ObjectiveDonkey hide is the sole legally designated raw material for the preparation of the traditional Chinese medicine Ejiao. The quality stability of donkey hide during preservation directly determines the efficacy and safety of Ejiao. This study focuses on the dynamic succession of microbial communities during the preservation of donkey hides from different origins, aiming to clarify the correlation between microbial biodiversity difference and the degradation profiles of hide collagen and critical biochemical components, thereby providing a theoretical foundation for developing targeted preservation strategies based on microbial regulation. MethodsDonkey hides originating from four different regions were subjected to an accelerated microbial aging assay to simulate the spoilage process. The microbial community succession was analyzed using high-throughput sequencing. Microstructure changes and pore structure characteristics were assessed by scanning electron microscopy and mercury intrusion porosimetry, respectively. Additionally, the content of major components, including lipids, proteins, and sugars were determined by biochemical methods. ResultsAfter 96 h of aging, the collagen fiber structure in Africa donkey hides (ADH) exhibited significant degradation and collapse, followed by Xinjiang donkey hides (XDH). Instead, the microstructure of Dong’e black donkey hides (DDH) and Peru donkey hides (PDH) remained relatively intact. The porosities of DDH, XDH, PDH, and ADH increased from 27.9%, 15.7%, 30.3%, and 46.2% to 36.5%, 52.6%, 42.8%, and 57.7%, respectively, during the aging process, which suggested that the originally compact fiber structure was disrupted by microbial aging. Fourier transform infrared spectrometer analysis revealed the amide bands in XDH exhibited relatively weak intensity, and no collagen amide I band was observed in ADH. Meanwhile, the lipid and protein contents decreased in all four types of donkey hides, indicating that these components served as the primary nutrient sources for the growth of microorganism. Notably, the most severe collagen degradation was observed in XDH and ADH. A substantial increase was detected in the total soluble sugar in PDH aging solution and hydroxyproline in the ADH aging solution, respectively. These results indicated that donkey hides exhibit distinct patterns of structural degradation and nutrient utilization. Furthermore, the viable cells number of donkey hides increased sharply after 48 h of aging. Metagenomic analysis revealed that the relative abundance of Euryarchaeota in ADH, PDH and XDH declining from initial 93.19%, 97.73% and 30.08% to 0.79%, 1.43% and 0.02% after 96 h, respectively. Conversely, a significantly increase was observed in the abundance of Bacillota, with a marked increase in ADH, peaking at 92.75%. Additionally, the abundance of Pseudomonadota in PDH increased from 0.10% to 87.84%, suggesting that Bacillota and Pseudomonadota may be key factors exacerbating donkey hide spoilage. Unlike the other three types of donkey hides, the dominant bacterial phylum in DDH shifted from Pseudomonadota to Bacteroidota, characterized by a substantial abundance increase of Bacteroidota from 0.13% to 44.22%. ConclusionRegional variation in origin significantly influence the microbial aging of donkey hides, leading to distinct patterns of structural deterioration and differential nutrient utilization. Therefore, implementing origin-specific preservation strategies, through the precisely controlling environmental factors to suppress harmful phyla such as Bacillota and Pseudomonadota, is crucial for enhancing the storage quality of donkey hides.

