Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.
Gene Targeting ; Green Fluorescent Proteins ; genetics ; MicroRNAs ; genetics ; Oryza ; genetics ; Plasmids ; Protoplasts ; metabolism ; RNA, Plant ; genetics ; Transfection

Objective To observe blood glucose values and serum insulin levels at different times after acupoint injection of insulin with PVP as a sustained release agent and analyze the correlation between them in SD Rats.Methods One hundred and forty-four male SD rats were randomized into groups A (acupoint injection of insulin alone), B (acupoint injection of sustained-release insulin) and C (control). At one hour after every group received an oral gavage of 40% glucose solution (2.2 g/kg), groups A and B were given point Zusanli injection and group C was not treated. In the three groups of rats, blood glucose values and serum insulin levels were measured at six time points: 0.5, 3, 6, 8, 10 and 24 hours after acupoint injection (in group C at the corresponding time points). In every group, the reaction of Point Zusanli region to the stimulation was observed by optical microscopy in at three time points: 3, 10 and 24 hours after acupoint injection.Results The glucose-lowering rate reached its peak at 3 hours after acupoint injection and then declined in groups A and B. The hypoglycemic effect tended to be stable at 6 hours after acupoint injection in group A and at 10 hours after acupoint injection in group B. The hypoglycemic effect was better in group B than in group A at 6 and 8 hours after acupoint injection (P<0.01). In group A, serum insulin reached its peak at 3 hours and tended to be stable at 6 hours after acupoint injection (P<0.05). Serum insulin levels were significantly lower in group B than in group A at four time pints: 0.5, 3, 6 and 8 hours after acupoint injection (P<0.05). An analysis of the correlation between rat serum insulin levels and the glucose-lowering rate showed a significant positive correlation in group A (P<0.01) and a low positive correlation in group B (P<0.05). In groups A and B of rats, clear stripes and complete structure of muscle fibers were seen with no obvious degeneration, necrosis and inflammatory reaction around point Zusanli at three time points: 3, 8 and 24 hours after acupoint injection; there were no obvious pathological changes compared with group C.Conclusions PVP as a sustained release agent can slow down the absorption rate of insulin in rat point Zusanli region. Meanwhile, it prolongs the continuous time of the hypoglycemic effect of insulin. There is a low correlation between serum insulin levels and the hypoglycemic effect, suggesting that acupoint injection with a sustained release agent can prolong the effect of acupoint injection of medicine.

Objective To investigate the effect of glucagon-like peptide-1(GLP-1) in a dose related manner on glucose metabolism after 65% hepatectomy in rats.Methods We determined the serum glucose levels of hepatomized rats at 0,5,10,20,and 30 minutes after an intravenous glucose load(IVGTT,0.5 g/kg glucose) on the first postoperative day,and the changes of blood glucose,serum insulin and glucagon concentrations of hepatomized rats that received the volum load with normal saline or 0.3 nmol/kg GLP-1,0.45 nmol/kg GLP-1 respectively.Blood was drawn for determination of glucose(glucose oxidase),insulin,glucagon,and GLP-1(radioimmunoassay).Results The peak glucose and 30-minute glucose levels and the area under the curve(AUC 0-30) were significantly higher in the hepatomized rats compared to the control rats,which had not undergone any operation and received a same intravenous glucose load(0.5 g/kg glucose with normal saline)(P0.05).Nevertheless the peak glucose and 30-minute glucose levels and AUC 0-30 of the hepatomized rats that received with 0.45 nmol/kg GLP-1 were significantly lower compared to the rats that received the same volum load with normal saline or 0.3 nmol/kg GLP-1 respectively after liver resection.There was an increasing postoperative serum concentration of glucose,insulin,glucagon on the first day,then,the serum glucose concentration was significantly lowered after infusion of GLP-1 in rats undergoing hepatectomy(P

0.05).Conclusions After hepatectomy,in rats is associated with an obvious insulin resistance,mainly due to the level of serum insulin was obviously decreased(P

