AIM:To explore the mechanism of ATP synthase mitochondrial F0 complex H+ transporting,sub-unit F6(ATP5J)in affecting the metastasis of hepatoma carcinoma cells by regulating mitochondrial function-mediated cy-toskeletal remodeling.METHODS:Hepatocellular carcinoma cells Li-7 were used to construct the ATP5J overexpression and knockdown models.JC-1 staining was used to detect the mitochondrial membrane potential in each group,reactive oxygen species(ROS)levels were examined by DCHF-DA,and mitochondrial ATP fluorescence probe was used to assess mito-chondrial function.Cytoskeletal remodeling was detected with a microfilament green fluorescent probe(Actin-Tracker Green-488).Transwell assay was used to assess cell invasion ability.The expression levels of ATP5J and translocase of outer mitochondrial membrane 20(TOMM20)were determined by Western blot.RESULTS:Overexpression of ATP5J up-regulated mitochondrial membrane potential and mitochondrial ATP fluorescence intensity,induced cytoskeletal re-modeling,promoted cell invasion and TOMM20 expression,and inhibited ROS production(P＜0.01).On the contrary,knockdown of ATP5J significantly decreased mitochondrial membrane potential and mitochondrial ATP fluorescence inten-sity,significantly decreased cell invasion ability and TOMM20 expression,promoted ROS production and blocked cyto-skeletal remodeling(P＜0.01).CONCLUSION:ATP5J regulates mitochondrial energy transformation in hepatocellular carcinoma cells,and affects metastasis of hepatoma carcinoma cells by regulating mitochondrial membrane potential and mitochondrial ATP production-mediated cytoskeletal remodeling through TOMM20.

Dachengqi Decoction is a classic prescription attacked by Yangming excessive syndromes in clinic, which has the effects of relieving heat, softening and dispersing knots, etc., and is often used in the treatment of gastrointestinal dysfunction caused by various diseases. This article reviewed the recent studies on the chemical compositions and pharmacological effects of Dachengqi Decoction in recent years. On this basis, combined with the "five principles" of TCM quality markers, the quality markers of Dachengqi Decoction were predicted and analyzed. It is suggested that emodin, Rhein, chrysophanol, aloe-emodin, synephrine, hesperidin, naringin, magnolol and magnolol can be used as quality markers of Dachengqi Decoction.

Objective To explore the role and underlying mechanism of type Ⅲ secretion system(T3SS)in regulating the internalization of Pseudomonas aeruginosa(PA)into pulmonary epithelial cells.Methods The human non-small cell lung cancer A549 cells were infected with or without PA strains,including wild-type PAO1(a standard experimental PA strain),△exsA(knockout of the critical activator for T3SS genes),△pscJ(T3SS secretion-defective strain)and PAO1-E(EGTA-induced high expression of T3SS genes).The A549 cells pretreated with ERK inhibitor U0126 or reactive oxygen species(ROS)inhibitor apocynin(APO)/N-acetyl-L-cysteine(NAC)were infected with PAO1 or PAO1-E strain.Thus,the experiment was grouped as follows:the mock-treated group,PAO1-or PAO1-E-infected group,inhibitor-treated group,and PAO1/PAO1-E plus inhibitor-treated group.Extracellular bacteria were killed by gentamicin,and the cell lysates were diluted and then plated on PA screening plates.Bacterial amounts were detected by counting colony-forming units(CFUs).The production of ROS was analyzed using fluorescent probe labeling and flow cytometry.The activation of the ERK pathway was detected by Western blotting.Results Compared with the PAO1-infected group,the intracellular bacteria and ROS level in △exsA-or△pscJ-infected cells were lower(P＜0.05,P＜0.01),so was the generation of ROS(P＜0.01);In contrast,those of the PAO1-E strain-infected cells displayed an opposite trend(P＜0.01).Compared with the PAO1-or PAO1-E-infected group,the cells pretreated with APO/NAC followed by PAO1 or PAO1-E infection showed reduced intracellular bacterial amounts(P＜0.01).Compared to the PAO1-infected A549 cells,the phosphorylation level of ERK was increased in the △exsA-or △pscJ-infected cells(P＜0.01),while that level was suppressed in the PAO1-E-treated cells(P＜0.01).Compared with the PAO1-infected group,the PAO1-infected cells pretreated with U0126 displayed reduced ERK activation,elevated ROS production,and increased intracellular counts of PAO1(P＜0.01).Conclusion T3SS-mediated inhibition of the ERK pathway promotes the production of ROS and the internalization of PA in lung epithelial cells.

