CRISPR/Cas9 gene-editing system has been broadly used in various fields of bioscience and medicine in recent years. The system can be guided by RNA to specific DNA site thus achieving targeted gene editing.Off-target effect and editing efficiency remain to be two crucial challeges to the system. Currently, a number of researches have been focused on the optimization of the system by reducing off-target effects and increasing editing efficiency, which may enhance its safety and expand its application.

Objective To clone the gene encoding protein of EspA and Stx2B from EHEC OI57:H7 by DNA recombinant technology, construct prokaryotic expression vector pET-28a ( + )-espAstx2B, express fusion protein of EspA-Stx2B and to analyze the biological and immunological characteristics of the fusion protein. Methods the sequence encoding the protein of EspA and Stx2B was amplified by PCR from the enterohemorrhagic Escherichia coli strain. The amplified products were connected with linker by recombinant technology and cloned into pET-28a( + ) vector. The vector was then transferred to the host cells E. Coli BL21 strain (DE3). Following, the protein expression was induced by IPTG. The expression quantities and style of fusion protein was then determined by SDS-PAGE. Its immunoreactivity was analyzed by Western blot. Finally, BALB/c mice were injected with the preliminarily purified recombination protein EspA-Stx2B, then oral challenged these mice with EHEC O157-SMR2 and counteracted toxic substances with O157 ultrasonic supernatant. Results The determination of the sequence encoding of the espA-stx2B fusion gene has 100% of consistency with the sequence from GenBank Sakai strain and contrivable linker. This fusion protein EspA-Stx2B was expressed as inclusion body formation and the percentage is approximately 40%. Western blot suggested the fusion protein has excellent immunoreactivity. Titer of antiserum of the mice to EspA-Stx2B increased evidently. EspA-Stx2B could not decrease bacterial number attached to the intestinal tract of mice based on fecal shedding of Oi57 in mice. In the test of death of BAI,B/c causing by conteracting toxic substances with O157 ultrasonic supernatant, immunoprotection of EspA-Stx2B rate was 66.7%. Conclusion A recombinant plasmid that has high performance on expression of EspA-Stx2B prorein was successfully constructed in present study, and the fusion protein has excellent immunoreactivity and immunogenicity. EspA-Stx2B could not decrease bacteria] number attached to the intestinal tract of mice based on fecal shedding of O157 in mice, but evidently decrease the mortality rate of the mice. The antiEspA and anti-Stx2B had immunoprotection effect by different means. These results may provide the foundation for the further development on EHEC O157:H7 double subunit vaccine.

Objective To investigate the clinical efficacy of intermittent liver-nourishing and mind-regulating acupuncture in preventive treatment of migraine. Method One hundred and twenty migraine patients were randomly allocated to treatment and control groups, 60 cases each. The treatment group received intermittent liver-nourishing and mind-regulating acupuncture and the control group took flunarizine hydrochloride capsules orally. The migraine score, the visual analogue scale (VAS) score and the headache severity rating score were recorded in the subjects before treatment and at the end of treatment and the 4-week, 8-week, 12-week, 16-week and 20-week follow-ups. The Self-Rating Anxiety Scale (SAS) score and the Self-Rating Depression Scale (SDS) were recorded before and after treatment.Result After one course of treatment, the total efficacy rate was 98.3% in the treatment group and 89.5% in the control group; there was a statistically significant difference between the two groups (P<0.05). There was a statistically significant difference in the migraine score between before treatment and at the end of treatment in the two groups (P<0.05) and between the two groups at the same follow-ups (P<0.05). There was a statistically significant difference in the VAS score between before treatment and at the end of treatment in the two groups (P<0.05) and between the two groups at the same follow-ups (P<0.05). There were statistically significant differences in the SAS and SDS scores between before treatment and at the end of treatment in the two groups (P<0.05) and between the two groups after treatment (P<0.05).Conclusion Intermittent liver-nourishing and mind-regulating acupuncture has marked efficacy in preventive treatment of migraine. It can increase the total efficacy rate and reduce the number of relapses.

