By using a yeast two-hybrid system, a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins, and its effects on the growth of yeast cells and the activation of reporter genes were investigated. Total mRNA extracted from Hela cells was reversely transcribed into cDNA. Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector. The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector were transformed into the yeast cell AH109, respectively. After they were cultured respectively in YPDA liquid medium and nutrition-deficient culture medium, their toxicity and transcriptional activation were tested by both the phenotype assay and the color assay. The bait plasmid HPV18 E6 was successfully obtained. After being cultured in YPDA liquid medium for 16h, the A (600 nm) values of two yeast fluids were 0.98+/-0.03 and 0.99+/-0.02, respectively. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector could grow to white colonies on SD/-Trp/X-alpha-gal plates, while no colony could survive on SD/-His/-Trp/X-alpha-gal, SD/-Ade/-Trp/X-alpha-gal plates, indicating that the bait plasmid pGBKT7-HPV18 E6 was constructed successfully and expressed correctly, and could not activate the transcription of reporter gene alone. The yeast two-hybrid GAL4 system 3 can be utilized to find HPV18 E6 interacting proteins.

Objective To evaluate the clinical efficacy of Xiaojianzhong Decoction plus Dachaihu Decoction in the treatment of chronic atrophic gastritis(CAG),and to provide scientific evidence for the clinical application of the formula.Methods The clinical observation was carried out in 80 CAG patients with spleen deficiency and stasis-heat syndrome of normal-people pulse type(the ratio of patients'pulse to the number of respirations within one minute being 4-5 evaluated by Changsangjun pulse-taking method)who attended the clinic of the Department of Gastroenterology of Shunde Hospital,Guangzhou University of Chinese Medicine,from January 2020 to December 2023.According to the treatment method,the patients were divided into the treatment group and the control group,with 40 cases in each group.The control group was given conventional western medicine treatment,and the treatment group was given Xiaojianzhong Decoction plus Dachaihu Decoction.Seven days constituted one course of treatment,and the treatment covered 6 months.The changes of traditional Chinese medicine(TCM)syndrome score,gastroscopy score,pathological score and gastric function indicators in the two groups were observed before and after the treatment.After treatment,the clinical efficacy and safety of the two groups were evaluated.Results(1)After 6 months of treatment,the total effective rate of the treatment group was 90.00%(36/40),and that of the control group was 60.00%(24/40).The intergroup comparison(tested by chi-square test)showed that the efficacy of the treatment group was significantly superior to that of the control group(P＜0.05).(2)After treatment,the scores of TCM syndromes in the two groups were significantly lower than those before treatment(P＜0.05),and the decrease of the scores in the treatment group was superior to that in the control group(P＜0.05).(3)After treatment,the gastroscopy scores of the two groups were significantly lower than those before treatment(P＜0.05),but there was no significant difference between the two groups after treatment(P＞0.05).(4)After treatment,the total pathological scores of the two groups and the scores of the gastric mucosal atrophy and intestinal metaplasia of the gastric antrum,gastric angle and gastric body in the treatment group were significantly improved compared with those before treatment(P＜0.05),and the improvement of the gastroscopy scores in the treatment group was significantly superior to that in the control group(P＜0.05).No statistically significant differences were presented in the scores of the gastric mucosal dysplasia and chronic inflammation of gastric antrum,gastric angle and gastric body in the two groups and in the scores of the gastric mucosal atrophy and intestinal metaplasia of gastric antrum,gastric angle and gastric body in the control group when compared with those before treatment(P＞0.05).(5)After treatment,the serum levels of gastric function indicators of pepsinogen Ⅰ(PGⅠ),pepsinogen Ⅱ(PGⅡ)and gastrin 17(G-17)in the two groups were significantly decreased compared with those before treatment(P＜0.05),and the decrease in the treatment group was significantly superior to that in the control group(P＜0.05).(6)There were no obvious adverse reactions occurring in the two groups during the treatment,with high safety.Conclusion Xiaojianzhong Decoction plus Dachaihu Decoction can significantly enhance the clinical efficacy of CAG patients with spleen deficiency and stasis-heat syndrome of normal-people pulse type,significantly improve the gastrointestinal function and pathological scores of the patients,and has high safety.

A rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometric (UPLC-ESI-MS/MS) method is developed for the qualitative identification of constituents in the flower buds of seven Lonicera species. The optimal condition of separation and detection were achieved on an AcQuity UPLC BEH C18 column with a gradient elution with acetonitrile and 0.1% acetic acid within 17 min. Among the 33 constituents detected, 6 caffeoylquinic acids (including caffeic acid), 8 flavonoids and 8 iridoid glycosides were characterized based on their fragmentation patterns in collision-induced dissociation (CID) experiments and/or by comparison with standard compounds. In addition, to statistically establish the correlation and discrimination of the Lonicera species, principle component analysis (PCA) was applied in this study. Lonicera samples were divided into well-defined groups directly related to their species based on PCA in terms of the log transformed relative contents of the major caffeoylquinic acids (including caffeic acid) as the variables. All of results indicated that the method presented here is able to classify the sample species and to reveal characteristic details of the chemical constituents of different Lonicera species.
Chromatography, Liquid ; methods ; Flowers ; chemistry ; Lonicera ; chemistry ; classification ; Principal Component Analysis ; Spectrometry, Mass, Electrospray Ionization ; methods ; Tandem Mass Spectrometry ; methods

The hematopoietic system of the mouse arises from extraembryonic mesoderm that migrate through primitive streak to the presumptive yolk sac at day 7.0 of gestation. However, the mechanisms regulating mesoderm commitment to hematopoietic lineages remain poorly understood. Previous studies demonstrated that the development kinetics and growth factor responsiveness of hematopoietic precursors derived from embryonic stem cells (ES cells) is similar to that found in the yolk sac, indicating that the onset of hematopoiesis within the embryoid bodies (EBs) parallels that found in the embryo. Furthermore, in vitro differentiation of ES cells to hematopoietic cells is valuable for establishment of therapeutic clone against a variety of hematological disorders. Despite the identification of multipotential hematopoietic progenitors in EBs, a subset of more primitive progenitors, identical to the high proliferative potential colony-forming cells (HPP-CFC) derived from human and murine hematopoietic tissues, have not been clearly identified regarding particular their replating potential in vitro. HPP-CFC is among the most primitive hematopoietic multipotent precursors cultured in vitro. In this study, our aim was to investigate the in vitro and in vivo hematopoietic capacity of HPP-CFC within the day 12 EBs, rather than the expansion of more committed progenitors. In this study the HPP-CFC could be detected within EBs differentiated for 5 to 14 days of murine ES cells, but the development dynamics of the HPP-CFC differed greatly among distinct serum lots. Qualitatively HPP-CFC is capable of forming secondary colonies. As to our expectation the ES cells-derived HPP-CFC demonstrated similar regeneration capacity to those from yolk sac, giving rise to secondary granulocyte, erythrocyte, macrophage and mast cells, however largely differed from the counterparts of adult bone marrow. In addition, by RT-PCR ES cells-derived HPP-CFC were found to express transcription factors associated closely with stem cell proliferation including SCL, GATA-2 and AML1 as well as various receptors of hematopoietic growth factors such as c-kit, GM-CSF receptor and interleukin 3 receptor et al. Finally, in order to understand the in vivo hematopoietic capacity of the ES cells-derived HPP-CFC, spleen colony-forming unit (CFU-S) assay was performed. Nevertheless, typical CFU-S was not observed after transplantation of the day 12 EB cells or HPP-CFC colonies into lethally irradiated adult murine. In conclusion the HPP-CFC differentiated from murine ES cells displayed robust hematopoietic activity in vitro, however their in vivo reconstitution ability was not detected. The difference between in vitro and in vivo hematopoietic activities of ES cells-derived primitive hematopoietic precursors deserves further investigation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Cell Differentiation ; genetics ; physiology ; Colony-Forming Units Assay ; Core Binding Factor Alpha 2 Subunit ; genetics ; Embryonic Stem Cells ; cytology ; GATA2 Transcription Factor ; genetics ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Mice ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Receptors, Interleukin-3 ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; T-Cell Acute Lymphocytic Leukemia Protein 1

OBJECTIVETo study the relationship of the incidence of bronchial dysplasia (bronchial anomalous origin and bronchial stenosis) with congenital heart disease.

METHODSA total of 185 children with congenital heart disease or bronchial dysplasia were enrolled. Bronchial dysplasia was identified by the 64-MSCT conventional scanning or thin slice scanning with three-dimensional reconstruction.