To gain insights into the function of potential post-translational modifications on the activity of the Cucumber mosaic virus (CMV)-encoded silencing suppressor protein 2b, one predicted phosphorylation site (S40) and two predicted ubiquitination/sumoylation sites (K22 and K39) in CMV-Q2b protein were individually or simultaneously mutated by site-directed mutagenesis methods. These Q2b mutants were inserted into plant expression vectors, expressed in plant leaves, and then analyzed for their silencing suppressor activities. The results showed that S40A mutation greatly impaired both the local and systemic silencing suppressor activity, and the K22R mutation has no significant effect on the suppressor activity, while the K22R/K39R double mutation reduced the systemic silencing suppressor activity. To test if the decrease of suppressor activity were due to protein accumulation changes, western blot were performed to monitor the protein level of Q2b mutants. The results indicated that mutations of both K22 and K39 to R or S40 to A all significantly reduced the accumulation of the Q2b protein in plants, while the single mutation of K22 to R did not alter the accumulation of Q2b protein, suggesting that two potential post-translational modification sites, K39 and S40, contribute to the suppressor activity and stability of 2b protein in plant cells.
Cucumovirus ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Gene Silencing ; Mutagenesis, Site-Directed ; Mutation ; Protein Processing, Post-Translational ; Protein Stability ; Viral Proteins ; genetics

OBJECTIVETo evaluate the use of alginate three-dimensional culture system for mandibular condylar chondrocytes culture in vitro from human osteoarthritic temporomandibular joint.

METHODSThe cultured mandibular condylar chondrocytes from the operatively removed cartilage from a patient with osteoarthritic temporomandibular joint were harvested by mechanical dissection and enzyme digestion. Partial chondrocytes were suspended in the aqueous sodium alginate beads with high seeding density and cultured for 4 weeks, while the others were cultured in monolayer culture condition for 1 week. Thereafter, the alginate beads were embedded in paraffin and sectioned, then studied immunologically with type II collagen antibody and aggrecan antibody, same studies were adopted for the monolayer cultures.

RESULTSThe monolayer cultures were confirmed as chondrocytes. The chondrocytes cultured in the alginate medium showed well. These cells exhibited the excellent differentiated phenotype after 4 weeks culture in alginate gel.

CONCLUSIONSThe condylar chondrocytes from human osteoarthritic temporomandibular joint were successfully cultured in vitro. The alginate three-dimensional culture system was successfully adopted for in vitro culture of condylar chondrocytes from human osteoarthritic temporomandibular joint, in which the chondrocytes exhibited the excellent differentiated phenotype.

Objective:To evaluate the reliability and validity of the Chinese version of the Personal and Social Performance scale (PSP-CHN) in patients with schizophrenia.Methods:Totally 165 out-patients and in-patients meeting the DSM-IV-TR criteria for schizophrenia were entered in the study.Ten of subjects was included the intra-rater reliability training.The Global Assessment of Functioning Scale (GAF) was regarded as the' gold standard' to investigate the validity of PSP-CHN,and the Positive and Negative Rating Scale was used to assess the severity of disease to explore the correlative validity,in the other 155 subjects.Five to seven days after the first PSP-CHN interview,the second PSP-CHN was evaluated by another investigator to assess the test-retest reliability among 66 subjects.Twenty-seven subjects with the score of Positive and Negative Syndrome Scale (PANSS) more than 60 were followed up for 8 weeks of standardized pharmacotherapy.By the end of 8 week of treatment,the PANSS and PSP-CHN were assessed again to explore the sensitivity of PSP-CHN.Results:The internal consistency (Cronbach α=0.84) and the inter-rater reliability (κ value=0.56,ICC=0.94 for PSP-CHN total score) were good.The test-retest reliability was high [intraclass correlation coefficient (ICC) of 0.95].The scale showed good construct validity with statistically significant correlations with the Global Assessment of Functioning Scale (GAF) (ICC of 0.95).The PSP-CHN score had a good negative correlation with the PANSS total score(r=-0.79,-0.57,-0.63 and -0.71,respectively,P<0.01).After 8 week treatment,PSP-CHN total score was increased with the improvement of PANSS,and the responder showed higher increasing of PSP-CHN total score (21.2) than those partial responder(10.2),significantly.Conclusion:The Chinese version of the PSP-CHN is a convenient and valid instrument to assess the personal and social functions of stabilized and acute patients with schizophrenia.

