Objective To investigate the impact of test volume of flow imaging microscopy(FIM) on detection performance,and to determine a FIM test volume with practical application value.Methods Two FIM platforms from different manufac-turers were employed to systematically evaluate the performance of four test volumes across three particle concentration levels,two sample matrices,and three particle types,in order to clarify the effect of test volume on FIM measurement results.Results The findings showed that within the practically operable test volume range,differences in measurement outcomes among different test volumes were generally small,indicating good methodological stability.Notably,a test volume of appro-ximately 200 μL provided the most favorable balance among repeatability,measurement stability,and test representative-ness,yielding the lowest relative standard deviation(RSD) values.Conclusion A test volume of 200 μL appears to be a suitable choice that balances repeatability and representativeness,although further optimization should be conducted based on sample-specific characteristics and related factors.This study provides valuable preliminary evidence to support the standardization and procedural development of FIM-based methods for insoluble particle testing.

Chinese patent medicines are the core preparations for disease prevention and treatment in clinical traditional Chinese medicine （TCM）. Their quality is the key to ensuring safe and effective medication and promoting the high-quality development of the TCM industry. At present， the quality evaluation of Chinese patent medicines faces prominent problems， such as inadequate control of quality consistency， over-reliance on single indicators， one-sided evaluation dimensions， unstable statistical methods， and poor implementability of research findings. These defects lead to insufficient scientificity of evaluation results， making it difficult to support the implementation of policies including centralized procurement， high quality， and favorable prices for Chinese patent medicines. According to the core requirements of quality evaluation for Chinese patent medicines， this study established a four-dimensional comprehensive evaluation system encompassing consistency index（CI）， content index（MI）， effect index（EI）， and preparation index（PI）. All evaluation indicators were normalized to achieve horizontal comparability. Meanwhile， an improved radar chart was introduced to realize the visualization of evaluation results. This method can systematically integrate four types of core quality attributes： Quality consistency， component content， biological activity， and preparation characteristics. It can effectively distinguish quality differences among products of the same specification from different manufacturers. The evaluation results are unique， stable， and not affected by the addition or reduction of samples， which takes scientificity， objectivity， and operability into account. This method can provide scientific and technical support for screening high-quality Chinese patent medicines and implementing policies of centralized procurement， high quality， and favorable prices. Moreover， it has important practical significance for improving the quality evaluation system of Chinese patent medicines and advancing the standard and modern development of TCM.

ObjectiveTo establish a comprehensive evaluation framework combining multi-component quantification with daily intake in response to the challenges posed by multiple coexisting dosage forms， single-faceted quality evaluation， and ambiguous clinical equivalence of Huoxiang Zhengqi preparations， and to compare the quality differences and superiority among eight commercially available dosage forms. MethodsA total of 75 batches of Huoxiang Zhengqi preparations from 25 manufacturers were investigated. Ultra-performance liquid chromatography-triple quadrupole-tandem mass spectrometry （UPLC-QqQ-MS/MS） coupled with gas chromatography-triple quadrupole-tandem mass spectrometry （GC-QqQ-MS/MS） was adopted to simultaneously quantify 20 core marker components. Normalization was performed based on equivalent raw herb concentrations to eliminate interferences from dosage forms， excipients， and specifications. Considering the specified clinical dosage， the actual daily intake level was calculated to comprehensively evaluate component retention， content uniformity， and potential differences in in vivo exposure among different dosage forms. ResultsThe quality of Huoxiang Zhengqi preparations differed greatly among dosage forms and manufacturers. On the basis of the recommended daily doses in the package inserts， the total daily intake of the 20 tested components ranged from 3.14 to 614.30 mg·d^-1， while that of hesperidin， a quality-control marker， ranged from 0.03 to 40.65 mg·d^-1. Atractylodin was detected only in tinctures and soft capsules， with concentrations ranging from （2.68±0.01） to （5.73±0.37） mg·L^-1. The soft capsules from manufacturer M22 had the highest atractylodin level， while atractylodin was not detected in the other dosage forms. Nucleosides were most abundant in the oral liquids and tinctures （M4 to M13）. The tincture from manufacturer M5 showed the highest nucleoside level of 29.12 mg·L^-1， whereas the granules had the lowest nucleoside levels， with that of M23 being only 3.48 mg·L^-1. Perillae Folium-related components varied greatly among batches， with an relative standard deviation （RSD） of 87.55%. The daily intake of hesperidin from all three granule products was below 3.6 mg·d^-1， the reference value calculated for Huoxiang Zhengqi Shui according to the 2025 edition of the Chinese Pharmacopoeia. ConclusionThe levels of active components differed significantly among different dosage forms of Huoxiang Zhengqi preparations. Tinctures contained more types and higher overall levels of active components， while soft capsules were better at preserving volatile components. Granules and dripping pills had lower overall levels of active components. The comprehensive evaluation method established in this study can provide a basis for the quality control， process optimization， and the selection of high-quality products.

