）-ACLF Ⅱ score in predicting the 90-day prognosis of ACLF patients， and to analyze the predictive gain of sarcopenia over traditional prognostic scoring models. MethodsA retrospective study was conducted among 219 patients with ACLF who were hospitalized in The Affiliated Hospital of Inner Mongolia Medical University， No. 2 Hospital of Hohhot， Hohhot First Hospital， and Inner Mongolia People’s Hospital from July 2018 to July 2024. L3 skeletal muscle index （L3-SMI） was measured and calculated based on abdominal computed tomography images， and sarcopenia was diagnosed based on L3-SMI. According to 90-day prognosis， the patients with ACLF were divided into survival group with 124 patients and death group with 95 patients. The chi-square test or the Fisher’s exact test was used for comparison of categorical data between two groups； the independent samples t-test was used for comparison of normally distributed continuous data between two groups， and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups. The multivariate Cox regression analysis （stepwise backward approach） was used to identify independent risk factors for 90-day mortality in ACLF patients. The Kaplan-Meier method was used for survival analysis， and the Log-rank test was used for comparison between groups. The receiver operating characteristic curve was plotted to evaluate the predictive performance of each model， and the DeLong test， bootstrap internal validation， net reclassification improvement （NRI）， and integrated discrimination improvement （IDI） were used to assess the predictive gain of sarcopenia. ResultsThe multivariate Cox regression analysis showed that presence of sarcopenia （hazard ratio ［HR］=3.385， 95% confidence interval ［CI］： 1.722 — 6.657， P<0.05）， older age （HR=1.036， 95%CI： 1.017 — 1.056， P<0.05）， elevated alkaline phosphatase （HR=1.006， 95%CI： 1.004 — 1.009， P<0.05）， elevated total bilirubin （HR=1.003， 95%CI： 1.001 — 1.005， P<0.05）， elevated potassium （HR=1.581， 95%CI： 1.160 — 2.155， P<0.05）， elevated serum creatinine （HR=1.015， 95%CI： 1.007 — 1.024， P<0.05）， and prolonged prothrombin time （HR=1.035， 95%CI： 1.015 — 1.055， P<0.05） were independent risk factors for 90-day mortality in ACLF patients， while elevated albumin （HR=0.885， 95%CI： 0.837 — 0.935， P<0.05） was an independent protective factor against 90-day mortality. MELD， MELD-Na， MELD 3.0， and COSSH-ACLF Ⅱ scores had an area under the ROC curve （AUC） of 0.569， 0.570， 0.632， and 0.659， respectively， for predicting the 90-day prognosis of ACLF patients， suggesting that these scores had unsatisfactory performance； after they were respectively combined with sarcopenia， the AUC was increased to 0.702， 0.687， 0.723， and 0.728， respectively. The DeLong test， NRI， and IDI analyses showed the combined models had significantly better predictive performance （all P<0.05）. ConclusionSarcopenia is an independent risk factor for 90-day mortality in patients with ACLF. Traditional scoring models have limited predictive performance for 90-day prognosis in ACLF patients， and the combination with sarcopenia significantly improves predictive value， suggesting that sarcopenia can be used as an important supplementary indicator for prognostic evaluation of ACLF in clinical practice.

[Objective] To explore the feasibility of using whole blood as a source of NK cells for allogeneic CAR NK cell therapy and activated NK cell reinfusion therapy, and initially construct a technical system for the separation and purification of NK cells from whole blood. [Methods] All peripheral blood mononuclear cells (PBMCs) were enriched from 400 mL of whole blood by manual separation and machine separation, respectively. The erythrocyte loss rate, PBMCs number, NK cell purity of the two methods were compared. NK cells were sorted from PBMCs by three separation and enrichment methods as immunomagnetic bead negative selection method, platelet lysate culture expansion and PERCOLL density gradient separation method, and the purity and yield of NK cells, the activity of NK cells and the tumor-killing ability of the three separation and enrichment methods were compared. [Results] The proportion of NK cells in the lymphocyte population was higher in the manual separation method than in the machine separation method[(13.16±5.16)% vs (8.56±3.92)%, P<0.05]; the number PBMCs was lower in the manual separation method than in the machine separation method[(4.09±1.80)×10⁸vs (6.49±2.16)×10⁸, P<0.05], and there was no difference in the red blood cell loss between the two methods (P>0.05). The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomagnetic was (96.77±2.31)%; the yield was (56.27±10.47)%; the inhibition of tumor proliferation was (38.67±14.05)%; and the tumor killing rate was (19.90±8.05)%. The purity of NK cells isolated and enriched from PBMCs by manual separation method using platelet lysis culture expansion method was the highest at day 7, which was (54.84±15.80)%; the cell expansion multiple could reach 16.92±6.28 at day 7; the in vitro tumor killing rate of NK cells was (15.83±5.5)%; the tumor inhibition rate was (44.33±13.5)%; and there was no difference in the toxicity and activity of NK cells between the two methods (P>0.05). The purity of NK cells isolated and enriched by PERCOLL density gradient separation method was (15.83±5.82)%, and the yield was (14±6.25)%, which was significantly lower than the other two methods. [Conclusion] PBMCs isolated from whole blood by manual separation and NK cells enriched by negative selection with immunomagnetic beads have the potential to provide NK cell materials for CAR-NK cell therapy, and NK cells enriched by platelet lysate-conditioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.