Objective To analyze the electrocardiography(ECG)data of pressure overload-induced cardiac hy pertrophy rats.Methods Pressure overload cardiac hypertrophy was induced by abdominal aorta constriction. Echocardiogram and heart weight measurement demonstrated the occurrence of cardiac hypertrophy.Standardized ECG parameters of limb and chest were measured and statistically analyzed.Results Two weeks after hypertrophy models were established,echocardiogram showed greater LVPWTd,IVSTd,LVDd.ECG showed that left axis deviation and higher R waves in V_A,V_B,V_C(P

[ ABSTRACT] AIM:To investigate the antipyretic effect of patchouli oil on lipopolysaccharide ( LPS)-induced fe-ver in rabbits.METHODS:Male rabbits (n=42) were randomly divided into 7 groups according to their body weight and basal body temperature, including control group, model group, western medical positive group, traditional Chinese medical positive group, and high, middle and low doses (2%, 1%and 0.5%) of patchouli oil groups.Subsequently, except the controls, the rabbits were injected with LPS at a dose of 1 mL/kg (2 mg/L) through marginal ear vein to establish rabbit fever model and the rabbits in control group received the same volume of NS.The rabbits in control group and model group were injected with 0.5%Tween-80 0.5 h late, and the rabbits in the other groups were treated with correspoonding drugs. The effect of patchouli oil on the body temperature was observed, and the levels of interleukin-1β( IL-1β) and tumor nec-rosis factor-α(TNF-α) in the serum, and prostaglandin E2(PGE2) and cyclic adenosine monophosphate (cAMP) in the hypothalamus were measured by radioimmunoassay.RESULTS: The body temperature and the levels of IL-1β, TNF-α, cAMP and PGE2 in model group were significant higher than those in control group.Patchouli oil notably inhibited the body temperature in the febrile rabbits.From 1.5 h to 5.5 h after administration, the body temperatures were increased by (1.06 ±1.55), (1.62 ±1.36), (1.38 ±1.22), (0.98 ±0.98) and (0.48 ±0.95) ℃in high patchouli oil group, re-spectively.From 3.5 to 5.5 h after administration, the body temperatures were elevated by ( 1.47 ±0.73 ) , ( 1.15 ± 0.68) and (0.63 ±0.54) ℃ in middle patchouli oil group, respectively.A tendency of downregulation of the elevated body temperatures was observed at every time point after administration in low patchouli oil group.Patchouli oil significantly decreased the levels of TNF-αin the serum and cAMP content in the hypothalamus, and attenuated the elevated tendency of the IL-1βlevel in the serum and PGE2 level in the hypothalamus.CONCLUSION:Patchouli oil evidently has antipyretic effect on LPS-induced fever in the rabbits.The antipyretic mechanism might be related to the inhibition of TNF-αlevel in serum and cAMP content in the hypothalamus.

425 halophilic bacteria strains were isolated by using six different media from thirty salt-soil samples collected from Heijing ancient salt mine,Yunnan. By the growth on NaCl grandient concentration and other screening methods,79 strains of extremely halophilic microorganisms were chosen for further research. Based on colonies color,shape and size,15 stains were selected to be sequenced. The sequencing results revealed that 11 of them were haloarchaea. According to the phylogenetic analysis,they belong to four genera of the family Halobacteriaceae. The blast results showed that the similarities of six sequences were higher than 97 % with the validly described species of the following four genera: Halorubrum,Natronococcus,Natrialba,Halalkalicoccus. The similarities of other five sequences with any validly described species were less than 97 %,therefore,the taxonomic positions of the following five strains YIM-ARC 0032,YIM-ARC 0036,YIM-ARC 0037,and YIM-ARC 0050 could be determined according to further polyphasic taxonomy data.The results indicated that there was a considerable diversity of haloarcheaea in salt-mine environment of Heijing ancient salt mine,Yunnan and it was worth continuing to research on this area.

The aim was to construct and identify the mammalian expression vector of pCAG-IRES-SHIP-GFP and to detect whether it could express in human acute leukemia cell line K562.The cDNA fragment of SHIP obtained by RT-PCR was inserted into pCAG-IRES-GFP.The recombinant plasmid was confirmed by restriction enzyme digesiton,PCR and DNA sequecing.pCAG-IRES-SHIP-GFP was transfected into K562 cells with lipofectamine 2000.The expression of SHIP was determined by GFP fluorescence and Western blot analysis.FQ-PCR was used to quantitate SHIP mRNA.The expression of p-Akt,Akt were determined by Western blot.PI were tested by flow cytometry and MTT to verify whether exogenous SHIP could inhibit proliferation of K562 cells.The results showed that the correct constrution of the recombinant plasmid pCAG-IRES-SHIP-GFP has been shown by restriction enzyme digestion,PCR and DNA sequencing.pCAG-IRES-SHIP-GFP could express SHIP protein in K562 cells.The K562 cells viability after transfected with SHIP gene droped down.Western blot analysis showed that phospha-Akt308 and Akt473 were reduced to 38.7% and 68% respectively.It was concluded that the vector of pCAG-IRES-SHIP-GFP has been successfully constructed and it can be expressed in K562 cells.The expression of exogenous SHIP gene can lead to apoptosis of K562 cells by down-regulating the p-Akt expression.What found here might be one of the mechanisms involved in the pathogenesis of leukemia.