Objective:To generate a chronic lymphocytic leukemia (CLL) mouse model with intact immune competence and short latency by adoptively transferring (AT) splenocytes from immunoglobulin heavy-chain enhancer-driven T-cell leukemia/lymphoma 1 (Eμ-TCL1) transgenic donors into wild-type (WT) recipients.Methods:Specific pathogen-free C57BL/6J WT mice and H11-Eμ-VH-TCL1-β-globin-PolyA knock-in mice were utilized. The H11-Eμ-VH-TCL1-β-globin-PolyA knock-in mice were generated using CRISPR/Cas9 technology, and their genotypes were confirmed by PCR. Experimental animals were randomly divided into an adoptive transfer (AT) group and a WT control group ( n=10 per group). Mice in the AT group received an intraperitoneal injection of splenocytes from H11-Eμ-VH-TCL1-β-globin-PolyA knock-in mice. The weight and general condition of the mice were monitored. Mice were euthanized by cervical dislocation at 9 weeks post-transplantation. The CLL model was validated using key indicators, including pathological manifestations, changes in peripheral blood leukocyte counts, and immunophenotype. Results:AT group mice exhibited significantly increased spleen weight [ (0.92±0.16) g vs (0.06±0.01) g in WT group, P<0.05] and liver weight [ (2.11±0.56) g vs (1.42±0.13) g in WT group, P=0.006], indicative of marked splenomegaly and hepatomegaly. The peripheral blood leukocyte count was significantly higher in the AT group [ (124.33±8.74) ×10 9/L] compared to the WT group [ (5.55±1.67) ×10 9/L] ( P=0.002). Similarly, the percentage of peripheral blood B lymphocytes was markedly increased in the AT group versus the WT group [ (69.13±6.88) % vs (39.78±5.94) %, P<0.05]. Histopathological examination revealed CLL manifestations in the spleen, lymph nodes, and bone marrow of AT group mice, with significant lymphocytic infiltration observed in the liver, lung, and kidney tissues. Flow cytometry analysis showed that the percentages of CD19 +CD5 + B lymphocytes among total lymphocytes in peripheral blood, bone marrow, and spleen of the AT group were (61.37±9.92) %, (28.61± 7.08) %, and (86.03±5.78) %, respectively. These were significantly higher (all P<0.05) than in the WT group [ (4.51±1.32) %, (5.58±1.46) %, and (14.33±3.20) %]. Furthermore, these CLL-like cells in the AT group were positive for CD43 and CD200, but showed lower expression of CD20, CD22, and CD79b compared to WT B cells. Conclusion:Adoptive transfer of splenocytes from Eμ-TCL1 transgenic mice successfully established a CLL mouse model with a relatively short latency period. This model represents a valuable preclinical tool for investigating CLL and related pathologies.

