OBJECTIVE To investigate the ameliorative effects and potential mechanism of total secondary ginsenosides （TSG） on hypertrophic changes of primary cardiomyocytes stimulated by angiotensin Ⅱ （Ang Ⅱ）. METHODS Primary cardiomyocytes were isolated from the hearts of neonatal SD rats and divided into the following groups： control group， AngⅡ group （2 µmol/L）， TSG group （7.5 µg/mL）， PFK-015 group [6-phosphofructo-2-kinase/fructose-2， 6-bisphosphatase 3 （PFKFB3） inhibitor， 10 nmol/L]， and TSG+PFK-015 group （TSG 7.5 µg/mL+PFK-015 10 nmol/L）. The surface area， protein synthesis， energy metabolism-related indicators [free fatty acid （FFA）， coenzyme A （CoA）， acetyl coenzyme A （acetyl-CoA）]， and the expressions of glycolysis-related factors [hypoxia-inducible factor 1α （HIF-1α）， glucose transporter protein 4 （GLUT-4）， lactate dehydrogenase A （LDHA）， pyruvate dehydrogenase kinase 1 （PDK1） and PFKFB3] in primary cardiomyocytes of each group were measured. RESULTS Compared with the control group， the surface area of primary cardiomyocytes and protein synthesis were significantly increased， the content of FFA， protein and mRNA expressions of HIF-1α， LDHA， PDK1 and PFKFB3 were significantly increased or up-regulated in the AngⅡ group， while the contents of CoA and acetyl-CoA， the protein and mRNA expressions of GLUT-4 were significantly decreased or down-regulated （P＜0.05）. Compared with the AngⅡ group， both TSG group and PFK-015 group showed significant improvements in these indexes， with the TSG+PFK-015 group generally demonstrating superior effects compared to either treatment alone （P＜0.05）. CONCLUSIONS TSG can reduce the surface area of AngⅡ-induced primary cardiomyocytes， decrease protein synthesis， and inhibit their hypertrophic changes. These effects may be related to improving energy metabolism and the inhibition of glycolysis activity.

AIM:Using bioinformatics analysis methods to identify the hub genes involved in myocardial isch-emia-reperfusion injury(MIRI).METHODS:Firstly,the rat MIRI related dataset GSE122020,E-MEXP-2098,and E-GEOD-4105 were downloaded from the database.Secondly,differentially expressed genes(DEGs)were screened from each dataset using the linear models for microarray data(limma)package,and robust DEGs were filtered using the robust rank aggregation(RRA)method.In addition,the surrogate variable analysis(SVA)package was used to merge all datas-ets into one,and merged DEGs were screened using the limma package.The common DEGs were obtained by taking the intersection of the two channels of DEGs.Next,the protein-protein interaction(PPI)network of common DEGs was con-structed,and the hub genes were identified using the density-maximizing neighborhood component(DMNC)algorithm.The receiver operating characteristic curve(ROC)was plotted to evaluate the diagnostic performance of the hub gene.Then,the mRNA and protein expression levels of hub genes were detected in the rat MIRI model,and the literature re-view analysis was carried out on the involvement of hub genes in MIRI.Finally,the gene set enrichment analysis(GSEA)was performed on hub gene to further reveal the possible mechanism in mediating MIRI.RESULTS:A total of 143 robust DEGs and 48 merged DEGs were identified.After taking the intersection of the two,48 common DEGs were obtained.In the PPI network of common DEGs,5 hub genes were screened out,namely MYC proto-oncogene bHLH transcription fac-tor(MYC),prostaglandin-endoperoxide synthase 2(PTGS2),heme oxygenase 1(HMOX1),caspase-3(CASP3),and plasminogen activator urokinase receptor(PLAUR).The ROC results showed that the area under the curve values for all hub genes were greater than 0.8.MYC,PTGS2,CASP3,and PLAUR showed high mRNA and protein expression in rat MIRI,while there was no difference in mRNA and protein expression for HMOX1.The literature review revealed that among the 5 hub genes,only PLAUR has not been reported to be involved in MIRI.The GSEA results for PLAUR indicat-ed that its functional enrichment mainly focused on pathways such as NOD-like receptor signaling pathway,P53 signaling pathway,Toll-like receptor signaling pathway,apoptosis,and fatty acid metabolism.CONCLUSION:MYC,PTGS2,CASP3,HMOX1,and PLAUR are involved in the pathological process of MIRI.PLAUR is a potential hub gene that can mediate MIRI by regulating pathways such as NOD like receptor signaling,P53 signaling,Toll like receptor signaling,cell apoptosis,and fatty acid metabolism.The results can provide reference for further investigation into the molecular mechanisms and therapeutic targets of MIRI.

