Metabolic reprogramming， as one of the core hallmarks of malignant tumors， plays a key role in the occurrence， development and treatment of breast cancer （BC）. Abnormal changes in glucose metabolism， amino acid metabolism， lipid metabolism， as well as the tricarboxylic acid （TCA） cycle and oxidative phosphorylation （OXPHOS） pathways significantly influence the pathogenesis and progression of BC. Studies have shown that various active components of traditional Chinese medicine （TCM） （such as berberine， matrine， quercetin， curcumin， etc.） and their compound formulations （e.g. Xihuang pill， Danzhi xiaoyao powder， Yanghe decoction， etc.） can inhibit the proliferation and migration of BC cells and induce apoptosis by regulating key metabolic pathways such as glycolysis， lipid synthesis， and amino acid metabolism. TCM demonstrates multi-target and holistic regulatory advantages in intervening in BC metabolic reprogramming， showing significant potential in modulating key molecules like hypoxia inducible factor-1α， hexokinase-2， pyruvate kinase M2， lactate dehydrogenase A， glucose transporter-1， fatty acid synthase， and signaling pathways such as AKT/mTOR. However， current researches still focus predominantly on glucose metabolism， with insufficient mechanistic studies on lipid metabolism， amino acid metabolism， the TCA cycle， and OXPHOS. The precise targets， molecular mechanisms， and clinical translation value of these interventions require further validation and clarification through more high-quality experimental studies and clinical trials.

Objective To investigate the role of STK4-AS1 in regulating the proliferation,invasion,and migration of esophageal squamous cell carcinoma(ESCC)cells through the MYG1/Notch signaling pathway.Methods Quantitative real-time PCR(qRT-PCR)was used to detect the expression of STK4-AS1 in ESCC cells.MTS assay,wound healing and Transwell assay were conducted to explore the proliferation,migration,and invasion abilities in each group in Eca109 and Kyse150 cells.mRNA sequencing(mRNA-seq)was used to detect the down-stream target genes of STK4-AS1.KEGG functional enrichment analyses were used to predict the possible biological processes and signaling pathways.qRT-PCR and western blot were performed to identify mRNA expression of MYG1 and the key downstream transcription factors HES1,HES5,and HEY1 of the Notch signaling pathway,as well as the protein expression of NICD1.Co-transfection plasmids(for over-expressing STK4-AS1 and MYG1)were used to detect the mRNA expression of HES1,HES5,and HEY1 and the protein expression of NICD1 which acted as the key downstream transcription factors in the Notch signaling pathway,as well as the effects on the proliferation,migration,and invasion abilities of ESCC cells.Results The expression of STK4-AS1 was decreased in ESCC cell lines(P<0.01).Over-expression of STK4-AS1 inhibited the proliferation,migration and invasion abilities in Eca109 and Kyse150 cells(P<0.05).STK4-AS1 negatively regulated the expression of MYG1(P<0.01),and the expression of MYG1 was increased in ESCC cell lines(P<0.01).Over-expression of MYG1 could partially reverse the effect of STK4-AS1 on the malignant biological behavior of Eca109 and Kyse150 cells(P<0.05),as well as the mRNA expressions of HES1,HES5,and HEY1 and the protein expression of NICD1(P<0.05).Conclusion STK4-AS1 affects the malignant biological behaviors of ESCC through the MYG1/Notch signaling pathway.