RESULTSForty-five children (25.3%) had coexisting bronchial dysplasia and congenital heart disease. The incidence rate of bronchial dysplasia in children with congenital heart disease associated with ventricular septal defect was higher than in those without ventricular septal defect (33.7% vs 15.0%; P<0.05). There were no significant differences in the incidence rate of bronchial dysplasia between the children with congenital heart disease who had a large vascular malformation and who did not.

CONCLUSIONSBronchial dysplasia often occurs in children with congenital heart disease. It is necessary to perform a tracheobronchial CT scanning with three-dimensional reconstruction to identify tracheobronchial dysplasia in children with congenital heart disease, especially associated with ventricular septal defect.

Adolescent ; Bronchi ; abnormalities ; embryology ; Child ; Child, Preschool ; Female ; Heart Defects, Congenital ; diagnostic imaging ; Humans ; Imaging, Three-Dimensional ; Infant ; Male ; Tomography, X-Ray Computed

OBJECTIVETo investigate the chemical constituents from the stems of Schisandra sphaerandra.

METHODCompounds were isolated from ethanolic extract of the titled herb by silica gel column chromatography, and their structures were elucidated by physical and chemical evidences and spectroscopic analysis.

RESULT12 compounds were obtained and identified as wuweizisu C (1), ganwuweizic acid (2), nigranoic acid (3), catechin (4), 2 alpha,24-dihytroxyursolic acid. (5), 3 beta-O-acetylursolic acid (6), ursolic acid (7), slyceryl 26-hydroxyhexacosanoate (8), slyceryl hexacosanoate (9), fat acids (10), beta-sitosterol (11), daucosterol (12), respectirely.

CONCLUSIONThree pentacyclic triterpene carboxylic acid (5-7) were isolated from Schisandreae for the first time.

Plant Stems ; chemistry ; Plants, Medicinal ; chemistry ; Schisandra ; chemistry ; Triterpenes ; chemistry ; isolation & purification

BACKGROUNDIt has been long suggested that abnormal clinical factors in the body, such as dyslipidemia and diabetes, can affect the presence of atherosclerosis. However, few studies on the effect of factors within the normal range, such as the loss of renal function with age, on the prevalence of atherosclerosis are few know in healthy individuals. The aim of this study was to investigate risk factors affecting the presence of asymptomatic carotid plaques in a middle-aged and elderly healthy population.

METHODSIn this regard, we prospectively evaluated 245 healthy individuals (98 males and 147 females) at baseline and after 5 years. Changes in the presence of carotid plaque between 2003 and 2008 were categorized into four groups, i.e. subjects without plaque at entry (n = 165): Group 1 (without plaque on two occasions, n = 129) and Group 2 (with nascent plaque at follow-up, n = 36); subjects with plaque at entry (n = 80); Group 3 (with plaque regression at follow-up, n = 29) and Group 4 (with plaque on two occasions, n = 51).

RESULTSUnivariate analysis showed that the positive rate of carotid plaques in males was higher than that in females at the baseline, and that a significantly inverse correlation existed between the prevalence rate of plaque and aging. Logistic regression analysis of cross-sectional research showed that independent risk factors for the prevalence of atherosclerosis were male gender, lower estimated glomerular filtration rate (eGFR) and higher low-density lipoprotein cholesterol (LDL-C) at the baseline, and older age and lower eGFR were involved in the presence of carotid plaques at follow-up point. However, logistic regression analysis of the longitudinal data showed that older age, decreased eGFR and increased systolic blood pressure (SBP) independently predicted the presence of carotid plaques after 5 years in subjects without plaque at entry. In addition, in subjects with plaque at entry, age, changes in eGFR and the baseline levels of serum albumin (ALB) and serum total bilirubin (BIL) dependently influenced the outcome of carotid plaque.

CONCLUSIONPhysiological decline of renal function, together with advancing age, was an independent risk factor which consistently affected the presence of carotid atherosclerosis in two categories of healthy individuals.

Adult ; Aged ; Aged, 80 and over ; Aging ; physiology ; Carotid Artery Diseases ; pathology ; physiopathology ; Female ; Glomerular Filtration Rate ; physiology ; Humans ; Kidney ; physiology ; physiopathology ; Male ; Middle Aged ; Prospective Studies ; Risk Factors

OBJECTIVETo evaluate the significance of somatic mutations of VHL gene and hypoxia-inducible factor-1alpha (HIF-1alpha) expression in primary renal clear cell carcinoma (RCC).