Sequencing is becoming increasingly important in the early detection, companion diagnostics, molecular subtyping, and prognosis assessment of malignant tumors. In this special column of'tumors and sequencing′, renowned experts in the field of laboratory medicine elaborate on cutting-edge concepts, technologies, and applications related to drug target gene and polymorphism analysis, novel techniques of liquid biopsy, and the clinical cases of mutated genes in solid tumors and hematoma. Notably, liquid biopsy techniques based on circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are advancing rapidly, offering the potential to serve as non-invasive diagnostic tools that facilitate precision medicine in oncology. To address the limitations of current ctDNA next-generation sequencing (NGS), future developments may focus on NGS targeting CTC-DNA for more precise tumor-targeted therapy. Despite the progress, challenges remain in integrating oncogene sequencing into clinical laboratories and advancing laboratory medicine. Addressing these challenges is crucial to improving public health.

OBJECTIVE To study the effects of Yishen daluo decoction on inflammatory factors and cyclic adenosine monophosphate（cAMP）/protein kinase A （PKA）/cAMP response element binding protein （CREB） signal pathway in experimental autoimmune encephalomyelitis （EAE） model mice by inhibiting the expressions of β-arrestin1， and to explore the mechanism of Yishen daluo decoction in the treatment of EAE. METHODS Sixty mice were randomly divided into normal group， model group， TCM group （Yishen daluo decoction 20 g/kg）， positive control group （prednisone acetate 3.9 mg/kg）， β-arrestin1 siRNA adeno- associated virus （AAV-β） group， AAV-β+TCM group， with 10 mice in each group. Except for normal group， EAE model was made in other groups. AAV-β group and AAV-β+TCM group were injected with AAV-β via tail vein to interfere with the expression of β -arrestin1 protein. Starting from the 8th day of modeling， they were given corresponding drug solution/normal saline intragastrically， once a day， for consecutive 14 days. The neurological function score of mice was detected； the pathological and morphological changes were observed in the brain and spinal cord tissues of mice； the serum levels of inflammatory factors [interleukin-2 （IL-2）， IL-23， interferon-γ （IFN-γ）] in mice were determined； the expressions of β-arrestin1， cAMP， PKA and CREB in brain and spinal cord were detected. RESULTS Compared with normal group， neurological function scores， serum levels of inflammatory factors， and protein expressions of β-arrestin1 in brain and spinal cord were significantly increased （P＜0.05 or P＜ 0.01）； protein expressions of PKA， CREB and cAMP in brain and spinal cord were decreased significantly（P＜0.05 or P＜0.01）. The deep staining of cellular shrinkage and aggregation of inflammatory cells were observed in most neurons of the brain and spinal cord， with varying degrees of demyelinating. Compared with model group， the neurological function scores， pathological changes in brain and spinal cord tissues， and most indicators （except for CREB and cAMP proteins in the brain tissue of AAV-β group） were significantly reversed （P＜0.05 or P＜0.01）.Compared with AAV- β group， the neurological function scores， the levels of IFN-γ in serum and β-arrestin1 in spinal cord were significantly decreased （P＜0.05 or P＜0.01）， PKA and cAMP in brain and spinal cord tissues were significantly increased in AAV- β +TCM group （P＜0.05 or P＜0.01）. CONCLUSIONS Yishen daluo decoction can inhibit the expression of β-arrestin1 in the central nervous system thus activating the cAMP/PKA/CREB signaling pathway， relieving nervous system inflammation， and ultimately alleviates the symptoms of EAE.

