In modern biology and biotechnology research, recombinant gene expression has been the most popular method to obtain the target protein. In recent years, many foreign genes have been efficiently expressed in Escherichia coli. However, proteins encoded by animal, plant or mesophilic microbial genes often lose activities or become denatured within a few hours at regular growth temperatures for E. coli; some other target proteins are toxic to host cells and therefore difficult to be over-expressed. The new T-vector, pEXC-T, was constructed by combining TA cloning and cold-shock induction to obtain high expression levels with low costs. This paper reports the construction of pEXC-T and optimization of induction techniques for gene expression. Two instable proteins were tested and successfully expressed in soluble form by using pEXC vector. The development of pEXC-T offers a convenient technique for the preparations of recombinant proteins to be used in structure/function studies, or as diagnostic markers and medicinal proteins.
Biotechnology ; Cold Temperature ; Escherichia coli ; genetics ; Gene Expression ; Genetic Vectors ; Plasmids ; genetics ; Recombinant Proteins

Objective To evaluate the value of combining PCT, BNP, D-Dimer and PCIS score in predicting the prognosis of pediatric severe pneumonia in the early stage. Methods 80 cases of children with severe pneumonia were selected, 49 cases were boys , 31 cases were girls, with a median age of 7.5 months (1 month to 156 months). According to the final outcome, the cases are divided into treatment group and poor prognosis group. The score of PCIS, PCT, BNP and D-dimer within 24 hours after admission were recorded. According to the indicators, ROC curve was drawn independently and integrated with the four indicators,and the corresponding areas under ROC curve (AUC) were calculated to compare the accuracy of the assessment. Results The AUC of PCIS, PCT, BNP and D-dimer were between 0.7 and 0.9. The ROC curve integrated the four indicators showed the AUC were 0.932, which improved sensitivity and specificity. Conclusions The integration of PCIS, PCT, BNP and D-Dimer could improve the accuracy of prediction in the prognosis of severe pediatric pneumonia.

Objective To detect the protective mechanism of trehalose in tracheal cryopreservation.Methods Inbred male Sprague-Dawley (SD) rats were sacrificed with intraperitoneal injection of ketamine(150mg?kg -1).The tracheas were removed and immersed immediately in the freezing medium of low potassium dextran (LPD) solution only(Group Ⅰ) ,containing with 10% dimethylsulfoxide(DMSO)(Group Ⅱ), containing with 0.15mol?L -1 trehalose (Group Ⅲ),and containing with 10% DMSO and 0.15mol?L -1 trehalose (Group Ⅳ) respectively. A sterile plastic tube containing a 1-cm-long trachea was filled with the freezing medium,sealed,and frozen to -80℃ at rate of -1℃ per minute in a programmable freezer.Then the tube was stored in liquid nitrogen(-196℃) for 20 days. Then the specimen was thawed in a 37℃ water bath and rinsed with physiologic saline solution 10 times.Histologic changes before cryopreservation and after thawing were examined in each group. After the specimens were embedded in paraffin,5-(m-thick sections were stained with hematoxylin and eosin.The epithelium and cartilage was assessed. We also observed Bcl-2 and Bax gene expression by immunohistochemistry. At last, some tracheas(SD) after cryopreservation were thawed and transplanted into the abdominal cavity of Wistar rats. The transplanted tracheas were retrieved and assessed histologically.Results Microscopic findings of the tracheas in Group Ⅲ and Group Ⅳ showed their structure were intact and Bax gene expression was lower in cartilage after cryopreservation(20d) compared with other groups,especially in Group Ⅳ.The tracheas in Group Ⅲ and Group Ⅳ grew well after they were transplanted into cavity of Wistar rats heterotopically,too.There were no significant differences among 4 groups in Bcl-2 gene expression.Conclusion In tracheal cryopreservation the trehalose can protect the trachea by protecting the tracheal cartilage.It is one of the protective mechanism that the trehalose inhibit the Bax gene expression of cartilage cells.The concomitant use of trehalose and DMSO has a synergistic effect.