Objective To establish a method for determination of pyriclobenzuron (PBU) residues in rice and paddy environments, and to determine the residual amounts and observe the degradation dynamics of PBU. Methods In July 2022, the paddies of Zhejiang Academy of Agricultural Sciences were selected as experimental fields, and were divided into the blank control group (no pesticide application), the 1-fold-concentration pesticide group (1 kg/667 m²), and the 5-fold-concentration pesticide group (5 kg/667 m²), with a 100 m² area in each group. At the early tillering stage of rice, 20% suspension of PBU sulfate was sprayed once in the 1-fold-concentration and 5-fold-concentration pesticide groups, and rice plants, paddy water and soil samples were collected 2 h, and 1, 2, 3, 5, 7, 11, 14, 21, 28, 35, 49 d and 63 d following spraying PBU, while rice straw, field soil, brown rice and rice husk samples were collected 98 d following spraying. PBU was extracted and purified in samples using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment technique, and the PBU contents were determined in samples using ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The solvent standard working solution and matrix standard working solution were prepared. A linear regression equation was fitted between PBU concentration (x-axis) and peak area (y-axis), and the ratio of the slope (k) of the matrix standard curve to the slope (K) of the solvent standard curve was calculated to evaluate the matrix effect of PBU in samples. According to the Guidelines for Pesticide Residue Testing in Crops (NY/T 788—2018), the addition levels of PBU were set at 0.005, 0.050, 5.000, 1 000.000 mg/kg in rice plants, 0.005, 0.050, 2.000, 10.000 mg/kg in paddy water, 0.005, 0.050, 2.000 mg/kg in soil, and 0.005, 0.050, 5.000 mg/kg in brown rice and rice husks. The recovery and relative standard deviation (RSD) of PBU addition were calculated to evaluate the effectiveness of UPLC-MS/MS for determination of PBU contents. The first-order kinetic equation of PBU concentration was fitted in samples at different sampling time points to analyze the trends in PBU degradation in rice plants, paddy water, and soil, and the half-life of PBU was calculated in different samples. Results There was a good linear relationship between the mass concentration and peak area of PBU at concentrations of 0.000 1 to 0.020 0 mg/kg under solvent and matrix conditions (R² = 0.985 8 to 0.999 7, t = -0.47 to 1.62, all P values < 0.01). The matrix effects of PBU were 70.26%, 65.42% and 65.12% in rice plants, brown rice and rice husks, indicating a matrix-inhibitory effect, and the matrix effect was 87.06% in soils, indicating a weak matrix effect. The recovery of PBU addition was 77.61% to 100.12% in different samples, with RSD of 1.43% to 6.74%, and a limit of quantification (LOQ) of 0.005 mg/kg, and the addition recovery and RSD met the requirements of the Guidelines for Pesticide Residue Testing in Crops (NY/T 788—2018), validating the effectiveness of UPLC-MS/MS assay. Following spraying PBU at a dose of 1 kg/667 m², the half-life of PBU was 6.24 d in rice plants and 3.43 d in paddy water samples, respectively. The final residues of PBU were lower than the LOQ of 0.005 mg/kg in brown rice and rice husk samples 98 d following spraying PBU. Following spraying PBU at a dose of 5 kg/667 m², the half-life of PBU was 15.75 d in rice plants and 7.62 d in paddy water samples, respectively. The final residue of PBU was lower than the LOQ of 0.005 mg/kg in brown rice 98 d following spraying PBU, and the final residue of PBU was 0.049 mg/kg in rice husks. Conclusions A simple, and highly accurate and precise UPLC-MS/MS assay has been developed for determination of PBU residues in rice plants and paddy environments through extraction and purification of PBU from matrix samples using QuEChERS pretreatment. After spraying PBU in paddies, the concentration of PBU gradually decreases in rice plants and paddy water over time, and the final residual concentration is low.