Objective:To investigate the relationship between the expression levels of serum glycogen synthase kinase-3β (GSK-3β) and mothers against decapentaplegic homolog 4 (SMAD4) in patients with acute coronary syndrome (ACS) and the severity and prognosis of coronary artery disease.Methods:A total of 192 ACS patients admitted to Shandong First Medical University Affiliated Qingdao Hospital from June 2020 to May 2022 were selected as the ACS group, while 192 non ACS patients admitted to the same hospital were selected as the non ACS group. Enzyme-linked immunosorbent assay method was applied to determine the expression levels of serum GSK-3β and SMAD4 in two groups. The Gensini score was applied to evaluate the degree of coronary artery disease in patients, Spearman method was applied to analyze the correlation between serum GSK-3β, SMAD4 expression levels and Gensini score in ACS patients. Receiver operating characteristic (ROC) curve was applied to analyze the predictive efficacy of serum GSK-3β and SMAD4 levels on the prognosis of ACS patients.Results:The serum levels of GSK-3β and SMAD4 in the ACS group were significantly higher than those in the non ACS group: (2.70 ± 0.40) μg/L vs. (2.24 ± 0.41) μg/L, (12.19 ± 2.10) μg/L vs. (9.79 ± 2.82) μg/L, and there were statistical differences ( P<0.05). The serum levels of GSK-3β and SMAD4 in ACS patients with mild, moderate and severe coronary artery disease increased sequentially ( P<0.05). Spearman analysis showed that serum GSK-3β and SMAD4 levels in ACS patients were positively correlated with Gensini total score ( r = 0.569 and 0.587, P<0.01). In ACS patients, 48 cases had poor prognosis, and the incidence of poor prognosis was 25.00% (48/192); 144 cases had a good prognosis. The serum levels of GSK-3β and SMAD4 in ACS patients with poor prognosis were significantly higher than those in ACS patients with good prognosis: (3.15 ± 0.53) μg/L vs. (2.55 ± 0.36) μg/L, (14.03 ± 2.08) μg/L vs. (11.58 ± 2.11) μg/L, and there were statistical differences ( P<0.05). The ROC curve result indicated that the area under the curve (AUC) for predicting poor prognosis in ACS patients with serum GSK-3β and SMAD4 levels alone and in combination was 0.799, 0.784 and 0.858, respectively, the AUC predicted by the combination of the two was obviously higher than the AUC predicted separately ( Z = 2.04 and 2.75, P = 0.041 and 0.006). Conclusions:The expression levels of GSK-3β and SMAD4 in the serum of ACS patients are abnormally elevated. They are positively correlated with the degree of coronary artery disease in ACS patients, and both have good predictive power for adverse prognosis in ACS patients, while the combined use of the two has better predictive performance.

The establishment of mother-infant bonding is closely related to maternal mental health and early growth and development of infants. This paper reviews the concept, evaluation tools, influencing factors and intervention measures of maternal-infant bonding, in order to provide a basis for promoting the normal establishment of postpartum maternal infant relationships and maternal infant health.

Objective To establish an animal model of lung adenoma in BALB/c mice based on dynamic characterization by micro-computed tomography(CT).Methods Eighty female SPF-grade BALB/c mice were divided randomly into four groups:model low dose group(1 mg/g urethane,iP,once),model medium dose group(1 mg/g urethane,ip,once a week,followed by 2 weeks),model high dose group(1 mg/g urethane,ip,once a week,followed by 4 weeks),and blank group(equal volume of saline).Growth of lung nodules in the mice was monitored regularly using Micro-CT.Three-dimensional images of the lungs were drawn using the Analyze 12.0 system,and lung tissues were taken for histopathological examination(hematoxylin and eosin).Results Lung nodules with round high-density shadows were observed at week 11 in all model groups compared with the findings in the blank group.The rate of nodule formation increased with increasing modeling weeks,with rates of nodule formation in the model high,medium,and low dose groups of 93.8%,93.8%,and 87.5%,respectively,at week 21.Most mice had two to four,followed by one,and one to two nodules,respectively.The average maximum diameter of the lung nodules in the low dose group was significantly higher than the diameters in the medium-and high-dose groups(P＜0.05),but there was no significant difference in lung nodule volume among the three groups.Regarding pathological type,hematoxylin and eosin staining revealed that the tumors in all the model groups were lung adenomas.Conclusions Lung adenomas were successfully induced in all urethane dose groups of mice and growth of the lung nodules could be characterized by micro-CT.The rate of nodule formation was highest in the medium dose group,which developed a moderate number of lung adenomas and provided a stable model,and was thus considered the most suitable model for the study of lung adenomas in mice.