Adolescent ; Anticonvulsants ; administration & dosage ; adverse effects ; therapeutic use ; Child ; Child, Preschool ; Epilepsy ; drug therapy ; Female ; Fructose ; administration & dosage ; adverse effects ; analogs & derivatives ; therapeutic use ; Humans ; Infant ; Male ; Time Factors ; Treatment Outcome

Based on the infrared spectra of Lophatheri Herba and Commelinae Herba, one-dimensional infrared spectra, second derivative spectra and two-dimensional correlated spectra were used to find out the differences between Lophatheri Herba and its imitations, respectively. The common peak ratio and variant peak ratio dual-indexes sequential were calculated and established according to infrared spectra of eleven batches of herbs. Infrared spectral data of Lophatheri Herba cluster analysis was applied to explore the similarity between each sample. The grouping results trend of sequential analysis of dual-indexes and cluster analysis was accordant. The results showed that the differences could be found by multi-level identification, and the source and the quality of the herbs could be effectively distinguished by the two analysis methods. Infrared spectroscopy, used in the present work exhibited some advantages on quick procedures, less sample required, and reliable results, which could provide a new method for the identification of traditional Chinese medicine with the imitations and adulterants, and the control of quality and origin.
Drugs, Chinese Herbal ; analysis ; chemistry ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Spectrophotometry, Infrared ; methods ; Spectroscopy, Fourier Transform Infrared ; methods

Background The establishment of diabetic animal model is a crucial step for the study about diabetic eye diseases. At present,the main modeling method include the injection of streptozotocin and alloxan. But the shortcoming of the former is an expensive price, and that of the later is high death rate of animals. Objective This experiment was to discuss the way which decrease the death of alloxan-injected animal and explore the effects of high blood glucose on the posterior capsular opacification (PCO). Methods Forty clean healthy male New Zealand white rabbits were randomly divided into 2 groups. 90mg/kg of alloxan were injected via ear vein once in 20 rabbits to create the diabetic animal models,and the equivalent amount of normal saline solution was injected at the same way as normal blood glucose group. The successful models were selected in the animals with the blood glucose level over 12. 0 mmol/L two weeks later, and PCO of lens were graded based on the method of Odrieh under the slit lamp. Extracapsular lens extraction was then performed on the right eye of rabbits in both groups, and the posterior capsules were obtained from these eyes at the 6th, 10th and 14th days after operation. The expression of proliferating cell nuclear antigen ( PCNA ) in posterior capsular lens epithelial cell was detected by immunohistochemistry. Results The modeling successful rate was 70% after injection of alloxan. The body weight of rabbits in high blood glucose group was significantly lowed and the blood glucose was significantly elevated in comparison with normal blood glucose group ( all P<0. 05). Two weeks after surgery ,2 eyes occurred 2 grade of PCO and only one eye showed the 1 grade of PCO in the high blood glucose group. However, 1 grade of PCO was found in 3 eyes in the normal blood glucose group. Biopsy revealed that PCNA was positively expressed in the cell nuclei of LECs in high blood glucose group rather than the normal blood glucose group from the 10th day after surgery. The proliferation index of PCNA was 0. 86±0. 04 and 0. 25±0. 03 respectively in high blood glucose group and normal blood glucose group, showing a significant difference between them (t = -16. 171 ,P = 0. 000). Conclusion Stable diabetic models of rabbits can be created by intravenous injection of 90 mg/kg alloxan. High blood glucose level is one of the important factors for the development of PCO.

