Construction a shuttle vector containing AaCPR100A epidermal protein gene of Aedes aegypti with two spore-producing-dependent promoters in Bacillus thuringiensis by in-fusion cloning / 中国热带医学

@#Abstract: Objective To construct a shuttle vector pHT315-AaCPR100A with two spore-producing-dependent promoters and the target gene AaCPR100A in Escherichia coli-Bacillus thuringiensis. Methods The forward promoter of Cry3A, named Pro-1 (+), was amplified by PCR using pSVP27A plasmid as the template, and the target gene AaCPR100A was amplified using Aedes aegypti RNA reverse conversion cDNA as the template. The plasmid pHT315 was linearized by digestion with Hind Ⅲ and Sal Ⅰ. The forward promoter and the target gene were inserted into the linearized vector pHT315 successively by in-fusion cloning according to the transcription direction. The synthesized plasmid containing the Cry3A reverse promoter sequence was used as the template, and the Pro-1 (-) reverse promoter was amplified by PCR. The intermediate vector containing the forward promoter and the target gene was linearized by EcoR I restriction enzyme, and the reverse promoter was inserted downstream of the target gene by in-fusion cloning in the direction of transcription. Results By agarose gel electrophoresis, the forward promoter, target gene AaCPR100A and reverse promoter bands were clear and of good quality, which could be used for in-fusion cloning experiments. The two spore-producing-dependent promoters and target gene fragments were connected by In-fusion cloning. The recombinant vector pHT315-AaCPR100A was verified by PCR. The forward promoter, target gene fragment and reverse promoter were successfully amplified in the recombinant vector. Nucleotide sequencing verified that the sequencing results of the bidirectional promoter sequence and the target gene sequence were basically consistent with the sequence alignment results, which met the requirements of the construction of vector elements and proved that the recombinant vector was successfully constructed. Conclusions Based on the above results, this study proves that the recombinant shuttle vector with two spore-producing-dependent promoters can be successfully constructed by in-fusion cloning technology, laying the foundation for the construction of engineered Bacillus thuringiensis expressing dsRNA of AaCPR100A.

Inhibitory effect of astragalus membranaceus injection on apoptosis and autophagy induced by enterovirus 71 / 中国生物制品学杂志

@#Objective To investigate the effect of astragalus membranaceus（AM）injection on apoptosis and autophagy of human gastric epithelial cell line（GES⁃1）induced by enterovirus 71（EV71）. Methods GES⁃1 cells were cultured in vitro and divided into infected group（EV71 infected at a MOI of 3 and control group（no virus infected）. The morpho⁃logical changes of EV71 infected cells were observed by inverted microscope. The level of VP1 in GES⁃1 cells infected with EV71 was detected by Western blot；CCK⁃8 assay was used to detect the viability of GES⁃1 cells infected with EV71；Nuclear staining with DAPI was used to observe the morphological changes of nuclear apoptosis infected with EV71. GES⁃1 cells were divided into control group（without virus infection），infection group and AM intervention group with final concentration of 1，2. 5，5 and 10 μg／mL，respectively. Western blot was used to detect the effect of AM intervention on the expression of apoptosis⁃related proteins Caspase⁃3，PARP and autophagy⁃related proteins LC3 and P62 in GES⁃1 cells infected withEV71. CCK⁃8 method was used to detect the effect of AM intervention on the viability of GES⁃1 cells infected with EV71. Results GES⁃1 cells were round，shrunken with nuclear pyknosis and uneven size；VP1 level increased（t = 41. 56，P < 0. 01），cell viability decreased（t = 19. 07，P < 0. 01），Caspase⁃3 and PARP proteins were cut off（t = 35. 29 and 3. 648， P < 0. 01 and 0. 021 8，respectively），LC3Ⅱ／LC3Ⅰ ratio increased（t = 10. 16，P = 0. 000 5）and P62 protein was degraded（t = 68. 68，P < 0. 01）；AM inhibited the degradation of Caspase⁃3，PARP and P62 proteins induced by EV71 （t = 52. 66，59. 60 and 40. 22，respectively，each P < 0. 01）and increased the ratio of LC3Ⅱ／LC3Ⅰ（t = 5. 521，P = 0. 005 3），andreducedtheinhibitoryeffectofEV71ontheviabilityofGES⁃1cells（t =4. 420，P =0. 0115）. Conclusion EV71 infection induced apoptosis of GES⁃ 1 cells and AM intervention inhibited EV71 induced apoptosis by inhibiting EV71 induced autophagy.