Objective To investigate the role of STK4-AS1 in regulating the proliferation,invasion,and migration of esophageal squamous cell carcinoma(ESCC)cells through the MYG1/Notch signaling pathway.Methods Quantitative real-time PCR(qRT-PCR)was used to detect the expression of STK4-AS1 in ESCC cells.MTS assay,wound healing and Transwell assay were conducted to explore the proliferation,migration,and invasion abilities in each group in Eca109 and Kyse150 cells.mRNA sequencing(mRNA-seq)was used to detect the down-stream target genes of STK4-AS1.KEGG functional enrichment analyses were used to predict the possible biological processes and signaling pathways.qRT-PCR and western blot were performed to identify mRNA expression of MYG1 and the key downstream transcription factors HES1,HES5,and HEY1 of the Notch signaling pathway,as well as the protein expression of NICD1.Co-transfection plasmids(for over-expressing STK4-AS1 and MYG1)were used to detect the mRNA expression of HES1,HES5,and HEY1 and the protein expression of NICD1 which acted as the key downstream transcription factors in the Notch signaling pathway,as well as the effects on the proliferation,migration,and invasion abilities of ESCC cells.Results The expression of STK4-AS1 was decreased in ESCC cell lines(P<0.01).Over-expression of STK4-AS1 inhibited the proliferation,migration and invasion abilities in Eca109 and Kyse150 cells(P<0.05).STK4-AS1 negatively regulated the expression of MYG1(P<0.01),and the expression of MYG1 was increased in ESCC cell lines(P<0.01).Over-expression of MYG1 could partially reverse the effect of STK4-AS1 on the malignant biological behavior of Eca109 and Kyse150 cells(P<0.05),as well as the mRNA expressions of HES1,HES5,and HEY1 and the protein expression of NICD1(P<0.05).Conclusion STK4-AS1 affects the malignant biological behaviors of ESCC through the MYG1/Notch signaling pathway.

Objective:To investigate the predicting value of serum miR-195 and miR-599 for the outcome of patients with acute ischemic stroke (AIS).Methods:Patients with AIS admitted to Danzhou People's Hospital from January 2018 to July 2020 were enrolled prospectively. The modified Rankin Scale was used to evaluate the outcome of patients at 14 d after onset or when they were discharged from the hospital. A score of 0-2 was defined as a good outcome and a score of >2 were defined as a poor outcome. Multivariate logistic regression analysis was used to determine the independent risk factors for poor outcome of patients with AIS. Receiver operating characteristic (ROC) curve analysis was used to determine the predictive value of serum miR-195 and miR-599 for the poor outcome of patients with AIS. Results:A total of 158 patients with AIS were enrolled. Their age was (65.80±12.36) years old, 105 were males (66.46); 95 patients (60.1%) had a good outcome, and 63 patients (39.9%) had a poor outcome. The age, total cholesterol, triglycerides, low-density lipoprotein cholesterol, baseline National Institutes of Health Stroke Scale (NIHSS) score, serum miR-195 and miR-599 levels in the poor outcome group were significantly higher than those of the good outcome group ( P<0.05). Multivariate logistic regression analysis showed that age (odds ratio [ OR] 1.984, 95% confidence interval [ CI] 1.315-3.617; P=0.036), low-density lipoprotein cholesterol ( OR 2.108, 95% CI 1.406-4.103; P=0.013), baseline NIHSS score ( OR 2.584, 95% CI 1.675-4.505; P=0.005), serum miR-195 ( OR 3.927, 95% CI 2.615-8.227; P<0.001) and miR-599 ( OR 2.952, 95% CI 1.973-6.114; P<0.001) were the independent risk factors for the poor outcome of patients with AIS. ROC curve analysis showed that the area under the curve (0.938, 95% CI 0.882-0.997) of serum miR-195 combined with miR-599 for predicting poor outcome was significantly higher than that predicted alone, and its predictive sensitivity and specificity were 97.0% and 87.4% respectively. Conclusions:The higher levels of serum miR-195 and miR-599 are associated with the poor outcome of patients with AIS. The combination of the both had good predictive value for the poor outcome of patients with AIS.

