Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.
CRISPR-Cas Systems ; DNA Ligase ATP ; genetics ; Gene Expression Regulation ; genetics ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Ku Autoantigen ; genetics

Objective:To analyze the subcellular localization of family of serine hydrolases 1 (FSH1) protein in Microsporum canis. Methods:The FSH1 and enhanced green fluorescent protein (EGFP) genes were amplified by PCR using the previously constructed plasmid containing the FSH1 gene and the recombinant plasmid pCAMBIA-LRP-EGFP as the template; the vector DNA was obtained by double-enzyme digestion of the recombinant plasmid pCAMBIA-LRP-EGFP with SnaBI/KpnI. Then, the EGFP expression plasmid and Ptrcp-FSH1-EGHP-Ttrcp fusion plasmid were constructed by inserting the amplified EGFP gene and EGFP-FSH1 gene into the vector DNA respectively, and identified by PCR and sequencing. The two recombinant plasmids were transformed into Microsporum canis by an Agrobacterium tumefaciens-mediated method, and the gene EGFP and fusion gene FSH1-EGFP were expressed integratedly in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc. The cellular localization of the fusion protein was observed by laser scanning confocal microscopy. Results:The Agrobacterium tumefaciens-mediated transformation system and EGFP expression vector in Microsporum canis were successfully constructed; the fusion gene FSH1-EGFP was expressed integratedly in Microsporum canis. Laser confocal microscopy showed that fluorescence signals of the FSH1-EGFP fusion protein were concentrated in the cytoplasm and nuclei of Microsporum canis, with a granular or cluster-like appearance. Conclusion:The FSH1-EGFP fusion protein was successfully localized in the cytoplasm and nuclei of Microsporum canis, providing a basis for further clarifying the function and pathogenic mechanisms of the FSH1 gene in Microsporum canis.

Objective To determine the location of PQ-LRP protein in Microsporum canis using the enhanced green fluorescent protein(EGFP)as a marker.Methods The total RNA was extracted from Microsporum canis,and reversely transcribed into cDNA.The PQ-LRP gene was amplified by PCR using the above cDNA as the template.The fusion gene of PQ-LRP gene and EGFP gene was linked to the plasmid pCAMBIA 1300.Microsporum canis was subjected to Agrobacterium tumefaciens-mediated transformation,in order to achieve the integrated expression of the fusion gene LRP-EGFP in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc.Laser-scanning confocal microscopy was conducted to determine the cellular localization of the fusion protein.Results The expression vector pCAMBIA-LRP-EGFP was successfully constructed,and the fusion gene LRP-EGFP was expressed integratedly in Microsporum canis.Laser-scanning confocal microscopy showed that fluorescence signals of LRP-EGFP were concentrated on the cell membrane of Microsporum canis,giving a granular or cluster-like appearance.Conclusion The infusion gene LRP-EGFP can be successfully expressed in Microsporum canis,and PQ-LRP protein is located on the cell membrane of Microsporum canis.

Objective To study the pros and cons of two methods of infusing itraconazole injectionand prevent the blockage of peripherally inserted central catheter (PICC) and improve patients' satisfaction with nursing technology.Methods 172 patients infusing itraconazole were divided into two groups by random digital table method.86 cases established an independent infusion pathway as the control group,another 86 cases using PICC for itraconazole injection and withdrawing plunger of the syringe about 0.5 ml before and after the infusion and then pulsing-flushing it with 10 ml normal saline as the experimental group.Then compared the blockage rate of PICC and the patients' satisfaction with nursing technology.Results The blockage rate of the two groups had no significant difference (x2 =0.206,P ＞ 0.05) while patients' satisfaction with nursing skills was distinct,and the experimental group's was 96.51％ (83/86),much higher than 16.28％ (14/86) of the control group.Conclusions Withdrawing taken before and after the infusion of itraconazole injection could effectively prevent catheter blockage and improve patients' satisfaction with nursing technology.

BACKGROUND:Reduction quality is essential for the healing and function of limb. Traditional X-ray and CT scans have some limitationsin quantitatively and visualy observing the reduction quality of the remnant fracture in three-dimensional displacements. Mimics have advantages in bone rapid modeling and three-dimensional measurement, which offers the chance in quantifying the reduction quality of the displacement in three-dimensional methods.
OBJECTIVE:To measure residual displacement in femoral neck fracture patients with satisfactory reduction by using three-dimensional measurement method, and to evaluate the association between residual displacement and femoral head necrosis.
METHODS:We enroled 141 patients with femoral neck fracture, who were treated in the Third Department of Joint, Affiliated Hospital of Logistics University of People’s Armed Police Force from January 2012 to December 2013. Al patients were divided into undisplaced fracture group (n=44; Garden1&2) and displaced fracture group (n=97; Garden3&4). Based on DICOM data of CT files, three-dimensional models were constructed in each patient. Three parameters were measured: displacement of femoral head’s center, fossa capitis femoris and rotational displacement. Femoral head necrosis was folowed up. The correlation between residual displacement and femoral head necrosis was explored.
RESULTS AND CONCLUSION:(1) The duration of folow-up in those patients ranged from 25 to 43 months. Femoral head necrosis occurred in 7 cases from the undisplaced fracture group and 21 cases from the displaced fracture group. (2) The three parameters (displacement of femoral head’s center, fossa capitis femoris and rotational displacement) were respectively (7.44±2.84) mm, (11.75±4.12) mm and (17.68±8.99)° in the undisplaced group, and (6.51±3.75) mm, (9.34±5.27) mm and (14.67±7.43)° in the displaced group. Except displacement of femoral head’s center, significant differences in the other two parameters were detected between the two groups (P< 0.05). (3) These findings suggest that poor reduction quality is the main factor causing femoral head necrosis. X ray is insufficient to describe the spatial shift and the angle, and has limitations in assessing the reductionquality of femoral neck fracture. Three-dimensional measurement has certain advantages to assess quality of fracture reduction.

