ObjectiveTo explore effect of Xianlian Jiedu prescription on the recurrence of colorectal cancer （CRC） and investigate the related mechanisms. MethodsA postoperative recurrence model was established in 25 Balb/c mice by injecting CT26 cells subcutaneously into the armpit， followed by surgical removal of 99% of the subcutaneous tumor. The mice were randomly divided into model group， low-dose Xianlian Jiedu prescription （XLJDP-L） group （6.45 g·kg^-1·d^-1）， medium-dose Xianlian Jiedu prescription （XLJDP-M） group （12.9 g·kg^-1·d^-1）， high-dose Xianlian Jiedu prescription （XLJDP-H） group （25.8 g·kg^-1·d^-1）， and 5-fluorouracil （5-FU） group （1×10^-3 g·kg^-1·d^-1）. The mice were euthanized after 14 days of continuous intervention， and recurrent tumor tissue was harvested. Hematoxylin and eosin （HE） staining was used to observe pathological and morphological changes in the recurrent tumor tissue. Immunohistochemistry （IHC） was employed to assess the expression of proliferating cell nuclear antigen （Ki67）， vascular endothelial growth factor （VEGF）， and platelet-endothelial cell adhesion molecule （CD31） in recurrent tumor tissue. The Western blot was used to detect the protein expression levels of angiopoietin-2 （ANG-2）， VEGF， phosphorylated-protein kinase B （p-Akt）， protein kinase B （Akt）， phosphorylated-phosphatidylinositol 3-kinase （p-PI3K）， and phosphatidylinositol 3-kinase （PI3K） in recurrent tumor tissue. ResultsBefore treatment， there were no statistical differences in tumor volume， tumor weight， and body mass among the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group compared to the model group， indicating model stability. After treatment， compared with those in the model group， the tumor volume and tumor weight in the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group were significantly reduced （P<0.01）， showing dose dependency. Meanwhile， there were no significant differences in body weight among the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group compared to the model group. HE staining showed that compared with that in the model group， tumor tissue in the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group had loosely arranged cells， increased intercellular spaces， small and shriveled nuclei， light staining， fewer mitotic figures and atypical nuclei， and increased necrotic areas. IHC showed that compared with those of the model group， the positive rates of Ki67， VEGF， and CD31 in the recurrent tumor tissue of the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group were significantly reduced （P<0.01） in a dose-dependent manner. Western blot results showed that compared with those of the model group， the protein expression levels of ANG-2 and VEGF in the recurrent tumor tissue of the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group were significantly downregulated （P<0.05， P<0.01）， and the p-Akt/Akt and p-PI3K/PI3K ratios were significantly decreased in a dose-dependent manner （P<0.05， P<0.01）. ConclusionXianlian Jiedu prescription significantly inhibits the recurrence of CRC in mice after subcutaneous tumor surgery. The mechanism may involve regulating the PI3K/Akt pathway and downregulating key angiogenic proteins such as ANG-2， VEGF， and CD31.

Objective To compare the differences in alkaloids between Nelumbinis Semen and Nelumbinis Plumula and their inhibitory effects on the proliferation of HepG2 cells, and investigate the material basis for their anti-cancer activity differences. Methods Simultaneous Thermal Analysis was used to preliminarily compare the component differences between Nelumbinis Semen and Nelumbinis Plumula. Alkaloids were extracted from them by both using reflux extraction, and their contents were measured by UV and HPLC methods. The CCK-8 method was used to assess the in vitro inhibitory effects of the alkaloids on the HepG2 cells, and to verify pharmacological differences. Results Simultaneous thermal analysis revealed distinct peak shapes, positions, and sizes in the thermal analysis curves of Nelumbinis Semen and Nelumbinis Plumula at respective temperature stages. The contents of total alkaloids showed as follows: the total alkaloids in Nelumbinis Plumula > total extract of Nelumbinis Plumula > the total alkaloids in Nelumbinis Semen. The total alkaloids in Nelumbinis Plumula effectively inhibited HepG2 cell proliferation, while the total alkaloids in Nelumbinis Semen showed no impact. Conclusion Differences in the composition and content of alkaloids may be key factors underlying the biological activities differences between Nelumbinis Semen and Nelumbinis Plumula. This study provided a basis for exploring the material foundation of the differential efficacy and properties of Nelumbinis Semen and Nelumbinis Plumula, which could support their rational clinical application.