Objective: To explore the clinical characteristics of follicular lymphoma (FL) in the era of rituximab combined with chemotherapy and the prognostic significance of the follicular lymphoma international prognostic index (FLIPI), follicular lymphoma international prognostic index 2 (FLIPI2), international prognostic index (IPI), revised international prognostic index (R-IPI), National Comprehensive Cancer Network international prognostic index (NCCN-IPI) among Chinese patients. Methods: 229 FL patients who were treated initially with rituximab combined with CHOP-like (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy from November 2008 to April 2018 were analyzed retrospectively and all were scored by the above clinical index. Univariate and multivariate survival analysis were performed on 201 patients who completed the treatment and were followed regularly. Results: In the univariate survival analysis, age>60 years, hemoglobin<120 g/L, elevated serumβ(2)- macroglobulin, involvement of bone marrow and elevated CRP were the risk prognostic factors for overall survival (OS) and progression free survival (PFS). Moreover, the analysis of the OS and PFS between rituximab (R) maintenance (RM) group and non-maintenance (non-RM) group showed that the OS and PFS of RM group were better than those of non-RM. In the multivariate analysis of OS, hemoglobin<120 g/L, involvement of bone marrow, elevated CRP and non-RM were independent prognostic factors. In the multivariate analysis of PFS, hemoglobin<120 g/L, CRP and non-RM were independent prognostic factors. When FLIPI2 was included in the multivariate analysis, CRP and FLIPI2 were independent prognostic factors in both OS and PFS, and non-RM was independent prognostic factors in PFS. Conclusion: FLIPI2 is the better risk stratification in FL patients in the era of rituximab.
Antineoplastic Combined Chemotherapy Protocols ; Cyclophosphamide ; Disease-Free Survival ; Doxorubicin ; Humans ; Lymphoma, Follicular/drug therapy* ; Prednisone ; Prognosis ; Retrospective Studies ; Rituximab/therapeutic use* ; Vincristine

OBJECTIVETo study the clinical features, neuroimaging presentations, diagnosis and differential diagnosis, and operative warning events of the solid cerebellar hemangioblastoma.

METHODSThe clinical data of 48 patients with solid cerebellar haemangioreticuloma were retrospectively analysed. Preoperative examinations included CT and MRI in all patients, DSA in 20, preoperative embolization of feeding arteries of tumor in 16, a preoperative ventriculoperitoneal shunt was placed in 5 cases. The lesion distribution of 48 patients was as follows: 14 lesions were situated in the left hemisphere of cerebellum with an extent into the cerebellopontine angle in 2 cases, 12 in right hemisphere of cerebellum, 8 in superior vermis, 7 in inferior vermis, 6 in left subtentorium, 1 in left cerebellar tonsil.

RESULTSThe tumors of 48 patients were totally removed. One patient occurred normal perfusion pressure breakthrough during surgery, and gastrointestinal hemorrhage on the second day after operation. Other patients all were intra- and postoperatively uneventful. Except for autoinfusion, no patients transfused heterogenous blood. There were no operative mortality and serious complications in this series.

CONCLUSIONSThe solid cerebellar hemangioblastoma was benign neoplasm. Abiding by properly operative techniques, the optimal results can be obtained.

Adolescent ; Adult ; Angiography, Digital Subtraction ; Cerebellar Neoplasms ; diagnosis ; surgery ; Child ; Diagnosis, Differential ; Female ; Hemangioblastoma ; diagnosis ; surgery ; Humans ; Magnetic Resonance Imaging ; Male ; Microsurgery ; Middle Aged ; Neurosurgical Procedures ; Retrospective Studies ; Tomography, X-Ray Computed