Changes of T cell subsets in major immune organs during sepsis / 中国小儿急救医学

Objective:To explore the changes in the proportion and number of T cell subsets in different immune organs during sepsis.Methods:Eight-week-old female C57BL/6 mice were randomly divided into sepsis group and sham group.The experimental sepsis model was constructed through cecal ligation and puncture, and the sham group just underwent sham operation.Then we detected the changes in the total number of lymphocytes and in the ratio and absolute number of CD4 + T cells, CD8 + T cells, CD4 + CD25 high Foxp3 + regulatory T cells(Treg) and CD4 + CD25 low Foxp3 - effector T cells(Teff) in the mouse spleen, axillary and inguinal lymph nodes and bone marrow by cell counting and flow cytometry 24 h and 16 d after modeling. Results:In the spleens of septic mice, the ratio and absolute numbers of CD4 + T cells and Teff, as well as the absolute number of CD8 + T cells were significantly reduced 24 h and 16 d after modeling.There was no significant change in the number of Treg 24 h after modeling, but a significant increase occurred 16 d after modeling.During sepsis, the changes of CD4 + T cells, CD8 + T cells and Teff in mouse lymph nodes were basically the same as those in the spleen; but the changes in Treg were different, with no significant change in the early stage and a significant decrease in the late stage.In addition, the absolute numbers of CD4 + T cells, CD8 + T cells, and Teff in the bone marrow did not change significantly in the 24 h model, but decreased significantly in the 16 d model.The proportion and absolute number of Treg during sepsis were significantly reduced. Conclusion:During different periods of sepsis, there is a large consumption of lymphocytes in the spleen, lymph nodes and bone marrow.In most cases, the trend of Treg changes is inconsistent or even opposite to that of other T cell subsets.There are differences in the changes of T cells among major immune organs, suggesting that the responses of different immune organs to sepsis are heterogeneous.

Changes of bone marrow vascular system and endothelial cells in the early stage of sepsis / 中国小儿急救医学

Objective:To clarify the changes in the bone marrow vascular system in the early stage of sepsis in animal model.Methods:A sepsis mouse model was established by cecal ligation and puncture (CLP), and HE staining, immunofluorescence staining, flow cytometry and real-time quantitative PCR were used to comprehensively analyze the varieties of bone marrow vascular system in structure, the relative proportion of vascular endothelial cells and the expressions of damage-related genes at mRNA level.Results:A series of adaptive changes occurred in the bone marrow vascular system in the early stage of sepsis.Histological analysis showed that the bone marrow vascular structure was significantly remodeled.The average density of bone marrow sinusoids in the CLP group was (410.43±72.63)counts/mm 2, which was significantly higher than that in the sham group[(294.43±68.94)counts /mm 2, P<0.01]. The area of luminal pixels accounted for (43.46±3.21)%, which was significantly higher than that in the sham group[(30.28±4.44)%, P<0.001]. The exudation amount of evans blue in the bone marrow tissue of the CLP group was (0.42±0.12)ng/mg tissue, which was significantly higher than that in the sham group[(0.24±0.09)ng/mg tissue, P<0.05], suggesting increased vascular permeability.The results of flow cytometry analysis showed that the EC in bone marrow of the CLP group mice was in a proliferative state, with the proportion of Ki67 + endothelial cell increasing[(1.91±0.65)% vs.(5.06±1.10)%, P<0.01]. The mRNA levels of some genes related to the activation of vascular endothelial cells were up-regulated. Conclusion:Sepsis changes the structure and function of the bone marrow vascular system, and has a significant impact on the bone marrow microenvironment.

Research progress of regulatory T cells in sepsis / 中国小儿急救医学

Sepsis is a common critical illness in clinic and a worldwide serious public health issue.Regulatory T cell(Treg) is a type of negative immune-regulatory cell that can affect the organism immune status through various pathways to inhibit inflammation.Mastering the research progress in related fields of Treg is of great significance for further understanding the pathogenesis of sepsis and exploring new diagnostic and therapeutic methods for sepsis.This review provided the recent research progress of Treg cells in sepsis.