Glycosylation modification is an important post-translational modification of proteins, which participates in regulating protein half-life, biological activity, and immunogenicity, thereby affecting their functions. Glycoproteins expressed in Pichia pastoris predominantly carry high-mannose type glycans, primarily composed of mannose residues, which starkly contrasts with the complex-type glycans synthesized by mammalian cells. This study aims to transform the high mannose glycosylation modification of P. pastoris into a hybrid glycosylation modification similar to that of mammalian cells through genetic engineering technology. We introduced the mannosidase Ⅰ gene (MDSⅠ) from Trichoderma viride and the human β-1,2-N-acetylglucosaminyltransferase I gene (GnTⅠ) into a previously constructed P. pastoris strain (∆och1) capable of producing Man₈GlcNAc₂ glycans. To precisely regulate the expression of MDSⅠ and GnTⅠ, we designed various promoter combinations, including the strong inducible AOX promoter and the constitutive GAP promoter. The receptor-binding domain (RBD, residues 377-588) of the spike protein from the Middle East respiratory syndrome coronavirus (MERS-CoV) was selected as the reporter protein for this investigation (MERS-RBD). The N-glycosylation profile of MERS-RBD was systematically analyzed using PNGase F digestion coupled with mass spectrometry. The results showed that after the knockout of och1 and the introduction of MDSⅠ and GnTⅠ genes with different promoter combinations, P. pastoris strains capable of producing GlcNAcMan₅GlcNAc₂ glycans were successfully generated. When the AOX promoter was used to control the MDSⅠ gene and the GAP promoter was used to control the GnTⅠ gene, the engineered strain exhibited the highest proportion of hybrid-type GlcNAcMan₅GlcNAc₂ glycans, which accounted for 68.38% of the total N-glycosylation. In conclusion, we successfully engineered a P. pastoris strain capable of synthesizing hybrid-type GlcNAcMan₅GlcNAc₂ glycans, establishing a foundation for subsequent research on the biosynthesis of complex-type N-glycans in P. pastoris.
Glycosylation ; Glycoproteins/genetics* ; Polysaccharides/metabolism* ; N-Acetylglucosaminyltransferases/metabolism* ; Pichia/metabolism* ; Humans ; Mannosidases/metabolism* ; Genetic Engineering ; Trichoderma/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales

Premature ovarian insufficiency(POI)is a complex endocrine disorder that significantly affects the physiological and repro-ductive functions of women,and it has become one of the main causes of infertility in women of childbearing age.The clinical features of POI include amenorrhea or oligomenorrhea,low estrogen or estrogen deficiency,and an increase in the level of gonadotropin.The in-cidence rate of POI tends to increase,yet its etiology and pathogenesis remain unclear.At present,the conventional treatment methods for POI have limited efficacy in fundamentally improving ovarian function or addressing the fertility issue.With the development of re-generative medicine,mesenchymal stem cells(MSCs)have become one of the research hotspots in POI therapy,and their exosomes have attracted wide attention as a promising"cell-free therapy".A number of animal experimental studies have shown that MSCs and their exosomes can exert a therapeutic effect on POI by affecting granulosa cell proliferation and apoptosis,promoting ovarian angiogen-esis,reducing oxidative stress and fibrosis,enhancing follicular development,and regulating immunity.In addition,related clinical studies have also made some progress.This article reviews the mechanisms and clinical effect of MSCs and their exosomes in the treatment of POI,in order to provide a reference for further research and bench-to-bedside translation of POI therapies.