Objective:To establish a modified one-stage clotting assay (mOSA) based on the STA-R Evolution coagulation analyzer for quantifying emicizumab (EMI) concentration and to preliminarily evaluate its analytical performance; meanwhile to explore the clinical utility of the standard one-stage clotting assay (sOSA) in indirectly predicting EMI levels through surrogate factor Ⅷ (FⅧ) activity.Methods:A total of 30 pediatric patients with hemophilia A (HA) treated with EMI in the Hemophilia Treatment Center of Shenzhen Children′s Hospital from January 2023 to March 2025 were enrolled, and 48 post-treatment plasma samples were collected. EMI standards (2.5~100 μg/ml) were prepared using FⅧ-deficient plasma to establish the mOSA detection system. The linearity, accuracy, and precision of the method were evaluated. Surrogate FⅧ activity was measured by sOSA to estimate EMI concentrations, and its correlation with mOSA-derived EMI concentrations was analyzed using Spearman correlation analysis. The equivalent FⅧ activity in patient plasma samples was measured using a human chromogenic substrate assay-based FⅧ activity detection reagent, and Spearman correlation analysis was employed to evaluate its correlations with both the EMI concentrations measured by the mOSA method and estimated by the sOSA method respectively.Results:The established mOSA method for EMI detection showed excellent linearity in the range of 2.5?100 μg/ml ( Y=1.047 X?1.033, R 2=0.995, P<0.001). Average spike recovery rates at 25, 50, and 75 μg/ml were 101.55%(25.39/25.00), 105.31%(52.66/50.00), and 98.20%(73.65/75.00), respectively. Coefficients of variations of within-and inter-batch were 3.47%?4.80% and 6.30%?8.96%, respectively. A prediction model for EMI concentration was established as follows: estimated EMI concentration (μg/ml)=0.095×[alternative FⅧ activity (%) measured by sOSA]+2.652 ( R2=0.999, P<0.001). Validation demonstrated a strong correlation between the EMI concentration measured by the mOSA method and the EMI concentration estimated by the sOSA method ( r=0.989, P<0.001), with good consistency ( Y=1.014 X+0.684, R2=0.972, P<0.001). Both the EMI concentration measured by the mOSA method and the EMI concentration estimated by the sOSA method showed extremely strong correlations with the equivalent FⅧ activity ( r=0.986 and 0.987, respectively; P<0.001 for both). Conclusions:The mOSA system established on the STA-R Evolution analyzer demonstrates robust linearity, accuracy, and reproducibility, fulfilling clinical requirements for therapeutic drug monitoring of EMI. The sOSA method provides reliable indirect estimation of EMI concentrations through surrogate FⅧ activity, offering critical support for emergency decision-making.

