Objective:To analyze the effects of espO gene knockout on the biological characteristics of enterhemorrhagic Escherichia coli (EHEC). Methods:Two-step methods mediated by the suicide plasmid pCVD442-Δ espO and plasmid pTrc99a were used to construct the espO gene-deleted strain (Δ espO) and the complemented mutant (CΔ espO), respectively. HeLa cells were infected with different EHEC strains to analyze the biological functions and lethal effects of espO gene during infection. Results:PCR, electrophoresis and gene sequencing showed that the Δ espO and CΔ espO mutants were successfully constructed. Compared with the wild-type strain, neither the Δ espO nor CΔ espO mutant showed significant difference in growth rate, indicating that the espO gene had no influence on the growth and replication of EHEC. Furthermore, EspO could activate the tumor necrosis factor receptor (TNF)-induced NF-κB signaling pathway, while the effector protein NleB could inhibit the process. EspO could not inhibit the death of HeLa cells induced by TNF or TNF-related apoptosis-inducing ligand (TRAIL) after EHEC infection. Conclusions:In this study, we successfully constructed the espO gene-deleted and complemented mutants of EHEC and preliminarily analyzed the interaction between espO gene and host cells and the effects of espO gene on cell apoptosis during infection, which provided reference for further research on the in vitro biochemical activity and in vivo pathogenic roles of EspO.

Objective To analyze the effect of the changes of hospital diagnosis and treatment mode on the treatment time in patients with acute ischemic stroke before and after the establishment of Cerebrovascular Disease Center. Methods A total of 103 consecutive patients with acute ischemic stroke admitted to the Department of Neurology,Changhai Hospital,the Second Military Medical University between June 2008 and December 2012 were enrolled retrospectively. Thirty-one of them were excluded because of incomplete medical records. Finally,72 patients were enrolled as a control group and received series diagnosis and treatment mode. A total of 210 consecutive patients with acute ischemic stroke admitted to the Cerebrovascular Disease Center,Changhai Hospital,the Second Military Medical University from September 2013 to February 2015 were enrolled retrospectively. Thirteen patients were excluded (4 patients with recurrent transient ischemic attack were treated with recombinant tissue-type plasminogen activator,9 without complete data were treated with intravenous thrombolysis),197 were enrolled as an observation group finally,and they were received series diagnosis and treatment mode. The patients of both groups were visited within 4. 5 h after onset and received rt-PA treatment. The time-consuming changes of each time period from onset-to-door,door-to-imaging,imaging-to-needle,door-to-needle,and onset-to-needle time between the control group and the observation group were compared and analyzed. Results Compared with the control group,the door-to-imaging,imaging-to-needle,door-to-needle and onset-to-needle time were significantly shorter in the observation group. There were significant difference between the 2 groups (24 ± 12 min vs. 60 ± 20 min,27 ± 12 min vs. 62 ± 31 min,51 ± 17 min vs. 122 ± 52 min,and 153 ± 69 min vs. 230 ± 81 min,all P < 0. 01). There was no significant difference for onset-to-door time between the observation group and the control group (P > 0. 05). Conclusion The establishment of cerebral vascular disease center and the improvement of the processes have shortened the treatment time in patients with acute ischemic stroke within time window. The time from onset-to-door is still longer,and the propaganda and education of stroke should be strengthened.

The intubation of conventional transesophageal echocardiography (TEE) probes into patients causes serious esophagus irritation,and thus the use of TEE probes in pediatric practice is limited.In this study,we aimed at the development of a special probe which could be inserted through the nasopharyngeal cavity into the esophagus to obtain the same high-quality echocardiography images as those obtained by conventional TEE and improve patients' experience.During the examination,the patients felt relaxed for a longer time and cooperated with the sonographers in the process of cardiac catheterization conducted in the surgery room or the intensive care unit (ICU),resulting in improved accuracy of the diagnosis and timely administration of appropriate treatment.Two years ago,Prof.Xin-fang WANG put theories into practice by inserting the probe through the nasal cavity and pharynx into the esophagus of volunteers to successfully detect the heart and great vessels at the retrocardiac space.Later,Prof.Ming-xing XIE performed the transnasal TEE examination in 12 atrial septal defect (ASD) patients and proved the safety and reliability of this method,which could become a new way for clinical diagnosis and treatment.