Data analysis models may assist the transmission of traditional Chinese medicine （TCM） experience and clinical diagnosis and treatment， and the possibility of constructing a “data-knowledge” dual-drive model was explored by taking gastric precancerous state as an example. Data-driven is to make clinical decisions around data analysis， and its syndrome-differentiation decision-making research relies on hidden structural models and partially observable Markov decision-making processes to identify the etiology of diseases， syndrome elements， evolution of pathogenesis， and syndrome differentiation protocols； knowledge-driven is to make use of data and information to promote decision-making and action processes， and its syndrome-differentiation decision-making research relies on convolutional neural networks to improve the accuracy of local disease identification and syndrome differentiation. The “data-knowledge” dual-driven model can make up for the shortcomings of single-drive numerical simulation accuracy， and achieve a balance between local disease identification and macroscopic syndrome differentiation. On the basis of previous research， we explored the construction method of diagnostic assisted decision-making platform for gastric precancerous state， and believed that the diagnostic and decision-making ability of doctors can be extended through the assistance of machines and algorithms. Meanwhile， the related research methods were integrated and the core features of gastric precancerous state based on TCM syndrome differentiation and endoscopic pathology diagnosis and prediction were obtained， and the elements of endoscopic pathology recognition based on TCM syndrome differentiation were explored， so as to provide ideas for the in-depth research and innovative application of cutting-edge data analysis technology in the field of intelligent TCM syndrome differentiation.

Objective To analyze the relationship between bone mineral density (BMD) of infants over 6 months of age and exclusive breastfeeding and calcium nutrition guidance during pregnancy in Baoding area, and to provide evidence for clinical application. Methods A total of 308 infants over 6 months of age were selected from Baoding Maternal and Child Health Hospital from January 2020 to January 2023, and their BMD was measured by ultrasound. The level of 25 (OH) D3 in subjects' blood was detected. spearman correlation test was used to analyze the correlation between infant bone mineral density and exclusive breastfeeding and calcium nutritional guidance during pregnancy, and logistics regression model was used to analyze the independent factors affecting infant bone mineral density. Results The level of serum 25 (OH) D3 in normal BMD group was significantly higher than that in abnormal BMD group (P<0.05). The rate of exclusive breastfeeding and the guidance rate of calcium nutrition during pregnancy in normal BMD group were significantly higher than those in abnormal BMD group (P<0.05). There was a significant positive correlation between different bone mineral density and exclusive breastfeeding and calcium nutrition guidance during pregnancy (P<0.05). Serum 25 (OH) D3 level, exclusive breastfeeding rate and calcium nutritional guideline rate during pregnancy were independent protective factors for bone mineral density (P<0.05). Conclusion Bone mineral density (BMD) of infants over 6 months of age is positively correlated with exclusive breastfeeding and calcium nutrition guidance during pregnancy, and exclusive breastfeeding and calcium nutrition guidance during pregnancy are independent protective factors affecting BMD of infants over 6 months of age.

