This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.
Humans ; Mice ; Animals ; Transcription Factors/genetics* ; DNA-Binding Proteins/genetics* ; Receptors, Cytoplasmic and Nuclear/genetics* ; Genes, Reporter ; Luciferases/genetics*

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons in the brain and spinal cord. One important aspect of ALS pathogenesis is superoxide dismutase 1 (SOD1) mutant-mediated mitochondrial toxicity, leading to apoptosis in neurons. This study aimed to evaluate the neural protective synergistic effects of ginsenosides Rg1 (G-Rg1) and conditioned medium (CM) on a mutational SOD1 cell model, and to explore the underlying mechanisms. We found that the contents of nerve growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor significantly increased in CM after human umbilical cord mesenchymal stem cells (hUCMSCs) were exposed to neuron differentiation reagents for seven days. CM or G-Rg1 decreased the apoptotic rate of SOD1^G93A-NSC34 cells to a certain extent, but their combination brought about the least apoptosis, compared with CM or G-Rg1 alone. Further research showed that the anti-apoptotic protein Bcl-2 was upregulated in all the treatment groups. Proteins associated with mitochondrial apoptotic pathways, such as Bax, caspase 9 (Cas-9), and cytochrome c (Cyt c), were downregulated. Furthermore, CM or G-Rg1 also inhibited the activation of the nuclear factor-kappa B (NF-κB) signaling pathway by reducing the phosphorylation of p65 and IκBα. CM/G-Rg1 or their combination also reduced the apoptotic rate induced by betulinic acid (BetA), an agonist of the NF-κB signaling pathway. In summary, the combination of CM and G-Rg1 effectively reduced the apoptosis of SOD1^G93A-NSC34 cells through suppressing the NF-κB/Bcl-2 signaling pathway (Fig. 1 is a graphical representation of the abstract).
Humans ; NF-kappa B/metabolism* ; Ginsenosides/pharmacology* ; Amyotrophic Lateral Sclerosis/genetics* ; Culture Media, Conditioned/pharmacology* ; Superoxide Dismutase-1 ; Neurodegenerative Diseases ; Neurons/metabolism* ; Apoptosis

In order to adapt to the needs of innovative medical personnel training,Zunyi Medical University conducted teaching reform and curriculum construction for human parasitology.Key teachers were trained by strengthening the construction of the teaching staff.Through the preparation of lessons,lectures,supervision and other measures,the teaching quality has been improved.Teaching reform was carried out by the introduction of humanities knowledge,scientific research training,and formative evaluation.Through the development of quality curriculum resources,standardized teaching archives,improved scientific research,and quality service for the society,the curriculum construction of human parasitology has been greatly improved.

Objective To observe humoral and cellular immune responses induced in mice by a TSOL18 recombinant Mycobacterium smegmatis (MS) vaccine of Taenia solium.Methods Sixty specific pathogen-free (SPF) Kunming mice (18-22 g,half male and half female) were divided into 3 groups by random number table method,20 mice in each group.One group was administered with recombinant MS-TSOL18 vaccine,one group was administered with MS as control,and one group was administered phosphate buffer saline (PBS) as control.Kunming mice were vaccinated once every two weeks for 2 times through intragastric administration.At 0,2,4,6,and 8 weeks after immunization,blood sample was collected and serum was separated.The levels of IgG and IgG2a in serum were detected using enzyme linked immunosorbent assay (ELISA).The spleen was separated to prepare spleen suspension.The proliferation of spleen lymphocyte with the stimulation of a specific antigen was detected by CCK-8.The levels of interleukin (IL)-2 and IL-4 in spleen cell culture supernatant with the stimulation of a specific antigen were detected by ELISA.Results Compared with MS control group (IgG:0.160 ± 0.019,0.187 ± 0.038,0.193 ± 0.050,0.170 ± 0.005;IgG2a:0.213 ± 0.010,0.198 ± 0.012) and PBS control group (IgG:0.159 ± 0.015,0.184 ± 0.029,0.191 ± 0.025,0.165 ± 0.018;IgG2a:0.198 ± 0.032,0.178 ± 0.025),the levels of specific IgG (0.310 ± 0.034,0.391 ± 0.029,0.443 ± 0.030,0.373 ± 0.021) in recombinant MS-TSOL18 vaccine group increased at 2,4,6,and 8 weeks after immunization (P ＜ 0.05),the levels of specific IgG2a (0.446 ± 0.056,0.339 ± 0.026) in recombinant MS-TSOL18 vaccine group increased at 6 and 8 weeks after immunization (P ＜ 0.05),and reached the highest level by the 6th week.After antigen stimulation,compared with MS and PBS control groups,the levels of spleen lymphocyte proliferation increased at 2,4,6,and 8 weeks after immunization (P ＜ 0.05),and reached the highest level by the 6th week.After antigen stimulation,compared with MS and PBS control groups,the levels of IL-2 and IL-4 in spleen lymphocyte culture supernatant increased at 2,4,6,and 8 weeks after immunization (P ＜ 0.05),and reached the highest level by the 6th and 4th weeks,respectively.Conclusion The recombinant MS-TSOL18 vaccine of Taenia solium might induce mice to produce humoral and cellular immune responses.