Objective To describe the clinical features of patients with primary sclerosing cholangitis(PSC)in China based on a nationwide multicenter patient cohort,and to investigate the risk factors for prognosis.Methods A retrospective cohort study was conducted among the patients with a confirmed diagnosis of PSC based on the electronic medical record system of seven grade A tertiary hospitals across the country,and related data were extracted.The Mann-Whitney U test was used for comparison of continuous data between groups,and the chi-square test was used for comparison of categorical data between groups.The Kaplan-Meier method was used to estimate liver transplant-free survival,and the log-rank test was used for comparison of survival rate between PSC patients with different features.The Cox regression model was used to identify independent risk factors for the prognosis of PSC patients and the interactions between key factors.Results A total of 107 patients were enrolled,among whom 55.6%(55/99)had large-duct PSC and 29.0%(31/107)had comorbidity with inflammatory bowel disease(IBD).The positivity rate of anti-neutrophil cytoplasmic antibody(ANCA)was 32.9%(24/73),and 50.0%(40/80)of the patients had an increase in IgG/IgM.The median symptom-to-diagnosis interval was 1 year(<1-4.0),and 38.3%(41/107)of the patients had progressed to decompensated cirrhosis at the time of diagnosis.The median liver transplant-free survival time was 114 months(95%confidence interval[CI]:62-166),with a 5-year survival rate of 65.7%.The multivariate analysis showed that an increase in total bile acid(TBA)(hazard ratio[HR]=1.006,95%CI:1.002-1.010,P=0.001)and a prolonged symptom-to-diagnosis interval(HR=1.252,95%CI:1.059-1.480,P=0.009)were independent risk factors for prognosis.The interaction analysis showed that compared with the female patients with TBA<50 μmol/L,both male and female patients with TBA≥50 μmol/L had a significant increase in the risk of liver transplantation or death(male:HR=16.563,95%CI:2.103-130.449,P<0.001;female:HR=17.009,95%CI:2.113-136.934,P<0.001),and compared with the patients with an age of<45 years and a TBA level of<50 μmol/L,the patients with an age of≥45 years and a TBA level of≥50 μmol/L had a significant increase in the risk of liver transplantation or death(HR=10.729,95%CI:1.325-86.859,P=0.026).Compared with the female patients with an symptom-to-diagnosis interval of≤2 years,the male patients with a symptom-to-diagnosis interval of>2 years had an increased risk of liver transplantation or death(HR=4.825,95%CI:1.725-13.644,P=0.003),and compared with the patients with an age of<45 years and a symptom-to-diagnosis interval of≤2 years,the patients with an age of<45 years and a symptom-to-diagnosis interval of>2 years had an increased risk of liver transplantation or death(HR=4.983,95%CI:1.366-18.173,P=0.015).Conclusion Compared with the reports from Western countries,large-duct PSC is also the main type of PSC in China,but with a relatively low proportion,and there is also a relatively low proportion of patients with IBD or positive ANCA.An increase in TBA and a prolonged symptom-to-diagnosis interval are independent risk factors for prognosis,with significant interactions with age and sex.This suggests that early screening and intervention should be enhanced to improve prognosis.

Objective To investigate the neuroprotective effects and mechanisms of the alcoholic extract of the Yi drug(Rubia yunnanensis)on the Alzheimer's disease(AD)model of Caenorhabditis elegans(C.elegans).Methods Based on a transgenic C.elegans model of AD,the efficacy and mechanism of alcoholic extracts of Rubia yunnanensis against β-amyloid(Aβ)-induced toxic effects were explored.C.elegans were synchronized and divided into a control group and low and high dose groups of the alcoholic extract of Rubia yunnanensis to study the effects of the alcoholic extract of Rubia yunnanensis on C.elegans paralysis,lifespan,lipofuscin levels,behavior,growth and development,heat stress and oxidative stress,ROS levels,and Aβ aggregation,as well as the key transcription factors in the insulin/insulin-like growth factor 1 signaling pathway were investigated by GFP reporter gene worm assay and RNAi assay DAF-16 expression.Results Compared with the Control group,the intervention of the alcoholic extracts of Rubia yunnanensis could effectively improve the paralyzed phenotype of C.elegans(P<0.001),prolong the lifespan of C.elegans and reduce the level of lipofuscin in C.elegans significantly(P<0.001),improve the mobility of C.elegans significantly(P<0.001),and improve its resistance to oxidative stress and heat stress damage significantly(P<0.001),and reduce the level of ROS in C.elegans significantly(P<0.001),and reduce the deposition of Aβ protein in the head of C.elegans significantly(P<0.01),and had no effect on the growth and development of C.elegans(P>0.05),and also promoted the nuclear translocation of DAF-16 in the reporter gene C.elegans TJ356,and the effect of the alcoholic extracts of Rubia yunnanensis in delaying the degree of paralysis of C.elegans was lost after silencing the expression of C.elegans DAF-16 by RNAi,whereas the L4440 empty vector control group in the administration of Rubia yunnanensis alcohol extract significantly reduced the level of C.elegans paralysis(P<0.001).Conclusion The alcoholic extract of Rubia yunnanensis can significantly ameliorate the paralyzed phenotype in the C.elegans model of Alzheimer's disease with certain antioxidant,anti-aging and anti-Aβ protein activities,and the mechanism may be related to the activation of DAF-16,a downstream transcription factor of the insulin signaling pathway.