Objective:To investigate the predictive value of circulating miR-143 and miR-182 for the short-term clinical outcomes in patients with acute ischemic stroke (AIS).Methods:Patients with AIS admitted to Danzhou People's Hospital from January 2018 to June 2020 were included prospectively. The modified Rankin Scale was used to evaluate the short-term clinical outcome at 14 d after onset or at discharge. 0-2 was defined as good outcome, and >2 was defined as poor outcome. Multivariate logistic regression analysis was used to identify the independent risk factors for poor short-term clinical outcomes in patients with AIS. The receiver operating characteristic (ROC) curve was used to evaluate the predictive value of miR-143 and miR-182 for the short-term clinical outcomes in patients with AIS. Results:A total of 158 patients with AIS, aged 65.80±12.36 years, were enrolled, including 105 males (66.46%), 95 patients with good outcome (60.1%) and 63 with poor outcome (39.9%). The age, total cholesterol, triglyceride, low-density lipoprotein cholesterol, baseline National Institutes of Health Stroke Scale (NIHSS) score, serum miR-143 and miR-182 level in the poor outcome group were significantly higher than those in the good outcome group (all P<0.05). Multivariate logistic regression analysis showed that age (odds ratio [ OR] 1.984, 95% confidence interval [ CI] 1.315-3.617; P=0.036), low-density lipoprotein cholesterol ( OR 2.108, 95% CI 1.406-4.103; P=0.013), baseline NIHSS score ( OR 2.584, 95% CI 1.675-4.505; P=0.005), miR-143 ( OR 3.205, 95% CI 2.370-6.180; P<0.001) and miR-182 ( OR 2.802, 95% CI 1.905-5.516; P<0.001) were the independent risk factors for poor outcomes in patients with AIS. ROC curve analysis showed that the combined area under the curve of miR-143 and miR-182 to predict the poor outcome in patients with AIS was 0.935 (95% CI 0.873-0.992), the sensitivity and specificity were 96.5% and 87.0% respectively. Conclusions:The increase of serum miR-143 and miR-182 was closely associated with the poor short-term outcomes in patients with AIS. The combination of the two has a good predictive value for the poor short-term outcomes in patients with AIS.

Objective:To investigate the expressions of serum microRNA-24 (miR-24) and microRNA-29b (miR-29b) in elderly patients with acute ischemic stroke (AIS) and their neural function prognostic value.Methods:A prospective study was conducted. 170 elderly patients with AIS admitted to department of neurology of Danzhou People's Hospital from January 1st, 2017 to March 31st, 2019 were enrolled. According to modified Rankin scale (mRS) score, the patients were divided into good neural function prognosis group (mRS score ≤ 2, n = 105) and poor neural function prognosis group (mRS score > 2, n = 65). According to National Institutes of Health stroke scale (NIHSS) score, the patients were divided into mild group (NIHSS score < 5, n = 50), moderate group (NIHSS score 5-20, n = 76) and severe group (NIHSS score > 20, n = 44). Sixty-five healthy volunteers in the same period were enrolled as the control group. The expressions of serum miR-24 and miR-29b were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Receiver operating characteristic (ROC) curve was plotted to analyze the value of serum expressions of miR-24 and miR-29b for predicting the poor neural function prognosis of elderly patients with AIS. Pearson correlation was used to analyze the correlation between the expressions of serum miR-24, miR-29b and NIHSS, mRS scores in elderly patients with AIS. Results:The expressions of serum miR-24 and miR-29b in the AIS group were significantly lower than those in the healthy control group [miR-24 (2 -ΔΔCt): 0.64±0.17 vs. 2.18±0.85, miR-29b (2 -ΔΔCt): 0.72±0.21 vs. 3.05±0.96, both P < 0.01]. The expressions of serum miR-24 and miR-29b in the poor neural function prognosis group were significantly lower than those in the good neural function prognosis group [miR-24 (2 -ΔΔCt): 0.20±0.05 vs. 1.16±0.48, miR-29b (2 -ΔΔCt): 0.18±0.03 vs. 1.41±0.56, both P < 0.01]. The expressions of serum miR-24 and miR-29b in the severe group were significantly lower than those in the mild and moderate groups [miR-24 (2 -ΔΔCt): 0.13±0.02 vs. 1.30±0.51, 0.56±0.14; miR-29b (2 -ΔΔCt): 0.09±0.01 vs. 1.52±0.60, 0.62±0.13; all P < 0.01], and they were significantly lower in the moderate group than those in the mild group (all P < 0.01). ROC curve analysis showed that the optimal cut-off values of serum miR-24 and miR-29b expressions for predicting poor neural function prognosis in elderly AIS patients were 0.53 and 0.48, respectively. The area under ROC curve (AUC) of the two combined prognoses was 0.920 [95% confidence interval (95% CI) was 0.861-0.982], and it was significantly higher than that of miR-24 (AUC was 0.802, 95% CI was 0.742-0.860) or miR-29b (AUC was 0.835, 95% CI was 0.778-0.890) alone ( Z values were 6.513 and 4.902, respectively, both P < 0.05), with sensitivity and specificity of 92.0% and 85.7%. Pearson correlation analysis showed that the expressions of serum miR-24 and miR-29b were negatively correlated with NIHSS score ( r values were -0.758 and -0.794, respectively) and mRS score ( r values were -0.817 and -0.860, respectively) in elderly AIS patients (all P < 0.01). Conclusion:The down-regulated expressions of serum miR-24 and miR-29b are correlated with the severity degree of neurological impairment and neural function prognosis of elderly AIS patients, and the two combined have certain value for predicting the neural function prognosis of elderly AIS patients.