Objective:To evaluate myocardial microcirculation perfusion with myocardial contrast echocardiography (MCE) in patients with acute myocardial infarction after percutaneous coronary intervention (PCI), and to explore the prognostic value of different types of myocardial microcirculation perfusion.Methods:This is a prospective cohort study. Patients with acute myocardial infarction who underwent successful PCI in Nanfang Hospital of Southern Medical University and Kanghua Hospital of Dongguan City from October 2019 to June 2021 were selected. All the enrolled patients completed MCE examination within 72 hours after PCI. According to the examination results, the patients were divided into normal microcirculation perfusion group, delayed microcirculation perfusion group, and blocked microcirculation perfusion group. Adverse cardiovascular events including all-cause death, cardiovascular death, and angina re-hospitalization were followed up, and left ventricular ejection fraction (LVEF) review results were collected at six months to one year after surgery. Kaplan-Meier survival curve was used to investigate the difference in the incidence of adverse cardiovascular events in different myocardial perfusion groups, and Cox regression analysis was used to evaluate the effect of myocardial perfusion on adverse cardiovascular events.Results:A total of 113 patients with acute myocardial infarction were included, aged (56.3±11.5) years, with 88(78%) males. There were 31 cases in the normal microcirculation perfusion group, 43 cases in the delayed microcirculation perfusion group and 39 cases in the blocked microcirculation perfusion group. LVEF was reviewed in 49 patients, and LVEF in the delayed microcirculation perfusion group was significantly improved compared with baseline at follow-up ((63.3±1.2) % vs. (58.6±1.8) %, P=0.043), and there was no statistically significant difference between the other two groups (all P>0.05). The median follow-up time was 473 days, during follow-up period 30 adverse cardiovascular events occurred. Kaplan-Meier survival curve analysis showed that there was a statistically significant difference in the incidence of adverse cardiovascular events among the three groups ( Plog-rank=0.029). Cox regression analysis showed that abnormal microcirculation perfusion (defined as delayed and blocked microcirculation perfusion) was an independent predictor of adverse cardiovascular events in patients with acute myocardial infarction after PCI ( HR=1.90, 95% CI1.16-3.12, P=0.011). Conclusions:Microcirculatory perfusion decrease or lost is common in patients with acute myocardial infarction after PCI. Timely restoration of blood flow reconstruction can save heart function when microcirculatory perfusion decreases. Microcirculatory perfusion is a predictor of adverse cardiovascular events in patients with acute myocardial infarction, and patients with poor myocardial perfusion are more likely to experience adverse cardiovascular events.

Objective:To analyze long-term prognosis and influencing factors of recurrence in vocal fold leukoplakia treated by endoscopic cordectomy with CO 2 laser. Methods:A retrospective review was conducted on 599 patients with vocal fold leukoplakia [566 males and 33 females, aged 17-84 years (median age 55 years)], undergoing endoscopic cordectomy by CO 2 laser under general anesthesia at the Department of Otorhinolaryngology Head and Neck Surgery of Beijing Tongren Hospital, Capital Medical University, from January 2000 to December 2023. The study analyzed the clinical features, pathological grade, surgical methods, postoperative voice function, recurrence, malignant changes and analyzed the long-term efficacy and and its influencing factors. The patients were followed up for a duration of 6-249 months postoperatively, with a median follow-up time of 48 months. SPSS 20.0 software was used for statistical analysis. Results:Among the 599 patients, the histopathological grades included simple squamous cell hyperplasia in ( n=264, 44.08%), mild dysplasia ( n=96, 16.03%), moderate dysplasia ( n=74, 12.35%), severe dysplasia( n=43, 7.81%), carcinoma in situ( n=35, 5.84%), and carcinoma in situ with microinvasion( n=87, 14.52%). The 3-year and 5-year overall recurrence rates were 12.91% and 16.00%, respectively. In patients with precancerous lesion, 3.91% of recurrences presented with an upgraded pathologic grade and 2.34% evolved into carcinoma. The risk of recurrence was higher in lesions involving the anterior commissure, larger lesions, significant reduction/absence of mucosal waves, neovascularization/suspected neovascularization, and pathology of dysplasia/carcinoma in situ/carcinoma in situ with microinvasion( F=44.76,21.54,11.55,8.78,23.20,respectively, P<0.05). Additionally, patients with recurrent disease exhibited higher reflux symptom index scores compared to those without recurrence. Exophytic lesion characteristics and the inability to cease smoking postoperatively were identified as independent risk factors for recurrence with an upgraded pathological grade, with odds ratios of 8.675 and 11.380 times greater than those with non-exophytic lesions and successful smoking cessation, respectively. At the 6-months postoperative assessment, patients who underwent subepithelial cordectomy (typeⅠ) demonstrated a statistically significant increase in fundamental frequency ( t=-3.38, P<0.05), and while other voice acoustic parameters were not significantly different.Conversely, those who underwent transmuscular cordectomy (TypeⅢ) exhibited significant alterations in multiple voice acoustic parameters when compared to preoperative values ( P<0.05). Furthermore, postoperative vocal fold adhesions developed in 84 patients with 13 of these individuals requiring surgical intervention for adhesion release. Conclusions:Lesions involving the anterior commissure, larger lesions, pathology of dysplasia/carcinoma in situ/carcinoma in situ with microinfiltration, significant reduction/absence of vocal fold mucosal waves, and neovascularization visible under NBI are indicative of an increased risk of recurrence, whereas exophytic lesions and the inability to cease smoking postoperatively significantly increase the risk of recurrence with elevated pathologic grade. Recovery of postoperative voice quality is procedure-dependent, underscoring the importance of surgical approach selection in the management of vocal fold leukoplakia.