ObjectiveHepatocellular carcinoma (HCC) represents 90% of all primary liver cancers. The main risk factors associated with HCC include viral hepatitis (B and/or C), alcohol abuse, and metabolic dysfunction-associated steatotic liver disease (MASLD), which progressively advance to liver fibrosis, cirrhosis, and ultimately evolve into HCC. Surgical resection represents the most effective treatment for HCC, while recent advances in immunotherapy, including immune checkpoint inhibitors and adoptive cell therapies, have provided improved treatment prospects for patients with unresectable HCC. However, the complex metabolic heterogeneity of HCC limits the therapeutic efficacy. Metabolic intermediates acyl-CoA not only provide energy and substrates for numerous biochemical reactions but also serve as donors for protein lysine acylation, a major class of post-translational modification (PTM). Therefore, a deeper understanding of the molecular mechanisms underlying protein lysine acylation and hepatocarcinogenesis is urgently needed. MethodsThe levels of protein lysine acylation and silence information regulator 5 (SIRT5) expression levels in clinical HCC samples were analyzed by Western blot. Quantitative malonylome and succinylome of HCC samples were analyzed by antibody-based affinity enrichment coupled with tandem mass spectrometry. The proliferation of HCC cells was analyzed with Cell Counting Kit-8 (CCK-8) assays, the apoptosis was quantified by Annexin V-FITC/propidium iodide (PI) staining coupled with flow cytometry, and the ability of cells to migrate was assayed by Transwell assays. The enzymatic activity of glutathione S-transferase Mu 1 (GSTM1) was quantified. Transgenic mice with hepatic overexpression of SIRT5 were constructed using CRISPR-Cas9, and primary hepatocarcinogenesis was induced by administration of diethylnitrosamine. ResultsWestern blot analysis indicated that the expression level of SIRT5 was elevated in clinical samples from HCC patients, and the levels of lysine malonylation, glutarylation, and succinylation were significantly reduced in HCC tissues. Knockout of SIRT5 in MHCC-97H and MHCC-97L hepatoma cells suppressed cell proliferation, and increased the percentage of apoptotic cells significantly. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially malonylome and succinylome of HCC samples revealed significant enrichment in two major classes of biological processes: core energy metabolism (e.g., glycolysis/gluconeogenesis, tricarboxylic acid metabolic process, fatty acid beta oxidation) and detoxification and oxidative stress response (e.g., response to toxic substance, chemical carcinogenesis, reactive oxygen species (ROS)). SIRT5 removes malonylation from lysine residues in GSTM1 and restores its detoxification activity, which is crucial for the survival of hepatocytes under stressed conditions. More importantly, in vivo experiment indicated that hepatic-specific overexpression of SIRT5 in mice accelerated diethylnitrosamine-induced liver fibrosis and hepatocarcinogenesis, indicating the critical role of SIRT5 in HCC progression. ConclusionThis study highlights the previously unrecognized SIRT5-GSTM1 axis as a key regulator in hepatocarcinogenesis, and suggests a potential target for the treatment of patients with HCC.

ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

Background: High-mobility group box protein 1 (HMGB1) is a chromatin-binding protein involved in arthritis, ischemia, sepsis, atherosclerosis, neurodegenerative disorders, meningitis, and cancer. HMGB1 exhibits dual roles in cancer, acting as either a tumor suppressor or oncoprotein depending on context. Objective: This research aimed to elucidate HMGB1’s functional significance in cutaneous squamous cell carcinoma (cSCC). Methods: We overexpressed HMGB1 in cSCC cell lines using recombinant adenovirus and examined its effects on cell proliferation, colony formation, and cell migration. Results: Immunohistochemical analysis revealed elevated HMGB1 expression levels in cSCC tissue relative to normal epidermis. To assess the influence of HMGB1, we employed recombinant adenoviruses expressing HMGB1 to transduce SCC cell lines (SCC12 and SCC13). Enhanced HMGB1 expression significantly promoted cellular proliferation and colony formation capacity.Notably, HMGB1 overexpression elevated the levels of proliferation regulators, including P63, SOX2, CDK4 and CDK6. Furthermore, HMGB1 overexpression substantially enhanced tumor invasiveness, accompanied by upregulation of epithelial-mesenchymal transition (EMT) biomarkers. Mechanistically, overexpression of HMGB1 enhanced transforming growth factor-β signaling by increasing phosphorylation of SMAD2/3, the key mediators of EMT. Conclusion These data imply that HMGB1 acts as a tumor-promoting factor in cSCC.