Objective To evaluate the regulatory role of azithromycin-induced persistent Chlamydia trachomatis (Ct) infection in the apoptosis of Hela229 cells.Methods Hela229 cells were firstly co-cultured with Ct for 22 hours,and then cultured with Dulbecco's modified Eagle's medium (DMEM) containing 0.08 mg/L azithromycin for 26 hours to establish a cell model of persistent Ct infection (persistent infection group).These infected Hela229 cells cultured with azithromycin-free DMEM served as a cell model of acute Ct infection (acute infection group).After 48-hour infection with Ct,azithromycin was removed,and infected Hela229 cells in the above 2 groups were successively cultured with DMEM for the resurgence of Ct.Immunofluorescence assay and electron microscopy were performed to verify the persistent Ct infection model.The Hela229 cells in the persistent infection group and acute infection group as well as uninfected Hela229 cells (control group) were treated with staurosporine (STS) for 4 hours to induce the apoptosis,and then cell apoptosis was detected by Hoechst 33258 staining,annexin V/propidium iodide staining and flow cytometry.Results After the treatment with azithromycin,atypical inclusions with aberrant reticulate bodies appeared in the Ct-infected cells.After removing azithromycin,cells were cultured until 96 hours after infection,and infectious elementary bodies reappeared in the Ct inclusions.After the treatment with STS,Hoechst staining showed that there was loose chromatin in the persistently infected cells,while chromatin condensation was observed in the uninfected cells.After 24-hour infection with Ct and 4-hour induction with STS,the apoptosis rate was significantly higher in the persistent infection group (45.567％ ± 2.631％) than in the acute infection group (38.567％ ± 1.701％,t =2.686,P =0.028),but significantly lower in the persistent infection group than in the uninfected group (69.800％ ± 2.835％,t =8.187,P ＜ 0.001).After 48-hour infection with Ct and 4-hour induction with STS,there was a significant difference in the apoptosis rate between the persistent infection group (46.700％ ± 5.257％) and acute infection group (61.767％ ± 1.815％,t =5.781,P ＜ 0.001),as well as between the persistent infection group and the uninfected group (68.667％ ± 3.156％,t =7.421,P ＜ 0.001).Conclusion This study showed that azithromycin-induced persistent Ct infection regulated the apoptosis of host cells,and this effect lasted 48 hours.

Teaching level is the core of teaching quality monitoring.Students' feedback to the quality ofteaching will help teachers to reflect and rectify the related factors of their teaching,so as to achieve the purpose of improving teaching quality.We have always insisted on doing this work.However,there are inconveniences in the "paper version" of the survey:complicated preparations and operations,inconvenient storage,and labor and material costs.Combined with the actual situation in our department,we are trying to reform and explore the digital survey for teaching quality ofhuman parasitology.Teachers will put the issue on the network,students can evaluate it at anytime,anywhere,and the platform automatically organize the information and quickly feed back to the teachers.The novel and effectiveway has been well received by students and teachers.

We expressed B cell epitopes of dengue virus envelope protein and NS1 protein in prokaryotic cells,and purified and evaluated for its serological activities.A recombinant multi-epitope chimeric gene named rE including eight B cell epitopes was connected by linker peptide (EAAAK)2 and cloned into prokaryotic expression vector pET-28a(+),and transformed into E.coli BL21(DE3) cells for expression under induction of IPTG.The expressed recombinant protein was purified with 6× His purification media,and identified by SDS-PAGE and Western blot,and its antigenicity was analyzed by using an indirect ELISA assay.The recombinant expression vector pET28a-rE was constructed and expressed in BL21 (DE3) successfully,but the recombinant proteins mainly appeared as inclusion bodies.The target protein was obtained with high purity through the purification of affinity chromatography.SDS-PAGE and Western blot analysis showed that the molecular weight of fusion protein was in the expected line.The established indirect ELISA has high accuracy.This recombinant peptide antigen expressed in E.coli has good potential for serum testing.