Objective:To observe the application effect of dexmedetomidine combined with esketamine nasal drops in pediatric flexible bronchoscopy (referred to as bronchoscopy) surgery with spontaneous respiration retained.Methods:A total of 96 children undergoing bronchoscopy in the Changsha Hospital for Maternal & Child Health Care from May 2022 to December 2023 were selected and divided into the dexmedetomidine nasal drop group (group D, 47 cases) and the dexmedetomidine combined with esketamine nasal drop group (group DE, 49 cases) by random number table method. The mean arterial pressure (MAP), heart rate (HR), blood oxygen saturation (SpO 2) and respiratory rate (RR) of the two groups were observed before nasal drops (T 0), 30 minutes after nasal drops (T 1), 5 minutes after surgery started (T 2), and 10 minutes (T 3), 30 minutes (T 4), 60 minutes (T 5) and 120 minutes (T 6) after surgery. The separation anxiety and mask cooperation scores at T 1, modified Aldrete scores and Pediatric Anesthesia Emergence Delirium Scale (PEAD) scores at T 3-T 6 were evaluated; the number of children with PEAD score>10 was recorded, as well as the time to postoperatively take food and clear water. Results:There was no statistically significant difference in MAP and HR at T 0 between the two groups (all P>0.05); the MAP and HR of Group DE at T 1-T 6 were significantly higher than those of group D (all P<0.05). There was no statistically significant difference in SpO 2 and RR between the two groups within the group or between the groups at each time point (all P>0.05), while the incidence of hypoxemia (SpO 2≤92%) in group D at T 2 was significantly higher than that in group DE ( P<0.05). The separation anxiety and mask cooperation scores of group DE at T 1 were significantly better than those of group D ( P<0.05). The modified Aldrete scores of group DE at T 3, T 4 and T 5 were significantly higher than those of group D (all P<0.05). The PEAD score of group DE at T 3 was significantly lower than that of group D ( P=0.001); there was no statistically significant difference in PEAD scores between the two groups at T 4, T 5 and T 6 (all P>0.05), and no child in either group had PEAD scores>10 at T 4, T 5 and T 6. The time to take clear water and food in group DE was significantly earlier than that in group D (all P<0.05). Conclusions:In pediatric bronchoscopy surgery with spontaneous respiration retained, dexmedetomidine combined with esketamine nasal drops can more effectively relieve children′s separation anxiety, meet surgical needs, improve recovery quality, shorten the postoperative time to take food, and better implement the concept of enhanced recovery after surgery (ERAS) compared with dexmedetomidine alone.

BACKGROUND: Several randomized controlled studies have suggested that the prophylactic use of proton pump inhibitors (PPIs) in intensive care unit (ICU) patients could not reduce the incidence of gastrointestinal bleeding (GIB) and may increase adverse events such as intestinal infection and pneumonia. Gut microbiota may play a critical role in the process. PPIs have been widely prescribed for GIB prophylaxis in patients with acute coronary syndrome (ACS). This study aimed to determine the short-term effects of PPI and histamine-2 receptor antagonist (H2RA) treatment on gut microbiota of ACS patients. METHODS: The study was designed as a single-blind, multicenter, three-parallel-arm, randomized controlled trial conducted at three centers in Beijing, China. We enrolled ACS patients at low-to-medium risk of GIB and randomized (2:2:1) them to either PPI ( n = 40), H2RA ( n = 31), or control group ( n = 21). The primary outcomes were the alterations in gut microbiota after 7 days of acid suppressant therapy. Stool samples were collected at baseline and 7 days and analyzed by 16S ribosomal RNA (rRNA) gene sequencing. RESULTS: There were no significant changes in the diversity of gut microbiota after the short-term use of acid suppressants, but the abundance of Fusobacterium significantly increased and that of Bifidobacterium significantly decreased, especially in PPI users. In addition, the abundance of some pathogenic bacteria, including Enterococcus and Desulfovibrio, was significantly elevated in the PPI users. The fecal microbiota of the PPI users included more arachidonic acid metabolism than that of control group. CONCLUSIONS: PPIs may increase the risk of infection by adversely altering gut microbiota and elevating arachidonic acid metabolism, which may produce multiple proinflammatory mediators. For ACS patients at low-to-medium risk of GIB, sufficient caution should be paid when acid-suppressant drugs are prescribed, especially PPIs. REGISTRATION www.chictr.org.cn (ChiCTR2000029552).
Humans ; Proton Pump Inhibitors/therapeutic use* ; Acute Coronary Syndrome/microbiology* ; Female ; Gastrointestinal Microbiome/drug effects* ; Male ; Middle Aged ; Histamine H2 Antagonists/therapeutic use* ; Aged ; Single-Blind Method