METHODSMutation of VHL gene and HIF-1alpha expression were detected by means of PCR, denaturing high-performance liquid chromatography (DHPLC), direct sequencing and immunohistochemistry in 32 samples from primary renal clear cell carcinoma patients.

RESULTSIn 32 RCC samples, 17 samples (53.1%) had and 32 samples of adjacent nonmalignant renal tissue had not mutations of VHL gene expression. Twelve RCC samples (70.6%) which had mutations of VHL gene expressed HIF-1alpha, and it had significant difference to 4 RCC (26.7%) samples which didn't have mutations of VHL gene (P < 0.05).

CONCLUSIONMutations of VHL gene may play a significant role in the tumorigenesis of RCC, and HIF-1alpha expression correlates with it.

Adenocarcinoma, Clear Cell ; genetics ; pathology ; Adult ; Aged ; Carcinoma, Renal Cell ; genetics ; pathology ; Chromatography, Liquid ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Immunohistochemistry ; Kidney ; chemistry ; metabolism ; pathology ; Kidney Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Mutation ; genetics ; Polymerase Chain Reaction ; Transcription Factors ; analysis ; genetics ; Tumor Suppressor Proteins ; analysis ; genetics ; Ubiquitin-Protein Ligases ; analysis ; genetics ; Von Hippel-Lindau Tumor Suppressor Protein

BACKGROUNDArrhythmogenic right ventricular cardiomyopathy (ARVC) is one of the leading causes of sudden cardiac death. Recent studies have shown that ARVC, which is an inheritable genetic change, results from mutations in genes encoding desmosomal proteins. Plakophilin-2 is an important component of the desmosome. Because the full range of genetic variations related to ARVC is unknown and no related studies of the Chinese population have been reported, we aimed to investigate the genetic variation of plakophilin-2 in ARVC patients from the Southern Region of China.

METHODSGenomic DNA was isolated from peripheral blood samples of all 34 ARVC patients, who were screened through a clinical evaluation. They were used to detect variations in the sequences of the plakophilin-2 genes by polymerase chain reaction amplification in combination with direct sequencing.

RESULTSIn exon-1 of the plakophilin-2 gene, a deletion mutation (c.145_148 del GACA) was found in one family pedigree. The mutation was also found in exon-2, 4, and 11 of the plakophilin-2 gene. The QT interval dispersion of the ECG was considerably longer in the mutation group than in the non-mutation group of ARVC patients, and this result was statistically significant (P < 0.05).

CONCLUSIONWe discovered a plakophilin-2 mutation that prolongs the QT interval dispersion in the southern Chinese ARVC population.

Adult ; Arrhythmogenic Right Ventricular Dysplasia ; genetics ; Asian Continental Ancestry Group ; genetics ; Child ; China ; DNA Mutational Analysis ; Exons ; genetics ; Genetic Predisposition to Disease ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; Plakophilins ; genetics

Objective To study the general mechanical behavior pattern of sensing angular velocity by 3 pairs of human semicircular canals.Methods Sinusoidal head rotation of a healthy subject was simulated by finite element method to analyze the dynamic responses in the above 3 pairs of canals in the left and right inner ear.Results Compared with the complicated node displacement distribution,the volumetric strain distribution of cupula was regular.Specifically,each pair of cupulae expanded or contracted within the same amplitude with the same response frequency to the angular velocity.The ratio of absolute volumetric strain among the horizontal semicircular canal cupula (HC cupula),the anterior semicircular canal cupula (AC cupula),and the posterior semicircular canal cupula (PC cupula),was approximately 1.00∶0.80 ∶ 1.72,which kept constant.In addition,the volumetric strain of HC cupula was in phase with that of AC cupula,but showing 14.4° out of phase with the angular velocity,and 180° out of phase with that of PC cupula.Conclusions The volumetric strain of cupula can preferably characterize the directional coding function of semicircular canal,and the amplitude,frequency and phase of the cupular volumetric strain can encode those of the stimulated angular velocity,respectively.These results will lay the foundation for establishing quantitative relationship of vestibulo-ocular reflex,and provide theoretical references for quantitative assessment for vestibular function by nystagmus examination.