ObjectiveTo evaluate the efficacy and safety of Xiaoji Hufei Formula in protecting children with close contact exposure to influenza， and to provide reference and evidence-based support for better clinical prevention and treatment of influenza in children. MethodsA multicenter， prospective， non-randomized， parallel， controlled trial was conducted from October 2021 to May 2022 in five hospitals， including Dongfang Hospital of Beijing University of Chinese Medicine. Confirmed influenza cases and influenza-like illness （ILI） cases were collected， and eligible children with close contact exposure to these cases were recruited in the outpatient clinics. According to whether the enrolled close contacts were willing to take Xiaoji Hufei formula for influenza prevention， they were assigned to the observation group （108 cases） or the control group （108 cases）. Follow-up visits were conducted on days 7 and 14 after enrollment. The primary outcomes were the incidence of ILI and the rate of laboratory-confirmed influenza. Secondary outcomes included traditional Chinese medicine （TCM） symptom score scale for influenza， influenza-related emergency （outpatient） visit rate， influenza hospitalization rate， and time to onset after exposure to influenza cases. ResultsA total of 216 participants were enrolled， with 108 in the observation group and 108 in the control group. Primary outcomes： （1） Incidence of ILI： The incidence was 12.0% （13/108） in the observation group and 23.1% （25/108） in the control group， with the observation group showing a significantly lower incidence （χ²=4.6， P<0.05）. （2） Influenza confirmation rate： 3.7% （4/108） in the observation group and 4.6% （5/108） in the control group， with no statistically significant difference. Secondary outcomes： （1） TCM symptom score scale： after onset， nasal congestion and runny nose scores differed significantly between the two groups （P<0.05）， while other symptoms such as fever， sore throat， and cough showed no significant differences. （2） Influenza-related emergency （outpatient） visit rate： 84.6% （11 cases） in the observation group and 96.0% （24 cases） in the control group， with no significant difference. （3） Time to onset after exposure： The median onset time after exposure to index patients was 7 days in the observation group and 4 days in the control group， with a statistically significant difference （P<0.05）. ConclusionIn previously healthy children exposed to infectious influenza cases under unprotected conditions， Xiaoji Hufei formula prophylaxis significantly reduced the incidence of ILI. Xiaoji Hufei Formula can be recommended as a specific preventive prescription for influenza in children.

ObjectiveTo evaluate the efficacy and safety of Xiaoji Hufei Formula in protecting children with close contact exposure to influenza， and to provide reference and evidence-based support for better clinical prevention and treatment of influenza in children. MethodsA multicenter， prospective， non-randomized， parallel， controlled trial was conducted from October 2021 to May 2022 in five hospitals， including Dongfang Hospital of Beijing University of Chinese Medicine. Confirmed influenza cases and influenza-like illness （ILI） cases were collected， and eligible children with close contact exposure to these cases were recruited in the outpatient clinics. According to whether the enrolled close contacts were willing to take Xiaoji Hufei formula for influenza prevention， they were assigned to the observation group （108 cases） or the control group （108 cases）. Follow-up visits were conducted on days 7 and 14 after enrollment. The primary outcomes were the incidence of ILI and the rate of laboratory-confirmed influenza. Secondary outcomes included traditional Chinese medicine （TCM） symptom score scale for influenza， influenza-related emergency （outpatient） visit rate， influenza hospitalization rate， and time to onset after exposure to influenza cases. ResultsA total of 216 participants were enrolled， with 108 in the observation group and 108 in the control group. Primary outcomes： （1） Incidence of ILI： The incidence was 12.0% （13/108） in the observation group and 23.1% （25/108） in the control group， with the observation group showing a significantly lower incidence （χ²=4.6， P<0.05）. （2） Influenza confirmation rate： 3.7% （4/108） in the observation group and 4.6% （5/108） in the control group， with no statistically significant difference. Secondary outcomes： （1） TCM symptom score scale： after onset， nasal congestion and runny nose scores differed significantly between the two groups （P<0.05）， while other symptoms such as fever， sore throat， and cough showed no significant differences. （2） Influenza-related emergency （outpatient） visit rate： 84.6% （11 cases） in the observation group and 96.0% （24 cases） in the control group， with no significant difference. （3） Time to onset after exposure： The median onset time after exposure to index patients was 7 days in the observation group and 4 days in the control group， with a statistically significant difference （P<0.05）. ConclusionIn previously healthy children exposed to infectious influenza cases under unprotected conditions， Xiaoji Hufei formula prophylaxis significantly reduced the incidence of ILI. Xiaoji Hufei Formula can be recommended as a specific preventive prescription for influenza in children.