Objective:To establish a rapid quantitative PCR (qPCR) technique for Mycobacterium marinum skin infections, and to analyze its clinical diagnostic efficiency. Methods:DNA was extracted from Mycobacterium marinum colonies and serially diluted (10 -1 to 10 -8). Twelve pairs of previously reported primers and probes, as well as 6 pairs of newly designed primers and probes in this study, were used for qPCR amplification to identify the most sensitive primers and probes for the detection of Mycobacterium marinum. Skin lesion tissues were collected from 72 patients with confirmed Mycobacterium marinum infections (experimental group) and 68 with other mycobacterial infections (control group) at Shandong Provincial Hospital for Skin Diseases & Shandong Provincial Institute of Dermatology and Venereology, Shandong First Medical University & Shandong Academy of Medical Sciences in 2021. These skin tissues were subjected to qPCR amplification, interferon-gamma release assay (IGRA), acid-fast staining, and tissue culture to evaluate the diagnostic efficacy. Results:The newly designed primers and probes targeting the mycobacterial enhanced infection locus 2 (Mel2) demonstrated the highest sensitivity, with a detection limit of 0.86 copies/μl (cycle threshold value = 37) ; the qPCR amplification with the Mel2 primers/probes did not yield positive results when used for the detection of other mycobacteria (including Mycobacterium leprae and Staphylococcus spp) . Among the 72 patients in the experimental group, 44 were positive for qPCR with a sensitivity of 61.1% (95% CI: 49.6% - 71.5%), and 47 were positive for culture with a sensitivity of 65.2% (95% CI: 53.8% - 75.3%) ; all the 68 controls were negative for both qPCR and culture, with their specificities both being 100%. Among 65 patients subjected to IGRA, 31 were positive with a sensitivity of 47.7% (95% CI: 36.0% - 59.6%), while 16 out of 25 controls were negative for IGRA with a specificity of 64.0% (95% CI: 44.5% - 79.8%). Among 58 patients subjected to acid-fast staining, 37 were positive with a sensitivity of 63.8% (95% CI: 50.9% - 74.9%), and 52 out of 66 controls were negative for acid-fast staining with a specificity of 78.8% (95% CI: 67.5% - 86.9%). The combination of qPCR and culture resulted in a sensitivity of 93% and a specificity of 100% for the detection of Mycobacterium marinum. Conclusion:In this study, a highly sensitive qPCR assay was developed for the detection of Mycobacterium marinum, and its combination with culture could further improve the detection sensitivity.

Taking chronic atrophic gastritis （CAG） as an example， the frontier technologies in data science have been introduced into the inheritance， innovation and development of traditional Chinese medicine （TCM）， providing reference for conducting real-world clinical research on specialized diseases of TCM. This paper put forward the construction of CAG data science system by elaborating the connotation of data science and its application value in TCM， and discussed the path to build CAG data science system， namely through "data acquisition-knowledge expression-knowledge reasoning" to establish CAG database， knowledge base and develop diagnosis platform differentiating diseases and syndromes. Besides， this paper analyzed the prospects of CAG data science in improving data governance ability and knowledge discovery efficiency， deepening the level of knowledge sharing， promoting interdisciplinary integration， and strengthening the integration process of industry， academia and research.