OBJECTIVE To explore the mechanism of Tingli dazao xiefei decoction on ventricular remodeling in model rats with heart failure after myocardial infarction. METHODS The rat model of heart failure after myocardial infarction was established by ligation of anterior descending branch of left coronary artery， which was divided into 8 groups： sham operation group， model group， A779 group （1 mg/kg）， A779 （1 mg/kg）+Tingli dazao xiefei decoction equivalent-dose group （0.8 g/kg）， A779 （1 mg/kg） +Tingli dazao xiefei decoction high-dose group （1.6 g/kg）， Tingli dazao xiefei decoction equivalent-dose group （0.8 g/kg）， Tingli dazao xiefei decoction high-dose group （1.6 g/kg） and losartan potassium group （10 mg/kg）. Each group was given equal volume of distilled water or corresponding drugs intragastrically for 4 weeks. Masson staining was used to determine the distribution of collagen fibers in rat myocardium. The content of hydroxyproline （Hyp） in myocardium was determined by alkaline hydrolyzation. The expressions of type Ⅰ and Ⅲ collagen （COLⅠ， COLⅢ）in myocardium were detected by immunohisto-chemistry. Myocardial fibrosis-related indexes such as matrix metalloproteinase-2 （MMP-2）， MMP-9， tissue inhibitor of metalloproteinase-1 （TIMP-1） and soluble suppression of tumorigenicity-2 （sST-2） were detected by ELISA. The protein expressions of angiotensin converting enzyme 2-angiotensin-（1-7）-Mas [ACE2-Ang-（1-7）-Mas] axis were detected by Western blot. RESULTS Compared with sham operation group， myocardial cells in model group and A779 group were disordered， collagen fiber deposition was significantly increased and myocardial fibrosis was obvious； the Hyp content and MMP-2， MMP-9， sST-2 levels were increased， and COL Ⅰ and COL Ⅲ positive expressions were significantly enhanced； TIMP-1 level， protein expressions of ACE2， Ang-（1-7） and Mas were significantly decreased （P＜0.05）. Compared with model group， above indexes of Tingli dazao xiefei decoction equivalent-dose and high-dose groups were improved to different extents. Compared with A779 group， A779+Tingli dazao xiefei decoction equivalent-dose and A779+high-dose groups could improve myocardial arrangement and collagen distribution， reduce the Hyp content and MMP-2， MMP-9 levels， reduce positive expressions of COL Ⅰ and COL Ⅲ （P＜0.05）， but couldn’t improve Ang-（1-7） and Mas protein expression. CONCLUSIONS Tingli dazao xiefei decoction can improve ventricular remodeling in myocardial failure model rats after myocardial infarction by improving the expression of ACE2- Ang（- 1-7）-Mas axis proteins.

OBJECTIVE：To investigate the effects of Shenfu yixin decoction on the utilization of fatty acid in primary hypoxic cardiomyocytes and its potential mechanism. METHODS ：The apical tissue of neonatal SD rats with 1-3 days old were collected ， and the primary cardiomyocytes were isolated ，cultured and identified. The cardiomyocytes were randomly divided into normal group，model group ，coenzyme Q 10 group（positive control ，1×10－4 mol/L），Shenfu yixin decoction low-dose and high-dose groups（0.25，0.5 mg/mL）. Except for normal group ，cells in other groups were cultured under 5％O2，5％CO2 and 90％N2 for 6 hours to induce hypoxic injury model. After 6 hours of hypoxia ，the content of ATP was detected by luciferase luminescence assay. Western blotting assay was adopted to detect the expression of FAT/CD 36，PPARα and PPARβ/δ. RESULTS：Compared with normal group ，the content of ATP and relative expression of FAT/CD 36 protein were decreased significantly in model group （P＜ 0.05）. Compared with model group ，the content of ATP was increased significantly in coenzyme Q 10 group and Shenfu yixin decoction high-dose group ，while the relative expression of FAT/CD 36 and PPARα protein in coenzyme Q10 group，the relative expression of FAT/CD 36 protein in Shenfu yixin decoction high-dose group as well as the relative expression of PPARα and PPARβ/δ protein in Shenfu yixin decoction groups were decreased significantly （P＜0.05）. CONCLUSIONS ：Shenfu yixin decoction can inhibit the utilization of fatty acid of primary hypoxic cardiomyocytes and improve their energy metabolism by inhibiting the expression of FAT/CD 36，PPARα and PPARβ/δ protein.