Effects of different treatments on seed germination and seedling growth of Bupleurum chinense / 中草药

Objective: To study the effects of different pretreatment methods and culture temperatures on Seed Germination and seedling growth of Bupleurum chinense, in order to provide scientific basis for artificial cultivation of B. chinense. Methods: Several physiological indexes such as the weight, pure rate, content of moisture, the rate of water absorption per thousand seeds were measured. Adopting a double-layer filter paper culture method, the pretreated seeds were cultured in the incubator at 20 ℃ in the 40% light. In different culture temperature treatment groups, the seeds were soaked in distilled water 24 h and then cultured in the corresponding temperature incubator. The germination energy, germination percentage, germination index, vigor index of seeds, and the root length and the shoot height of the seedling were recorded. Results: The pure rate of seeds was 91.51%, the weight of one thousand seeds was (2.83 ± 0.03) g and the weight of seed after absorbing water was about 2.24 times more than the weight of naturally dried seed. The content of moisture of seeds was 7.3%. Different hormones had different effects on seeds, among them the effect of 0.6 mg/L 6-BA treatment was the best. Water bath could increase the seedling vigor index, and 40 ℃ of its temperature significantly promoted the seed germination and seedling growth. The germination rate of seeds was the highest under the condition of 15 ℃, but the root length and seedling height were lower. Conclusion: The suitable condition for seed pretreatment and culture temperature of B. chinense established in this research can effectively improve the germination rate and seedling rate, which is of great significance for seed breeding and artificial cultivation of B. chinense.

Research progress on medicinal plant resources of bupleurum l / 中草药

Bupleuri Chinensis Radix, as traditional Chinese medicine, has great use amount in the past dynasties, and the amount of the existing wild resources is not enough to provide market demand. In order to protect the wild resources and expand the source of medicinal materials, the research on the chemical composition, pharmacological action, clinical application and other aspects of Bupleuri Chinensis Radix and other plants in the same genus have been widely carried out. The paper reviews the study of medicinal plant resources of Bupleurum L. in China from the distribution and species, chemical composition of medicinal parts, resources protection and utilization of Bupleurum L. medicinal plants, etc, to provide new clues for Bupleurum L. resources development.

Influence of group psychological training on the mental health and self consistency of nurses from operating rooms / 现代临床护理

Objective To explore the effect of group psychological training on the mental health and self consistency of nurses from operating rooms.Methods 52 nurses of an operating room were randomized into the treatment group(n=25)and the control group(n=27): The latter received humane care and instructions for mental self care and the former received group psychological training for 8 weeks based on the same treatment as in the control group.Symptom checklist 90(SCL-90)and self consistency and congruence scale(SCCS)were used to assess and compare the difference of the two groups before and after training.Results The intra-and inter-group comparisons between pre-and post-intervention showed statistical differences in terms of the total score by SCL,interpersonal sensitivity,depression and hostility,total score by SCCS and the score on the subscale of inconsistency between themselves and their experience(all P<0.05 or P<0.01).Conclusion Group psychological training can effectively improve the level of mental health and self consistency of OR nurses and thus can improve the total psychological status of the OR nurses.

Magnetic resonance imaging of the extraocular muscles and corresponding cranial nerves in patients with special forms of strabismus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>With the technical advances, magnetic resonance imaging (MRI) is now sensitive enough to detect subtle structural abnormalities of ocular motor nerves arising from the brainstem and orbits of living subjects. This study was designed to delineate the MRI characteristics in patients with special forms of strabismus.</p><p><b>METHODS</b>A total of 29 patients with special forms of strabismus underwent orbital and intracalvarium MRI. Imaging of the ocular motor nerves in the brainstem was performed in 0.8 mm thickness image planes using the three-dimensional fast imaging employing steady-state acquisition (3D-FIESTA) sequence. Nerves to extraocular muscles (EOMs), EOMs and their associated connective tissues were imaged with T1 weighting in tri-planar scans by dual-phased coils within 2.0 mm thick planes.</p><p><b>RESULTS</b>Patients with congenital fibrosis of the extraocular muscles exhibited hypoplasia of the oculomotor (CN3), abducens (CN6), trochlear (CN4) nerves, and the EOMs; hypoplasia of CN6 in the brainstem and an extra branch of the inferior division of CN3 to the lateral rectus were the most common but not the only presentation of Duane's retraction syndrome. Hypoplasia of CN6, facial (CN7) and hypoglossal (CN12) nerves were revealed in patients with Möbius syndrome. In a rare case of bilateral synergistic convergence and divergence, an enlarged branch of CN3 to the medial rectus and a questionable branch of CN3 to the inferior rectus bilaterally were found.</p><p><b>CONCLUSION</b>MRI can reveal subtle structures of the ocular motor nerves and their corresponding EOMs. This can provide valuable information regarding pathogenesis in some special forms of strabismus.</p>