Objective To investigate the effect of electroacupuncture (EA) pretreatment on neurological function, and the expression of Toll-like receptor 4 (TLR4) and nuclear factorκB (NF-κB) protein in ischemic penumbra after cerebral ischemia-reperfusion (CIR) injury. Methods A total of 36 male Sprague-Dawley rats were randomly divided into sham group (n=12), model group (n=12) and EA group (n=12). The later two groups were occluded their right middle cerebral arteries for two hours and reperfused. The EA group was pretreated with EA at Baihui (GV20) for two weeks before modeling. They were assessed with modified Neurological Severity Scores (mNSS), the injury in ischemic brain was detected with HE staining, and the expression of TLR4 and NF-κB protein in ischemic penumbra were detected with Western blotting, 24 hours after reperfusion. Results The score of mNSS decreased (P<0.05), the injury of brain tissue ameliorated, and the expression of TLR4 and NF-κB decreased in ischemic penumbra (P<0.05) in EA group compared with those in the model group. Conclu-sion EA pretreatment can reduce the injury and improve the neurological function in rats after CIR by down-regulating the expression of TLR4 and NF-κB protein in ischemic penumbra.

Objective To explore the association between diabetes mellitus type Ⅱ (DM) and deglutition function of amyotrophic lateral sclerosis (ALS) patients.Methods Seventy-five ALS patients older than 45 years and admitted to our hospital from August 2008 to November 2015 were selected into this study;67 of them were without DM and 8 were with DM.Water swallow test,amyotrophic lateral sclerosis severity scale-swallow (s-ALSSS),amyotrophic lateral sclerosis functional rating scale (ALSFR-R) and videofluoroscopic swallow study (VFS) were performed to evaluate the deglutition functions of these ALS patients with or without DM,and the results of the two groups were compared.Results (1) Patients with DM had significantly higher s-ALSSS scores,ingurgitation part in ALSFRS-R scores and in parts of VFS scores,such as transportation to pharyngeal,pharyngeal transit,flow into pharyngeal before reflexion,epiglottic vallecula residue,and piriform sinus residue than patients without DM (8.88±1.34 vs.7.54±1.47,3.50±0.54 vs.2.96±0.77,2.88±0.35 vs.2.16±0.69,2.75±0.46 vs.2.09±0.69,2.88±0.35 vs.2.42±0.56,2.88±0.35 vs.2.39±0.58,P＜0.05).(2) Scores of Kubota drinking test,ALSFRS-R,VGF (including oral phase,pharyngeal phase and aspiration degree) in patients without DM were 2.15±1.12,7.18±1.41(1.78±0.69,1.69±0.60 and 3.72±0.65),and those in patients with DM were 1.88±1.34,8.00±0.93(2.13±0.64,2.00±0.53 and 4.00±0.00);no significant differences were noted between the two groups (P＞0.05),but there was a trend showing that DM patients had higher scores in these evaluations.Conclusion As compared with ALS patients without DM,ALS patients with DM get more mildly impaired deglutition function.

Objective To explore the clinical and electrophysiological features and pulmonary function of 8 Chinese patients with Kennedy's disease (KD) and to enhance the understanding for the disease.Methods Eight patients with KD,admitted to out hospital from July 2010 to December 2013 and confirmed by gene examination,were chosen in our study; their clinical and electrophysiological features,and laboratory characteristics and pulmonary function were compared with those in the healthy volunteers.Results The average age of onset in the 8 patients was (45.13±17.47) years,and the average age of diagnosis was (55.63±12.11) years.The most common complaint was leg weakness.All patients presented hemifacial spasm,tongue muscle atrophy and fibrillation,amyotrophy and gynaecomastia; creatine kinase level was elevated and endocrine disorder appeared in different degrees.The electromyogram characteristics included widespread neurogenic changes accompanied with/without sensory or motor conduction abnormalities.The forced vital capacity,forced expiratory volume in first second,maximal voluntary ventilation and peak expiratory flow rate were significantly lower,and the residual volume in the KD patients was statistically higher than that in the healthy volunteers (P＜0.05).Respiratory muscle strength (maximum inspiratory pressure and maximum expiratory pressure) decreased in the KD patients.Conclusion KD is a degenerative disease with slow clinical progression which has its own characteristics of inheritance pattern and natural course; the age of onset,repeat number of CAG sequences,pulmonary function and respiratory muscle strength may be valuable for illness evaluation.