Objective:To compare the efficacy and safety of crisaborole ointment 2% versus pimecrolimus cream 1% in the treatment of mild to moderate atopic dermatitis in children aged 2 years or older.Methods:A multicenter, randomized, open-label, controlled clinical trial was conducted. A total of 120 pediatric patients aged 2 - 17 years with mild to moderate atopic dermatitis were enrolled from departments of dermatology of 8 hospitals in China between March 2022 and February 2023. The participants were randomly assigned in a 1∶1 ratio to the crisaborole group and the pimecrolimus group, and received the treatment with crisaborole ointment 2% and pimecrolimus cream 1% respectively, twice a day for 4 weeks. Visits were scheduled at baseline/on day 1, as well as on days 8, 15, and 29. The primary efficacy outcome was the percentage of patients achieving the Investigator's Static Global Assessment (ISGA) success (defined as clear [0] or almost clear [1] on the ISGA scale, combined with ≥ 2‐grade improvement from baseline) on day 29. The secondary efficacy outcomes included changes in the Eczema Area and Severity Index (EASI) total scores from baseline to day 29, percentages of patients achieving ISGA improvement (defined as clear [0] or almost clear [1] on the ISGA scale), as well as changes in the Peak Pruritus Numerical Rating Scale (NRS) scores, Dermatology Life Quality Index (DLQI) /Infants' Dermatology Life Quality Index (IDLQI) /Children's Dermatology Life Quality Index (CDLQI) scores, and in the Dermatitis Family Impact (DFI) scores. Drug safety was evaluated according to the incidence of adverse events. Categorical data were compared using the chi-square test. Since measurement data did not follow a normal distribution, the rank sum test was used for comparisons of measurement data between groups.Results:A total of 106 children with mild to moderate atopic dermatitis were included in the per-protocol analysis set, with 52 in the crisaborole group (26 males and 26 females) and 54 in the pimecrolimus group (27 males and 27 females). There were no significant differences in age, disease duration, ISGA and EASI scores at baseline between the two groups (all P > 0.05). On day 29, 22 patients (42.31%) in the crisaborole group and 25 (46.30%) in the pimecrolimus group achieved ISGA success, with no significant difference between the two groups ( χ2 = 0.17, P = 0.68) ; 35 patients (67.31%) in the crisaborole group and 45 (83.33%) in the pimecrolimus group achieved ISGA improvement, also with no significant difference between the two groups ( χ2 = 3.68, P = 0.06) ; additionally, there were no significant differences in the EASI, pruritus NRS, DLQI/IDLQI/CDLQI, or DFI scores between the two groups (all P > 0.05). Adverse reactions to the two topical agents were mainly local reactions such as mild to moderate pain, itching, or worsening of itching, and no obvious systemic adverse reactions occurred. The incidence of drug-related adverse reactions was 46.15% (24 cases) in the crisaborole group and 37.04% (20 cases) in the pimecrolimus group, with no significant difference between the two groups ( χ2 = 0.91, P = 0.34) . Conclusion:The efficacy of crisaborole ointment 2% was comparable to that of pimecrolimus cream 1% in the treatment of mild to moderate atopic dermatitis in children aged ≥ 2 years, and it yielded early and rapid improvement in the quality of life of patients and their families, with good safety and tolerability profiles.

Objective:To compare the efficacy and safety of crisaborole ointment 2% versus pimecrolimus cream 1% in the treatment of mild to moderate atopic dermatitis in children aged 2 years or older.Methods:A multicenter, randomized, open-label, controlled clinical trial was conducted. A total of 120 pediatric patients aged 2 - 17 years with mild to moderate atopic dermatitis were enrolled from departments of dermatology of 8 hospitals in China between March 2022 and February 2023. The participants were randomly assigned in a 1∶1 ratio to the crisaborole group and the pimecrolimus group, and received the treatment with crisaborole ointment 2% and pimecrolimus cream 1% respectively, twice a day for 4 weeks. Visits were scheduled at baseline/on day 1, as well as on days 8, 15, and 29. The primary efficacy outcome was the percentage of patients achieving the Investigator's Static Global Assessment (ISGA) success (defined as clear [0] or almost clear [1] on the ISGA scale, combined with ≥ 2‐grade improvement from baseline) on day 29. The secondary efficacy outcomes included changes in the Eczema Area and Severity Index (EASI) total scores from baseline to day 29, percentages of patients achieving ISGA improvement (defined as clear [0] or almost clear [1] on the ISGA scale), as well as changes in the Peak Pruritus Numerical Rating Scale (NRS) scores, Dermatology Life Quality Index (DLQI) /Infants' Dermatology Life Quality Index (IDLQI) /Children's Dermatology Life Quality Index (CDLQI) scores, and in the Dermatitis Family Impact (DFI) scores. Drug safety was evaluated according to the incidence of adverse events. Categorical data were compared using the chi-square test. Since measurement data did not follow a normal distribution, the rank sum test was used for comparisons of measurement data between groups.Results:A total of 106 children with mild to moderate atopic dermatitis were included in the per-protocol analysis set, with 52 in the crisaborole group (26 males and 26 females) and 54 in the pimecrolimus group (27 males and 27 females). There were no significant differences in age, disease duration, ISGA and EASI scores at baseline between the two groups (all P > 0.05). On day 29, 22 patients (42.31%) in the crisaborole group and 25 (46.30%) in the pimecrolimus group achieved ISGA success, with no significant difference between the two groups ( χ2 = 0.17, P = 0.68) ; 35 patients (67.31%) in the crisaborole group and 45 (83.33%) in the pimecrolimus group achieved ISGA improvement, also with no significant difference between the two groups ( χ2 = 3.68, P = 0.06) ; additionally, there were no significant differences in the EASI, pruritus NRS, DLQI/IDLQI/CDLQI, or DFI scores between the two groups (all P > 0.05). Adverse reactions to the two topical agents were mainly local reactions such as mild to moderate pain, itching, or worsening of itching, and no obvious systemic adverse reactions occurred. The incidence of drug-related adverse reactions was 46.15% (24 cases) in the crisaborole group and 37.04% (20 cases) in the pimecrolimus group, with no significant difference between the two groups ( χ2 = 0.91, P = 0.34) . Conclusion:The efficacy of crisaborole ointment 2% was comparable to that of pimecrolimus cream 1% in the treatment of mild to moderate atopic dermatitis in children aged ≥ 2 years, and it yielded early and rapid improvement in the quality of life of patients and their families, with good safety and tolerability profiles.

Metabolomics covers a wide range of applications in life sciences,biomedicine,and phytology.Data acquisition(to achieve high coverage and efficiency)and analysis(to pursue good classification)are two key segments involved in metabolomics workflows.Various chemometric approaches utilizing either pattern recognition or machine learning have been employed to separate different groups.However,insufficient feature extraction,inappropriate feature selection,overfitting,or underfitting lead to an insufficient capacity to discriminate plants that are often easily confused.Using two ginseng varieties,namely Panax japonicus(PJ)and Panax japonicus var.major(PJvm),containing the similar ginsenosides,we integrated pseudo-targeted metabolomics and deep neural network(DNN)modeling to achieve accurate species differentiation.A pseudo-targeted metabolomics approach was optimized through data acquisition mode,ion pairs generation,comparison between multiple reaction monitoring(MRM)and scheduled MRM(sMRM),and chromatographic elution gradient.In total,1980 ion pairs were monitored within 23 min,allowing for the most comprehensive ginseng metabolome analysis.The established DNN model demonstrated excellent classification performance(in terms of accuracy,precision,recall,F1 score,area under the curve,and receiver operating characteristic(ROC))using the entire metabolome data and feature-selection dataset,exhibiting superior advantages over random forest(RF),support vector ma-chine(SVM),extreme gradient boosting(XGBoost),and multilayer perceptron(MLP).Moreover,DNNs were advantageous for automated feature learning,nonlinear modeling,adaptability,and generalization.This study confirmed practicality of the established strategy for efficient metabolomics data analysis and reliable classification performance even when using small-volume samples.This established approach holds promise for plant metabolomics and is not limited to ginseng.