AIM:To observe the efficacy and safety of 1g/L bromfenac sodium hydrate ophthalmic solution in the partial substitution of glucocorticoid after laser subepithelial keratomileusis (LASEK).METHODS: Totally 104 cases (208 eyes) were received LASEK, which were selected and divided into study group and control group.The study group were adopted 1g/L bromfenac sodium hydrate ophthalmic solution combined with tobramycin dexamethasone eye drops and fluorometholone eye drops, the control group were adopted tobramycin dexamethasone eye drops and fluorometholone eye drops.The changes of visual acuity and intraocular pressure of two groups were recorded before and after surgery, and the score of painness and the occurrence of haze were observed after surgery.RESULTS: At postoperative 1, 2, 3d, respectively, the pain score of the study group were 1.70±0.35, 1.25±0.34, 0.82±0.32, the pain score of the control group were 2.30±0.43, 1.68±0.44, 1.12±0.33, the differences were significant (P<0.05).Before and at 2wk, 1, 3mo after surgery, respectively, uncorrected visual acuity of study group were 0.035±0.02, 0.71±0.13, 0.89±0.17, 0.88±0.18, while which of control group were 0.037±0.015, 0.73±0.15, 0.87±0.14, 0.86±0.15 (P>0.05), and the differences were not significant(P>0.05).At preoperative and postoperative 1, 2wk, 1 and 3mo of surgery, respectively, the intraocular pressure of study group were 17.33±1.58, 7.54±1.28, 7.23±1.58, 7.26±1.47, and 7.30±1.36 mmHg;the intraocular pressure of control group were 17.53±1.43,7.57±1.32,7.73±1.55,7.80±1.38,7.86±1.43 mmHg,the differences were not significant between before and at 2wk after surgery(P>0.05), the differences were significant between the two groups at 2wk, 1mo and 3mo (P<0.05).At postoperative 1, 3mo of surgery, the score of haze level of the study group were 91.7% and 93.3%, respectively and which in control group were 92.0% and 92.9%, respectively, and the differences were not significant between the two groups at every time point(P>0.05).CONCLUSION: It is safe and effective that 1g/L bromfenac sodium hydrate ophthalmic solution in the partial substitution of glucocorticoid after laser subepithelial keratomileusis.The patient has a lower intraocular pressure, has similar therapeutic effect as glucocorticoid in vision and antiinflammatory.

Objective To verify the association between common breast cancer susceptibility loci which have been confirmed in European and Asian populations and breast cancer susceptibility in sporadic breast cancer among the Han nationality in Henan province , and to analyze their genotypes in the internal type of breast cancer . Methods In 253 breast cancer patients ( the case group ) and 343 patients who had benign breast lesions ( the control group), rs2046210(6q25.1), rs2981582(EGFR2), rs889312(MAP3K1), and rs3803662(TOX3/TNRC9)were genotyped by SNP im-LDR technique.According to estrogen receptor(ER), progesterone receptor (PR), human epidermal growth factor receptor 2(HER2)and Ki67, breast cancer are divided into 5 types:Lu-minal A, Luminal B, HER2-enrich, Luminal HER2, and triple negative breast cancer ( TNBC).Results rs2046210(6q25.1), rs2981582(EGFR2), rs889312(MAP3K1)had no statistical differences between the case group and the control group(P=0.421, 0.459, and 0.468), but the genotype of rs3803662(TOX3/TNRC9)in the case group and the control group had statistical difference (P=0.037).The allelic frequencies of rs3803662 between the case group and control group were different in codominant inheritance ( OR=2.19, 95%CI:1.19-4.02)and recessive genetic models (OR =2.06,95% CI:1.15 -3.70).Compared with AA and GA, GG in-creased the risk of breast cancer ( P =0.012, 0.015 ).The genotypes of rs2046210 ( 6q25.1 ), rs2981582 (EGFR2), rs889312(MAP3K1), and rs3803662(TOX3/TNRC9)had no difference in different types of breast cancer.Conclusions Four common breast cancer susceptibility loci from GWAS are not entirely associated with breast cancer risk among the Han nationality in Henan province .Only rs3803662(TOX3/TNRC9)is confirmed to increase the risk of breast cancer .Different genotypes of 4 loci distribute equally in different types of breast cancer .

Small interfering RNA (siRNA) is the initiator of RNA interference and inhibits gene expression by targeted degradation of specific messenger RNA. siRNA-mediated gene regulation has high efficiency and specificity and exhibits great significance in the treatment of diseases. However, the naked or unmodified siRNA has poor stability, easy to degrade by nuclease, short half-life, and low intracellular delivery. As an emerging non-viral nucleic acid delivery system, ionizable lipid nanoparticles play an important role in improving the druggability of siRNA. At present, one siRNA drug based on ionizable lipid nanoparticles has been approved for the treatment of rare disease. This review introduces the research progress in ionizable lipid nanoparticles for siRNA delivery, focusing on the effect of each component of lipid nanoparticles on the efficiency of siRNA-mediated gene silencing, which provides new references for the studies on ionizable lipid nanocarriers for siRNA delivery.

Child ; Epiphyses ; Fracture Fixation, Internal ; Humans ; Lower Extremity