OBJECTIVE： To observe the effects of Shenfu yixin decoction on reactive oxygen species （ROS） and energy metabolism in primary hypoxic cardiomyocytes. METHODS： After isolation， culture and identification， primary cardiomyocytes of neonatal SD rats were randomly divided into normal group， model group， positive control group （coenzyme Q10， 0.1 mmol/L） and Shenfu yixin decoction low-dose and high-dose groups （0.25， 0.5 mg/mL）. Except for normal group， other groups were cultured with 5％O2， 5％CO2 and 90％N2 for 6 h to induce hypoxic injury model. After 6 hours of hypoxia， ROS contents in cardiomyocytes and mitochondria of each group were detected by ROS probe and flow cytometry. Luciferase luminescence and Western blotting were used to detect ATP content and CK protein expression of each group. Transmission electron microscope was used to observe ultrastructure of cardiomyocytes in each group. RESULTS： Compared with normal group， the expression of ROS in primary hypoxic cardiomyocytes and mitochondria as well as the content of ROS were increased significantly， while the content of ATP and expression levels of CK protein were decreased significantly （P＜0.05）； there were swelling of endoplasmic reticulum and mitochondria， dissolution or even disappearance of mitochondrial ridge， obvious cardiomyocytes injury. Compared with model group， the expression of ROS in primary hypoxic cardiomyocytes and mitochondria of administration groups， the contents of ROS in primary hypoxic cardiomyocytes of positive control group and Shenfu yixin decoction high-dose group as well as the content of ROS in primary hypoxic cardiomyocytes mitochondria of administration groups were all decreased significantly， while ATP contents in primary hypoxic cardiomyocytes of positive control group and Shenfu yixin decoction high-dose group as well as expression levels of CK protein in primary hypoxic cardiomyocytes of administration groups were all increased significantly （P＜0.05）. The primary hypoxic cardiomyocytes injury was relieved significantly in positive control group and Shenfu yixin decoction high-dose group. CONCLUSIONS： Shenfu yixin decoction can improve primary hypoxic cardiomyocytes， down-regulate the expression of ROS in cardiomyocytes and mitochondria and also improve its energy metabolism.

Over the past decade, the management model of cancer patients has gradually shifted to individual mode based on molecular mutation detection. Epidermal growth factor receptor (EGFR) gene mutation is an important driving factor in non-small cell lung cancer (NSCLC). Compared with traditional chemotherapy, EGFR-targeted therapy shows significant safety and efficacy. However, not all patients with EGFR mutations are eligible for EGFR-targeted therapy, and different types of mutations often indicate different clinical outcomes, such as the sensitive mutations EGFR 19-Del, L858R, and the resistance mutation. In addition, the third-generation TKI drugs Osimertinib (AZD9291) and Rociletinib (CO-1686) have been developed to further benefit patients with primary TKI resistance caused by T790M mutation of EGFR. Therefore, detection of the EGFR mutation status of patients before treatment, and continuously monitoring the mutation of drug resistance genes during the treatment process is useful for the management of targeted drugs in NSCLC patients. In recent years, the rapid development of "liquid biopsy" technology has made it possible to use non-invasive methods to monitor drug resistance mutations in real time. In this paper, we reviewed the clinical application of various non-invasive detection techniques for EGFR mutations in NSCLC in different liquid samples.
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Animals ; Antineoplastic Agents ; administration & dosage ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; genetics ; metabolism ; DNA Mutational Analysis ; methods ; ErbB Receptors ; genetics ; metabolism ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; Mutation

Objective This study was designed to determine the effect of transient receptor potential vanilloid type 4(TRPV4)on angiotensin Ⅱ(Ang Ⅱ)-induced renal injury in TRPV4-null mutant(TRPV4 -/-)mice. Methods The mice were divided into sham group and Ang Ⅱ-treated group. Ang Ⅱ was infused systemically into wild type(WT)and TRPV4 -/- mice via a miniosmotic pump for 4 weeks, and the sham mice were given with normal saline. Systolic blood pressure,urinary excretion of albumin and 8-isoprostane, serum creatinine, and the pathological changes in the kidney tissues were assayed after the 4-week treatment. Results Compared with corresponding sham mice,Ang Ⅱ infusion led to enhanced systolic blood pressure,increased urinary excretion of albumin and 8-isoprostane,increased serum creatinine(P< 0.05),and enhanced glomerulosclerosis degree and renal tubulointerstitial injury index(P< 0.05)in the WT and TRPV4 -/- mice. The result were associated with enhanced collagen levels in the kidney(P< 0.05). All of them were attenuated by the deletion of TRPV4 in the absence of alteration in blood pressure(P< 0.05). Conclusions Deletion of TRPV4 could alleviate renal injury during Ang Ⅱ-induced hypertension, suggesting that TRPV4 may contribute to the pathophysiology of angiotensin Ⅱ-induced renal injury.

BACKGROUND: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. METHODS: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. RESULTS: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, P<0.0001). CONCLUSIONS: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.
Adult ; DNA/*blood/standards ; Female ; Healthy Volunteers ; Humans ; Male ; Middle Aged ; Real-Time Polymerase Chain Reaction/*methods/standards ; Reference Values ; Wounds and Injuries/blood