Objective: To analyze the risk factors and the predictive value of LDL/HDL ratio in acute ischemic stroke recurrence.
Methods: A total of 143 patients with acute ischemic stroke treated in our hospital were studied. The patients were followed-up for 2 years, the risk factors and the predictive value of LDL/HDL ratio for stroke recurrence were analyzed.
Results: Single factor analysis indicated that lacunar infarction, hypertension, blood pressure, the levels of LDL, HDL, ratio of LDL/HDL, stroke scale score, life ability score were the risk factors of ischemic stroke recurrence. Multivariate analysis presented that hypertension, ratio of LDL/HDL, TG level were the independent risk factors for ischemic stroke recurrence. LDL and ratio of LDL/HDL were positively related to ischemic stroke recurrence, HDL was negatively related to ischemic stroke recurrence, P<0.05. The speciifcity and sensitivity of LDL/HDL for predicting ischemic stroke recurrence were at 81.16%and 95.65%respectively.
Conclusion: The predictive value of LDL/HDL ratio could help us to identify the patients with high risk of ischemic stroke recurrence which is important for the early clinical intervention.

Rotundine (1 micromol L(-1)) was incubated with a panel of rCYP enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remained parent drug in incubates was quantitatively analyzed by an Agilent LC-MS. CYP2C19, 3A4 and 2D6 were identified to be the isoenzymes involved in the metabolism of rotundine. The individual contributions of CYP2C19, 3A4 and 2D6 to the rotundine metabolism were assessed using the method of total normalized rate to be 31.46%, 60.37% and 8.17%, respectively. The metabolites of rotundine in incubates were screened with ESI-MS at selected ion mode, and were further identified using MS2 spectra and precise molecular mass obtained from an Agilent LC/Q-TOF-MSMS, as well as MS(n) spectra of LC-iTrap-MS(n). The predominant metabolic pathway of rotundine in rCYP incubates was O-demethylation. A total 5 metabolites were identified including 4 isomerides of mono demethylated rotundine and one di-demethylated metabolite. The results also showed that CYP2C19, 2D6 and 3A4 mediated O-demethylation of methoxyl groups at different positions of rotundine. Furthermore, the ESI-MS cleavage patterns of rotundine and its metabolites were explored by using LC/Q-TOF-MSMS and LC/iTrap-MS(n) techniques.

To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.
Cloning, Molecular ; Endothelial Cells ; cytology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Solubility ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor Receptor-1 ; genetics ; isolation & purification ; metabolism

We constructed and expressed an anti-CD3/anti-Pgp (P-glycoprotein) diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli (E. coli) 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry (FCM) was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37 degrees C in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.
ATP Binding Cassette Transporter, Sub-Family B ; immunology ; Antibodies, Bispecific ; biosynthesis ; chemistry ; genetics ; immunology ; Binding, Competitive ; CD3 Complex ; immunology ; Cell Line ; Disulfides ; chemistry ; Drug Stability ; Escherichia coli ; genetics ; metabolism ; Humans