Objective To investigate the effects of oral probiotics on gut microbiota diversity,colony structure,and intergroup differences in mice with subcutaneous colon cancer tumors,based on 16S rRNA sequencing technology.Methods Twenty-four 6-week-old male BALB/c mice were divided randomly into normal control group(NC group,n = 8),model group(M group,n = 8),and probiotic + model group(PM group,n = 8)after adaptive feeding for 1 week.Mice in the PM group were given 200 μL probiotic mixed solution(Bifidobacterium longum and Lactobacillus delbrueckii subsp.bulgaricus mixed lyophilized powder,2×108 colony-forming units)by gavage three times/week for 7 weeks,while the M group and PM group received 200 μL normal saline.At 10 weeks old,0.2 mL CT26.WT cell suspension(1×107/mL)was inoculated subcutaneously into the left hind limbs of M group and PM group,while NC group were inoculated with 0.2 mL normal saline.The general state of mice was observed,the growth of subcutaneous tumor was monitored,and the changes of intestinal flora structure were detected by 16S rRNA sequencing.Results The subcutaneous tumors of the M group were prominent,and the subcutaneous tumor volume and weight of the PM group were significantly reduced(P<0.05).Compared with NC group,Alpha diversity index was lower in the M group,and a significant difference of Beta diversity inter groups(P<0.01).And supplementation of probiotics had a certain effect on gut microbiota diversity in the M group.Compared with M group,the relative abundance of Bacteroidetes,Proteobacteria,Muribaculaceae,Bacteroides were higher in the PM group,while the relative abundance of Firmicutes,Desulfobacterota,Lachnospiraceae_NK4A136_group,Alistipes were lower in the PM group.LEfSe analysis showed that Muribaculaceae and Bacteroides in the PM group were different species with high abundance(LDA values>4).Conclusions Oral probiotics may improve the gut microbiota by increasing the relative abundance of beneficial Muribaculaceae and Bacteroides in subcutaneous tumors in mice with colon cancer.

Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase(tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
Animals ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Oryzias/genetics* ; RNA, Guide/genetics* ; RNA, Transfer/genetics*

Recombinant human interferon α2b(rhIFNα2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNα2b is complex.In this study,an anti-rhIFNα2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNα2b.RhIFNα2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNα2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNα2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNα2b detection assay had a limit of detection of 1 μg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other com-mercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.

OBJECTIVE To establish a method for simultan eous determination of 5 kinds of pentacyclic triterpenoids as 3-O- acetyl-pomolic acid in Chaenomeles speciosa ，and to analyze the difference in the contents of C. speciosa from different producing areas by different processing technologies . METHODS HPLC method was adopted . The determination was performed on COSMOSIL 5 C18-MS-Ⅱ column with mobile phase consisted of acetonitrile -0.005 mol/L ammonium dihydrogen phosphate solution （pH value adjusted to 6.5 with phosphoric acid ）（70∶30，V/V）at the flow rate of 1.0 mL/min. The column temperature was set at 30 ℃. The detection wavelength was set at 210 nm，and sample size was 20 μL. The contents of the other four pentacyclic triterpenoids were calculated according to quantitative analysis of multi -components by single -marker（QAMS）using oleanolic acid as internal reference . The results were compared with those determined by external standard method . The total content of oleanolic acid and ursolic acid ，the total content of 5 components in C. speciosa from different producing areas and different processing technologies were compared . RESULTS The linear range of 3-O-acetyl-pomolic acid ，betulinic acid ，oleanolic acid ，ursolic acid and 3-O-acetyl ursolic acid were 4.06-81.2，2.12-42.4，9.62-192.3，7.77-155.4，4.21-84.1 μg/mL，respectively（R2＞0.999）. RSDs of precision ，reproducibility and stability （24 h）tests were all lower than 3%. The average recoveries were 98.29%-101.38% （RSDs＜3%，n＝6）. The mass fraction of 3-O-acetyl-pomolic acid ，betulinic acid ，ursolic acid and 3-O-acetyl ursolic acid measured by QAMS w ere 0.023%-0.060%，0.044%-0.528%，0.101%-0.368%，0.067%-0.221%，respectively；the deviations from the results measured by external standard method were all within 8%. The total content of oleanolic acid and ursolic acid， the total content of 5 components in C. speciosa processed by fresh -cut technology from the same producing area were higher than those in C. speciosa processed by traditional technology ，and the total content of 5 components in C. speciosa from Chongqing Qianjiang were significantly higher than those from other producing areas （P＜0.05）. CONCLUSIONS QAMS method is established for the simultaneous determination of 3-O-acetyl-pomolic acid ，betulinic acid ，oleanolic acid ，ursolic acid and 3-O-acetyl ursolic acid in C. speciosa. Established method is simple ，rapid and accurate . The total content of 5 components in C. speciosa produced in Chongqing Qianjiang is higher ，and the total content of C. speciosa processed by fresh -cut technology from the same origin is higher than C. speciosa processed by traditional technology .