Research of hepatic pathology and clinical characteristics of patients chronic infected with hepatitis B virus in immune tolerant stage / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the characteristics of the hepatic pathological and clinical features of patients with hepatitis B virus (HBV) in immune tolerant stage and find the better measure of diagnosing patients chronic infected by HBV in immune tolerant phase.</p><p><b>METHODS</b>135 patients with HBV chronic infection and persistently normal serum alanine aminotransferase (ALT) levels were involved in this study, whose serum HBeAg and HBV DNA were positive. Statistical analysis included the ages, sex, serum levels of HBVDNA, ALT and histological grade. The grades of inflammation and fibrosis were obtained through hepatic biopsy performed on all the patients.</p><p><b>RESULTS</b>Mean age in those patients was 22.61 +/- 8.95 years old. All those patients were divided into two groups according to histological grade: low- histological grade group, G < or = 1 and S < or = 1; and high-histological grade group, G > or = 2, S > or = to 2. Levels of histological grade were low in most of patients (99/135). Patients of low-histological grade had no difference in age, sex and family history of HBsAg carriers. Compared with low-normal ALT (ALT less than 30U/L), those with high-normal ALT (ALT > or = 30U/L) had a greater frequencies of high-histological grade. Compared with high HBVDNA (HBVDNA > or = 10(7) copies/ml), those with low HBVDNA (HBVDNA less than 10(7) copies/ml) had a greater frequencies of high-histological grade.</p><p><b>CONCLUSION</b>Levels of histological grade were low in most of patients with HBV chronic infection, serum HBeAg and HBVDNA positive, persistently normal serum ALT levels, but some of them were high-histological grade. It showed those patients were not all in immune tolerant stage of chronic HBV infection. Examination of ALT and HBVDNA are helpful to evaluate hepatic pathological damage for them.</p>

Detection of coagulation factor V in patients with severe hepatitis and its clinical significance / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the prognostic significance and role of coagulation factor V (CFV) levels in clinical diagnostic criteria for severe hepatitis.</p><p><b>METHODS</b>The CFV level and prothrombin activity (PTA) were tested by turbidimetry for 129 times in 58 patients with severe hepatitis. Comparative studies and clinical significance of CFV and PTA were analyzed by SPSS and SDAS softwares.</p><p><b>RESULTS</b>1. The levels of CFV and PTA were 15.3%+/-9.7% and 23.5%+/-10.0%, respectively, at the onset of severe hepatitis. 2. The mortality of severe hepatitis gradually increased with the gradual decrease of CFV or PTA during the most severe stage of the illness (P=0.000). 3. The levels of CFV and PTA decreased continually and rapidly in patients who died but gradually increased in survivors. The decrease or increase of PTA preceded that of CFV on the exacerbation or convalescent stage. 4. Hepatic encephalopathy occurred in 14 cases (24.14%). In 10 cases, it occurred in the terminal stage of the illness, far later than the time of the decrease of CFV. 5. The level of CFV was closely related to PTA (the correlation coefficient was 0.812), the level of CFV was almost consistent with that of PTA.</p><p><b>CONCLUSION</b>1. The level of CFV is an important prognostic indicator in severe hepatitis and is more specific than PTA. 2. Simultaneous determination of CFV and PTA may be helpful in earlier and more accurate diagnosis of severe hepatitis. 3. Possible use of CFV as one of the criteria for liver transplantation in patients with severe hepatitis should be studied.</p>