Objective To explore the diagnostic value of exhaled volatile organic compounds (VOCs) for cystic fibrosis (CF). Methods A systematic search was conducted in PubMed, EMbase, Web of Science, Cochrane Library, CNKI, Wanfang, VIP, and SinoMed databases up to August 7, 2024. Studies that met the inclusion criteria were selected for data extraction and quality assessment. The quality of included studies was assessed by the Newcastle-Ottawa Scale (NOS), and the risk of bias and applicability of included prediction model studies were assessed by the prediction model risk of bias assessment tool (PROBAST). Results A total of 10 studies were included, among which 5 studies only identified specific exhaled VOCs in CF patients, and another 5 developed 7 CF risk prediction models based on the identification of VOCs in CF. The included studies reported a total of 75 exhaled VOCs, most of which belonged to the categories of acylcarnitines, aldehydes, acids, and esters. Most models (n=6, 85.7%) only included exhaled VOCs as predictive factors, and only one model included factors other than VOCs, including forced expiratory flow at 75% of forced vital capacity (FEF75) and modified Medical Research Council scale for the assessment of dyspnea (mMRC). The accuracy of the models ranged from 77% to 100%, and the area under the receiver operating characteristic curve ranged from 0.771 to 0.988. None of the included studies provided information on the calibration of the models. The results of the Prediction Model Risk of Bias Assessment Tool (PROBAST) showed that the overall bias risk of all predictive model studies was high, and the overall applicability was unclear. Conclusion The exhaled VOCs reported in the included studies showed significant heterogeneity, and more research is needed to explore specific compounds for CF. In addition, risk prediction models based on exhaled VOCs have certain value in the diagnosis of CF, but the overall bias risk is relatively high and needs further optimization from aspects such as model construction and validation.

A novel analytical method was developed in this study by combining online solid phase extraction with ultra performance liquid chromatography-tandem mass spectrometry(Online SPE-UPLC-MS/MS)for simultaneous determination of 50 kinds of steroid hormones in surface water.Specifically,after high-speed centrifugation of 4 mL water samples,the supernatant was directly injected into an Oasis HLB online SPE column for enrichment and purification.Subsequently,the target compounds were transferred to the analytical column via valve switching for separation and analysis.The chromatographic separation was performed on a Thermo Acclaim RSLC C18 column(100 mm×2.1 mm,2.2 μm),using a mobile phase composed of 5 mmol/L ammonium fluoride aqueous solution and acetonitrile.Mass spectrometric detection was conducted in positive ion mode,utilizing multiple reaction monitoring(MRM)with quantification achieved by the internal standard method.The method validation demonstrated that the limits of detection(LOD)for the 50 kinds of steroid hormones ranged from 0.02 to 0.50 ng/L,while the limits of quantification(LOQ)were between 0.08 and 1.67 ng/L.The average recoveries in surface water samples at spiked concentrations of 5,20 and 200 ng/L were between 74.1％and 119％,with relative standard deviations(RSDs)of 0.2％to 9.9％.This method was applied to analyze 11 surface water samples collected from sites surrounding a pharmaceutical and chemical industrial park.A total of 44 kinds of steroid hormones were detected,with concentrations ranging from 0.11 to 88.6 ng/L,revealing the presence of hormone contamination in the environmental waters surrounding industrial areas.Compared with the traditional offline SPE methods,the proposed online SPE technique significantly reduced sample volume requirements and pretreatment time,while minimizing the loss of target compounds during the pretreatment process.Moreover,compared to reported online SPE techniques,this method achieved high-throughput analysis of multiple classes of steroid hormones,with lower detection limits and higher recoveries.Overall,this method provided rapid sample preparation,high sensitivity,and excellent stability,making it suitable for the direct analysis of trace steroid hormones in surface water.

The prelimbic cortex (PL) plays a critical role in processing both the sensory and affective components of pain. However, the underlying molecular mechanisms remain poorly understood. In this study, we observed a reduction in hyperpolarization-activated cation current (I_h) in layer V pyramidal neurons of the contralateral PL in a mouse model of spared nerve injury (SNI). The expression of hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channels was also decreased in the contralateral PL. Conversely, microinjection of fisetin, a partial agonist of HCN2, produced both analgesic and anxiolytic effects. Additionally, we found that cyclin-dependent kinase 5 (CDK5) was activated in the contralateral PL, where it formed a complex with HCN2 and phosphorylated its C-terminus. Knockdown of CDK5 restored HCN2 expression and alleviated both pain hypersensitivity and anxiety-like behaviors. Collectively, these results indicate that CDK5-mediated dysfunction of HCN2 in the PL underlies nerve injury-induced mechanical hypersensitivity and anxiety.
Animals ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism* ; Hyperalgesia/metabolism* ; Cyclin-Dependent Kinase 5/metabolism* ; Neuralgia/metabolism* ; Male ; Anxiety/metabolism* ; Mice ; Potassium Channels/metabolism* ; Mice, Inbred C57BL ; Disease Models, Animal ; Pyramidal Cells/metabolism*