Objective To compare the levels of indoleamine 2,3-dioxygenase (IDO) in the peripheral blood mononuclear cells(PBMCs) from HIV-1 infected and HIV-1 negative individuals and in human tumor cells in the presence or absence of TLR ligand stimulation.Methods TaqMan probe real-time RT-PCR method for human IDO mRNA was established; IDO mRNA levels in the PBMCs from HIV-1+ and HIV-1-individuals were tested; IDO mnRNA levels in mucosal origin(T84,Caco-2,Hela) and leukocyte origin(THP-1,MT-4) tumor cells before and after exposure to agonists for TLR4,TLR7/8 and TLR9 were examined.Results It was found that a high level of IDO mRNA could be found in HIV-1+ individuals( 103.42 copy IDO mRNA/106 copy GAPDH mRNA) ; however,some high risk HIV-1-individuals may have also a high level of IDO mRNA.Some of the tumor cells could express higher level of IDO mRNA after exposure to TLR agonist.Conclusion This study indicated a role for IDO in the viral persistence and tumor formation in HIV/AIDS and further studies were warranted.

AIDS-Related Opportunistic Infections ; diagnosis ; microbiology ; Acquired Immunodeficiency Syndrome ; diagnosis ; microbiology ; Bronchoalveolar Lavage Fluid ; microbiology ; Female ; Humans ; Immunohistochemistry ; Male ; Methenamine ; Pneumocystis carinii ; isolation & purification ; Pneumonia, Pneumocystis ; diagnosis ; microbiology ; Polymerase Chain Reaction ; Silver Staining ; methods

BACKGROUNDPeroxisome proliferator-activated receptor-gamma (PPARgamma) is a kind of ligand-activated transcription factors binding to peroxisome proliferator response element (PPRE), a specific recognition site. It is thought to play a critical role in glucose and lipid metabolism and in inflammation control. The aim of this study was to establish a new cellular model for the quick screening of lipid-lowering drugs, which may be effective as PPAR-gamma ligands on the PPRE-mediated pathway regulatory system.

METHODSTwo plasmids were constructed: pXOE-PPARgamma, in which the human PPARgamma gene was in the downstream of TFIIIA gene promoter, and pLXRN-PPRE-d2EGFP, in which the enhanced green fluorescent protein (EGFP) gene was subcloned into PPRE. The xenopus oocytes were injected with these two plasmids, and consequently treated with prostaglandin E1, pioglitazone, and different kinds of lipid-lowering drugs. After 3 days, the oocytes were observed under a fluorescence microscope. To confirm the drug action,we injected pXOE-PPARgamma plasmid into the oocytes, which then treated with prostaglandin E1 and Hawthorn flavonoids. The mass of expressed lipoprotein lipase (LPL) in the cells was determined by enzyme labeling linked immunosorbent assay (ELISA).

RESULTSThe expression of EGFP was only induced by prostagalandin E1, pioglitazone, Hawthorn flavonoids. A concentration-response relationship was seen between expressed EGFP and Hawthorn flavonoids. The levels of LPL in both Hawthorn flavonoids groups and PPARgamma ligand prostagalandin E1 group injected with pXOE-PPARgamma plasmid increased significantly (< 0.001) compared with controls, and a concentration-response relationship was observed between LPL mass and Hawthorn flavonoids.

CONCLUSIONSIt is possible to establish a PPRE regulatory EGFP reporter system in xenopus oocytes to monitor the activity of PPARgamma ligand. Hawthorn flavonoids can increase the expression of gene downsteam of PPRE by effect on the PPRE pathway regulatory system.

Alprostadil ; pharmacology ; Animals ; Crataegus ; Female ; Hypolipidemic Agents ; pharmacology ; Lipoprotein Lipase ; biosynthesis ; Medicine, Chinese Traditional ; Oocytes ; metabolism ; PPAR gamma ; physiology ; Peroxisome Proliferators ; pharmacology ; Plasmids ; Response Elements ; physiology ; Xenopus

This study aims to investigate the effect of total flavones of Fructus Chorspondiatis (TFFC) on the mRNA and protein expression of collagen type I and III of rat cardiac fibroblasts (CFs) induced by angiotensin II (Ang II), and explore its anti-myocardial fibrosis molecular mechanism. Neonatal rat CFs were prepared from Sprague-Dawley rats (1-3 d after birth). The expression of collagen type I and III mRNA and protein were measured by RT-PCR and Western blotting, respectively. The study showed that stimulation of neonatal rat CFs with 100 nmol.L-1 of Ang II for 72 h resulted in a significant increase of the expression of collagen type I and III mRNA and protein. The changes on the expression level were blocked by TFFC. The results demonstrated that TFFC can inhibit myocardial fibrosis induced by Ang II in rats, which is probably associated with the collagen type I and III mRNA and protein levels up-regulated by Ang II, and TFFC was shown to decrease the expression levels of collagen type I and III mRNA and protein.
Anacardiaceae ; chemistry ; Angiotensin II ; pharmacology ; Animals ; Animals, Newborn ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Fibroblasts ; cytology ; metabolism ; Flavones ; administration & dosage ; isolation & purification ; pharmacology ; Fruit ; chemistry ; Myocardium ; cytology ; metabolism ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley

Objective:To observe the effect of cognitive training based on PASS theory on cognition in patients with post-stroke cognitive impairment (PSCI). Methods:From August, 2018 to June, 2019, 42 patients with PSCI were randomly divided into control group (n = 21) and intervention group (n = 21). The intervention group accepted computer-assisted cognitive training based on the PASS theory, and the control group accepted routine computer-assisted cognitive training, for four weeks. They were assessed with Mini-Mental State Examination (MMSE) and Lowenstein Occupational Therapy Cognitive Assessment (LOTCA) before and after training. Results:There were 18 patients in each group finishing the trail. After training, the scores of MMSE and LOTCA increased in both groups (t > 8.831, P < 0.001), and increased more in the intervention group than in the control group (t > 2.198, P < 0.05). For the LOTCA factors scores, it increased in orientation, visual perception, spatial perception, motion praxis, visuomotor organization and thinking operations in both groups (t > 2.122, P < 0.05) after training, and increased more in the intervention group than in the control group in visual perception, spatial perception and thinking operations (t > 2.356, P < 0.05), and the differences of visuomotor organization was more in the intervention group than in the control group (t = 2.354, P < 0.05). Conclusion:Cognitive training based on PASS theory can improve cognition for patients with PSCI, especially for visual perception, spatial perception, thinking operations and visuomotor organization.

This study was aimed to investigate the anti-leukemia activity of Tel03 in vivo. The K562 xenografted leukemia model was established and mice were divided randomly into three groups. Mice of different group were treated with PBS (control), 5 mg/kg Tel03 or 15 mg/kg Tel03 (ip, twice a week) respectively. Tumor volume, body weight and other behavior were observed regularly. Cell apoptosis was detected with TUNEL assay and the expression levels of Bcl-2 and Bax were detected by Western blot. The results indicated that Tel03 exerted anti-leukemia activity in mouse model. Tel03 significantly reduced tumor volume in Tel03-treated group compared with control. In addition, 5 mg/kg Tel03 induced cell apoptosis without exerting apparent toxicity in mice. After Tel03 treatment, the expression of Bcl-2 was inhibited, however, the expression of Bax was up-regulated. It is concluded that G-quadruplex ligand Tel03 can induce cell apoptosis in leukemia mouse model, and this agent may be a potential anticancer drug.
Animals ; Apoptosis ; Female ; G-Quadruplexes ; Humans ; K562 Cells ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the effect of hypoxia and hypoxia-inducible factor-1alpha (HIF-1alpha) on the expression of multidrug resistance gene-1 (mdr-1) and its coded p-glyeoprotein (P-gp) as well as the chemotherapeutic sensitivity of human ovarian cancer cells to paclitaxel and its mechanism.

METHODSThe mRNA and protein levels of HIF-1alpha, mdr-1 and p-gp were studied by immunocytochemistry, semiquantitative reverse transcription-ploymerase chain reaction (RT-PCR) and Western blot analysis in human ovarian cancer cells (A2780) in 5% CO2 + 1% O2 hypoxic culture and 21% O2 normoxic culture, respectively. Methyl thiazolyl tetrazolium (MIT) was used to evaluate the chemotherapeutic sensitivity of A2780 cells to paclitaxel by inhibition rate. RNA interference technique was used and small hairpin RNAs (shRNAs) eukaryotic expression vector targeting HIF-1alpha was constructed as pSiHIF-1alpha, and transfected into A2780 cells. RT-PCR and Western blot were used to detect gene silencing effect on HIF-1alpha, the expressions of mdr-1 and p-gp. The inhibition rate was observed after HIF-1alpha gene silence.

RESULTSHIF-1alpha at both mRNA and protein levels was induced significantly under hypoxia. The HIF-1alpha expression at mRNA level was oxygen gradient-independent, while HIF-1alpha expression at protein level was oxygen gradient-dependent. The inhibition rate of paclitaxel to hypoxic A2780 cells in 5% CO2 + 1% O2 was significantly lower than that in normoxic A2780 cells (P <0.05). The shRNAs plasmid targeting HIF-1alpha was constructed successfully and HIF-1alpha gene was silenced in A2780 cells efficiently followed by mdr-1 and p-gp down-regulation. The inhibition rate was greatly increased in hypoxic A2780/siHIF-1alpha cells.

CONCLUSIONHypoxia can decrease the chemotherapeutic sensitivity of human ovarian cancer A2780 cells to paclitaxel through HIF-1alpha regulating the expression of mdr-1 and p-gp.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Antineoplastic Agents, Phytogenic ; pharmacology ; Blotting, Western ; Cell Hypoxia ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Paclitaxel ; pharmacology ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection