The treatment of thyroid associated ophthalmopathy (TAO) is still a worldwide problem.The conventional glucocorticoid therapy can not benefit all the patients.In recent years,new immunosuppressive agents,such as tumor necrosis factor inhibitors and anti-CD20 monoclonal antibody have been emerging and serving as new targets in treating TAO to remedy the insufficiency of glucocorticoid treatment.In this paper,recent advances in immunotherapy of thyroid associated ophthalmopathy are reviewed.

Objective To establish a detection method of Salmonella in laboratory animals based on a high-throughput sequencing technology, and to apply it in detection of Salmonella in laboratory animals.Methods DNA samples were extracted from mouse feces.Universal primers for 16S rDNA, 23S rDNA, 16S-23S rDNA, 23S-5S rDNA region, gyrB preferred area were designed, respectively.Each primer was tested and analyzed to determine the best amplification conditions and build a database.Forty-two samples of Salmonella were assayed by Illumina high-throughput sequencing technology and evaluated the specificity and stability of this method.Results The species preferred region of Salmonella was gyrB gene region.The primers for gyrB gene were FP5 ’-AACCACCGCAATCAGACCTT3‘ and FP5 ’-AGCCACGAAACCTTCACYA-3’.The primers were optimized and determined, through a high-throughput sequencing, and the sequence analysis detected very small amount of Salmonella in the 42 samples, indicating that this detection method is stable, highly sensitive, and the limit of detection reached to 0-102 CFU.Conclusions We have established a complete detection system for detection of Salmonella in laboratory animals based on a high-throughput sequencing technology, This system can detect trace amounts of Salmonella in laboratory animals, and this detection method is stable and highly sensitive, which can be also used in detection of other kinds of pathogenic microorganism in laboratory animals.

Objective To investigate the clinical efficacy and safety of ultra pulse CO2 laser combined with collagen dressing in treatment of cutaneous amyloidosis.Methods A total of 68 cases of primary cutaneous amyloidosis patients according to the different treatment methods was divided into two groups:the control group (n =33 cases) that was given acitretin capsules oral and topical compound flumetasone ointment packet treatment,and observation group (n =35 cases) with ultra pulse CO2 laser combined with collagen dressing external treatment.Two groups were observed in patients with curative effect and adverse reaction.Results Disease of integral observation group after treatment was significantly lower than the control group(t =3.12,P ＜0.05).The observation group had the efficiency of [88.6％ (31/35)] that was significantly higher than that of control group[17/33(51.5％),x2 =11.23,P ＜.0.01].The observation group had significantly shorter healing time [(5.38 ± 1.81) d] compared to the control group [(10.75 ±2.06)d,t =11.39,P ＜ 0.01].The observation group did not have recurrence,which was significantly different from the control group (x2 =4.90,P ＜ 0.05).Conclusions Ultra pulse CO2 laser combined with collagen dressing in treatment of primary cutaneous amyloidosis is a kind of effective treatment methods with remarkable curative effect and less adverse reactions.It is worthy of clinical application.

This review summarized the research status on DNA molecular identification of plants in the genus Chrysanthemum. Some case studies on representative DNA molecular identification techniques, which included ge-nomic in situ hybridization (GISH), DNA molecular markers and DNA barcoding, were described. Simultaneously, the merits, demerits and development of the techniques were discussed. The above work provides evidence for the identi-fication and resource utilization of plants in the genus Chrysanthemum.

Objective To study the distribution characteristics and drug resistance of non-fermenting bacterial infection in intensive care unit (ICU) at a tertiary hospital during seven consecutive years,and to provide evidence for rational use of antibiotics in ICU.Methods A retrospective analysis was conducted.The related data about non-fermentative bacteria obtained from clinical specimens,collected from lower respiratory tract,blood,urine,bile and other secretions of ICU patients admitted to Binzhou Medical University Hospital from January 2009 to December 2015 were retrospectively analyzed.The distribution characteristics and drug resistance of non-fermentative bacteria,and isolation rate of multiple drug resistance (MDR) strains were analyzed.Results 2 672 strains of nonfermentative bacteria were isolated during seven consecutive years,accounting for 57.9％ gram negative (G-) bacilli (2 672/4 613),and 35.2％ of all bacteria (2 672/7 587).The top five were Acinetobacter baumannii (38.4％),Pseudomonas aeruginosa (34.6％),Onion burkholderia cepacia (9.9％),Stenotrophomonas maltophilia (6.2％),and Pseudomonas fluorescens (5.6％).Non-fermentative bacteria were mainly isolated from the lower respiratory tract (60.9％).Isolation of the non-fermentative bacteria accounted for over 50％ of G-bacilli during seven consecutive years,and the isolation rate of the top five types of bacteria showed no obvious change,while positive rate of Acinetobacter baumannii showed a tendency to increase (obviously from 26.5％ in 2009 to 50.2％ in 2015),and a lowering trend of positive rate of Onion burkholderia cepacia,Stenotrophomonas maltophilia,and Pseudomonas fluorescens was obvious (from 15.6％,10.6％,13.0％ in 2009 to 5.6％,7.4％,1.4％ in 2015 respectively) was observed.The isolation rate of Pseudomonas aeruginosa was stable (about 30％) during seven consecutive years.The drug susceptibility results showed that the resistant rates of Acinetobacter baumannii against imipenem,meropenem,aminoglycosides and third-generation cephalmsporins were all higher than 70％,while its resistant rate to cefoperazone-sulbactam was relatively lower (40.2％-68.1％)with relatively higher sensitivity rate (23.6％-46.0％).In contrast,the resistant rates of Pseudomonas aeruginosa against antibiotics were low,while the sensitivity rate to fourth-generation cephalmsporins cefepime (58.3％-87.7％)and third-generation cephalmsporins was high (ceftazidime:55.6％-79.3％,piperacillin-tazobactam:62.5％-86.2％,cefoperazone-sulbactam:46.0％-89.8％).From 2009 to 2015,the incidence of MDR strains of Acinetobacter baumannii showed an obvious increasing tendency (from 68.0％ to 84.1％);in contrast,the incidence of MDR strains of Pseudomonas aeruginosa did not show an obviously increase in incidence from 2009 to 2012,on the other hand,it showed a decreasing tendency from a peak 68.6％ in 2012 to 23.5％ in 2015.Conclusions The isolation rate of non-fermentative bacteria was high and the drug resistance situation was serious.Therefore,it is important to grasp the knowledge regarding distribution characteristics,drug resistance and variation of non-fermentative bacteria in ICU.It is not only beneficial for both rational use of antibiotics,improve efficacy but also helpful in reducing the emergence of drug resistance stains.

Chloroplast genome sequences have comprehensive application prospects in DNA barcoding and chloroplast engineering in traditional Chinese medicine. The complete chloroplast genome of Magnolia officinalis sequenced by high-throughput pyrosequencing and a sequencing procedure was established. Fourteen contigs were obtained after de nove assembly. The sequencing percent of coverage was 99.99%. The chloroplast genome is 160 183 bp in size, and has a typical quadripartite structure with the large (LSC, 88 210 bp) and small copy (SSC, 18 843 bp) regions separated by two copies of an inverted repeat (IRs, 26 565 bp each). chloroplast genes were successfully annotated, of which 17 genes located in each IR region. The chloroplast genome features in Magnolia officinalis are nearly identical to those from other Magnoliid chloroplast genomes. Phylogenetic analyses were performed based on 81 shared coding-genes for a total of 9 Magnolia samples of 5 closely related species. Results showed that distinguishing among species was generally straightforward at the species and population level. This study confirmed the effectiveness of our chloroplast genome sequencing procedure. The chloroplast genome can provide distinguishing differences to help identify Magnolia officinalis and its closely related plants.

This study was aimed to show the extraction research of cpDNA in Gentiana straminea Maxim. and G. crassicaulis Duthie ex Burk., which will increase the knowledge about the chloroplast genome sequence characteris-tics, understand its genetic diversity and improve the efficiency of breeding schemes. G. straminea Maxim. and G. crassicaulis Duthie ex Burk. were taken as study objects. The improved sucrose-gradient centrifugation and purifica-tion methods were used to obtain the cpDNA, measure the concentration, detect the OD value, and amplify the ITS2 sequence to verify the nucleus pollution. The results showed that the high quality and purity cpDNA (0.1 - 0.4 μg/10 g) was demonstrated without other pollution after ITS2 sequence amplification, which met to the subsequent effi-cient sequencing of chloroplast genomes. The purity and mass have an important influence on the accuracy and cred-ibility of the cp genome sequencing. It was concluded that this study improved the sucrose-gradient centrifugation and purification with great effect of the purity and mass of cpDNA, and guaranteed the obtaining of complete cpDNA of Gentiana.

METHODS:Lentiviral vectors carrying bFGF and BMP-2 were constructed to transfect sheep bone marrow mesenchymal stem cells. cells were divided into four groups:bFGF group, BMP-2 group, co-transfection group BACKGROUND:Basic fibroblast growth factor (bFGF) can promote the proliferation of bone marrow stromal cells, and bone morphogenetic protein-2 (BMP-2) has an important significance in the induction of new bone formation.
OBJECTIVE:To analyze the effects of bFGF, BMP-2 and their co-transfection on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and to compare the relative expressions of col agen I, osteocalcin and osteopontin before and after celltransfection, thereby providing theoretical implications for seed cells in the construction of tissue-engineered bone.
and control group. The RNA was extracted using real-time quantitative PCR to detect mRNA levels of col agen I, osteocalcin, and osteopontin.
RESULTS AND CONCLUSION:Significant difference in non-specific osteogenic gene expressions was found among the four groups (P<0.05). bFGF and BMP-2 showed an interaction (P<0.05). Expressions of col agen I and osteocalcin in the co-tranfection group were higher than those in the other three groups (P<0.05), but osteopontin expression exhibited no difference (P>0.05). In vitro experiments showed that the relative expression of col agen I, osteocalcin and osteopontin were higher in the co-transfection group, indicating the cells from the co-transfection group have strongest osteogenic capacity that are suitable for seed cells for bone tissue engineering.

Objective To evaluate the diagnostic and staging value of orbital structures quantitative measurement with 3T mag-netic resonance imaging in patients with Graves’ophthalmopathy (GO).Methods Twenty-three GO patients (patient group)and eighteen healthy volunteers (contrast group)were enrolled.Quantitative measurement of orbital structures including exophthalmos values,fatty tissue thickness of the inner side of eye balls,cross-sectional areas of extraocular muscles and signal intensity ratios of extraocular muscles to the ipsilateral temporal muscles (SIR values),and they were compared between two groups.Correlations be-tween the quantitative values and clinical active score (CAS)were accessed.Receiver operating characteristic (ROC)analysis was performed to evaluate the most relevant quantitative parameter and its diagnostic value in discriminating active from inactive GO pa-tients.Results There were significant differences between the GO group and contrast group regarding to all the quantitative parame-ters (P <0.05).Strong correlation was found between SIR values and CAS (r =0.730,P <0.001 ).Significant difference of SIR was found between patients with active GO and inactive GO (P =0.002),and a cut off value of 3.25 might be the critical threshold value,with diagnostic sensitivity of 81.8% and specificity of 83.3% respectively.Conclusion Quantitative measurement of orbital structures with 3T MR imaging could provide assistant in diagnosing and staging of GO.

Objective To observe the clinical effect of the club exercise designed ourselves on the prevention of should-hand syndromeafter stroke. Methods 100 stroke patients in early stage, whose motor function of upper limb were lower than Grade Ⅲ evaluated byBrunnstrom assessment and without shoulder pain and swelling of hand, were randomly divided into control group (n=50) and observationgroup (n=50). All of them received routine rehabilitation therapy, and the observation group received club exercise in addition. The incidenceof shoulder pain, the active range of motion of shoulder, the motor function of upper limb and activities of daily living (ADL) of 2groups were evaluated before and 2 courses (60 d) after treatment. Results There was no statistical difference between 2 groups before thetreatment (P>0.05). After 2-course treatment, the incidence of shoulder pain was significantly lower in the observation group than in the controlgroup (P<0.001), the active range of motion of shoulder, motor function of upper limb and ADL were significantly better in the observationgroup than in the control group (P<0.001). Conclusion The club exercise can prevent the pain of shoulder joint, increase the activerange of motion of shoulder joint and improve the motor function of upper limb and ADL in patients after stroke.