Echinococcosis is an age-old disease that causes serious damage to the animal husbandry and the human health perennially. As a newly discovered species of Echinococus, E. shiquicus has the potential public health significance and could be a potential parasitic zoonosis. In this review, its etiology, life cycle, epidemiology, detection and diagnoses, public health etc. are discussed or summarized. Also, a series of comparisons among E. granulosus, E. multilocularis and E. shiquicus are made.

Biofilms are complex three-dimensional bacterial assemblages that attach to biotic or abiotic solid surfaces, and frequently embed within a self-produced matrix of extracellular polymeric substances. Biofilm formation is a microbial defense response to biotic and abiotic stresses, and a key factor for survival in adverse environments. A wide variety of microorganisms can colonize distant tissues of higher plants, such as leaves, vascular network and roots, and adhere to the surface of the tissues to form biofilms. The dynamic processes in forming biofilms in response to plant internal environment are key steps required for full virulence of phytopathogenic bacteria. Exploring the mechanisms involved in regulation of bacterial biofilms is important for understanding the plant-pathogens interactions. In this review, we summarized the research progresses related to the biofilms of bacterial phytopathogens, including biofilm characteristics, essential regulatory mechanisms and key signals affecting the transition between a planktonic lifestyle and multicellular behavior.

Objective:To explore the cutoff value for assessing small airway dysfunction in children with asthma.Methods:A total of 364 asthmatic children aged 5 to 14 years, with normal ventilatory function, followed up at the Asthma Clinic of the Children′s Hospital of Capital Institute of Pediatrics from January 2017 to January 2018, were selected as the case group. Concurrently, 403 healthy children of the same age range and without any symptoms in the community were chosen as the control group, and pulmonary function tests were conducted. The values of forced expiratory volume in 1 second (FEV 1), forced vital capacity (FVC), forced expiratory flow at 50% of FVC (FEF 50), forced expiratory flow at 75% of FVC (FEF 75) and maximum mid-expiratory flow (MMEF) were compared between case group and control group. Statistical tests such as t-test, χ2 test, or Mann-Whitney U test were used to analyze the differences between the groups. Receiver operating characteristic (ROC) curves were constructed, and the maximum Youden Index was utilized to determine the optimal cutoff values and thresholds for identifying small airway dysfunction in asthmatic children. Results:This study comprised 364 children in the case group (220 boys and 144 girls) and 403 children in the control group (198 boys and 205 girls). The small airway parameters (FEF 50%pred, FEF 75%pred, MMEF%pred) in the asthmatic group were significantly lower than in the control group (77% (69%, 91%) vs. 95% (83%, 109%), 67% (54%, 82%) vs. 84% (70%, 102%), 76% (66%, 90%) vs. 97% (86%, 113%), Z=12.03, 11.35, 13.66, all P<0.001). The ROC curve area under the curve for FEF 50%pred, FEF 75%pred, MMEF%pred was 0.75, 0.74, and 0.79, respectively. Using a cutoff value of 80% for FEF 50%pred achieved a sensitivity of 56.9% and specificity of 81.4%. A cutoff value of 74% for FEF 75%pred resulted in a sensitivity of 67.3% and specificity of 69.2%. Finally, using a cutoff value of 84% for MMEF%pred achieved a sensitivity of 67.9% and specificity of 77.2%. Conclusion:In the presence of normal ventilatory function, utilizing FEF 50<80% predicted or MMEF<84% predicted can accurately serve as criteria for identifying small airway dysfunction in children with controlled asthma.