BACKGROUND: This study aimed to investigate programmed death-ligand 1 tumor proportion score in predicting the safety and efficacy of PD-1/PD-L1 antibody-based therapy in treating patients with advanced non-small cell lung cancer (NSCLC) in a real-world setting. METHODS: This retrospective, multicenter, observational study enrolled adult patients who received PD-1/PD-L1 antibody-based therapy in China and met the following criteria: (1) had pathologically confirmed, unresectable stage III-IV NSCLC; (2) had a baseline PD-L1 tumor proportion score (TPS); and (3) had confirmed efficacy evaluation results after PD-1/PD-L1 treatment. Logistic regression, Kaplan-Meier analysis, and Cox regression were used to assess the progression-free survival (PFS), overall survival (OS), and immune-related adverse events (irAEs) as appropriate. RESULTS: A total of 409 patients, 65.0% ( n = 266) with a positive PD-L1 TPS (≥1%) and 32.8% ( n = 134) with PD-L1 TPS ≥50%, were included in this study. Cox regression confirmed that patients with a PD-L1 TPS ≥1% had significantly improved PFS (hazard ratio [HR] 0.747, 95% confidence interval [CI] 0.573-0.975, P = 0.032). A total of 160 (39.1%) patients experienced 206 irAEs, and 27 (6.6%) patients experienced 31 grade 3-5 irAEs. The organs most frequently associated with irAEs were the skin (52/409, 12.7%), thyroid (40/409, 9.8%), and lung (34/409, 8.3%). Multivariate logistic regression revealed that a PD-L1 TPS ≥1% (odds ratio [OR] 1.713, 95% CI 1.054-2.784, P = 0.030) was an independent risk factor for irAEs. Other risk factors for irAEs included pretreatment absolute lymphocyte count >2.5 × 10 9 /L (OR 3.772, 95% CI 1.377-10.329, P = 0.010) and pretreatment absolute eosinophil count >0.2 × 10 9 /L (OR 2.006, 95% CI 1.219-3.302, P = 0.006). Moreover, patients who developed irAEs demonstrated improved PFS (13.7 months vs. 8.4 months, P <0.001) and OS (28.0 months vs. 18.0 months, P = 0.007) compared with patients without irAEs. CONCLUSIONS A positive PD-L1 TPS (≥1%) was associated with improved PFS and an increased risk of irAEs in a real-world setting. The onset of irAEs was associated with improved PFS and OS in patients with advanced NSCLC receiving PD-1/PD-L1-based therapy.
Humans ; Carcinoma, Non-Small-Cell Lung/metabolism* ; Male ; Female ; Retrospective Studies ; Middle Aged ; Lung Neoplasms/metabolism* ; Aged ; B7-H1 Antigen/metabolism* ; Programmed Cell Death 1 Receptor/metabolism* ; Adult ; Aged, 80 and over ; Immune Checkpoint Inhibitors/therapeutic use*

In this paper, near-infrared spectroscopy(NIRS) was employed to analyze 129 batches of commercial products of Reduning Injection. The batch reporting rate was estimated according to the report of Reduning Injection in the direct adverse drug reaction(ADR) reporting system of the drug marketing authorization holder of the Center for Drug Reevaluation of the National Medical Products Administration(National Center for ADR Monitoring) from August 2021 to August 2022. According to the batch reporting rate, the samples of Reduning Injection were classified into those with potential risks and those being safe. No processing, random oversampling(ROS), random undersampling(RUS), and synthetic minority over-sampling technique(SMOTE) were then employed to balance the unbalanced data. After the samples were classified according to appropriate sampling methods, competitive adaptive reweighted sampling(CARS), successive projections algorithm(SPA), uninformative variables elimination(UVE), and genetic algorithm(GA) were respectively adopted to screen the features of spectral data. Then, support vector machine(SVM), logistic regression(LR), k-nearest neighbors(KNN), naive bayes(NB), random forest(RF), and artificial neural network(ANN) were adopted to establish the risk prediction models. The effects of the four feature extraction methods on the accuracy of the models were compared. The optimal method was selected, and bayesian optimization was performned to optimize the model parameters to improve the accuracy and robustness of model prediction. To explore the correlations between potential risks of clinical use and quality test data, TreeNet was employed to identify potential quality parameters affecting the clinical safety of Reduning Injection. The results showed that the models established with the SVM, LR, KNN, NB, RF, and ANN algorithms had the F1 scores of 0.85, 0.85, 0.86, 0.80, 0.88, and 0.85 and the accuracy of 88%, 88%, 88%, 85%, 91%, and 88%, respectively, and the prediction time was less than 5 s. The results indicated that the established models were accurate and efficient. Therefore, near infrared spectroscopy combined with machine learning algorithms can quickly predict the potential risks of clinical use of Reduning Injection in batches. Three key quality parameters that may affect clinical safety were identified by TreeNet, which provided a scientific basis for improving the safety standards of Reduning Injection.
Spectroscopy, Near-Infrared/methods* ; Drugs, Chinese Herbal/administration & dosage* ; Machine Learning ; Algorithms ; Humans ; Quality Control