Objective To evaluate the efficiency and safety of ultrasonic debridement therapy (UDT) and intelligence negative-pressure wound therapy(INPWT) for diabetic foot ulcers with Wagner 2-3degree.Methods Eighty patients with diabetic foot ulcers with Wagner 2-3 degree were divided into two groups according to the treatment method.The UDT and INPWT group (INPWT group) had 53 cases.Routine treatment group(routine group) had 27 cases.Both groups were given blood sugar and blood pressure control,anticoagulation,anti-infection and supportive treatment.The efficiency and complications were compared between two groups.Results In INPWT group,29 cases were healed,14 cases showed obvious effectiveness,7 cases were effective and 3 cases were inefficacy.The total effective rate was 94.34％(50/53).For routine group,they were 7,8,6,6 cases and 77.78％(21/27) respectively.The total effective rate between two groups had significant difference (P ＜0.05).Pain was found in 9 cases and hemoglobin decreased in 1 case in INPWT group.Conclusion INPWT after UDT for diabetic foot ulcers with Wagner 2-3 degree shows a good efficiency and safety.

Objective To study the effect of small diameter graft (0.8 cm) splenocaval or mesocaval shunts combined with pericardial devascularization in the treatment of portal hypertensive variceal bleeding. Methods Splenocaval shunts were performed in 14 patients and mesocaval shunts were done in 24 patients, in combination with pericardial devascularization. Results The average decrease of free portal pressure was 6.6±1.2 cm. There was no significant changes in liver function postoperatively (P>0.05). Platelet counts and leukocyte counts were back to normal in splenocaval shunt patients postoperatively (P< 0.05). Operative mortality was 3%. Pyrexia developed in 4 patients, intractable ascites in 1 patient, chylons ascites in 1 patient, hepatic encephalopathy in 1 patient, intraabdominal infection in 1 patient and stress ulceration in 1 patient. All patients recovered after expectant treatment except one who died from severe intraabdominal infection. 35 patients received follow-up between 6 months and 3 years, total effective rate was 89%, 2 patients died from recurrent variceal bleeding, the shunt potency rate was 80% in 1 year and 75% in 3 years. Esophagogastric varices disappeared or alleviated as shown by endoscopy in 25 patients on 6 months postoperatively. Conclusions Small diameter portosystemic graft shunts combined with poricardial devascularization is an effective therapy for bleeding esophagogastric varices with a low rate of hepatic encephalopathy. Splenocaval shunt alleviates hypersplenism concurrently.

The expression of musclin gene in skeletal muscle of rats fed with high-fat diet and its correlation to insulin resistance.diabetes mellitus and GLUT4 were explored.The results showed that musclin expressin was up-regulated(1.39±0.36 vs 0.31±0.07,P<0.01)in skeletal muscle of rats fed with high-fat diet, suggesting that this finding may be related with insulin resistance and involvement of GLUT-4 expression(1.27 4± 0.32 vs 1.84±0.24.P<0.01).

To investigate the effects of rennin angiotensin system blockade on the microvessel density in islets of diabetic rats and its relationship with islet function, diabetes model was created by feeding of high-caloric laboratory chow plus intraperitoneal injection of a small dose of streptozotocin (30 mg/kg). After 8 weeks intervention with perindopril (AE, n=10) or valsartan (AR, n=10), the islet function of the animals was evaluated by intravenous insulin release test (IVIRT). The pancreases were immunohistochemically stained to analyze the content of insulin and vascular endothelial growth factor (VEGF) in the islets. The microvessel density (MVD) of islets was detected by counting CD34 positive cells. The hypoxia inducible factor (HIF)-1alpha mRNA expression in the islets was detected by RT-PCR. Compared with normal control group (NC, n=10), the area under the curve for insulin from 0 to 30 min (AUCI(0-30)) of diabetes group (DM, n=8) was decreased by 66.3%; the insulin relative concentration (IRC) of betacell was decreased significantly; the relative content of VEGF was increased obviously [(-4.21+/-0.13) vs (-4.06+/-0.29)]; MVD in islets was decreased by 71.4%; the relative expression of HIF-1alpha mRNA was increased by 1.19 times (all P<0.01). Compared with DM group, the AUCI(0-30) of AE and AR group was increased by 44.6% and 34.9% respectively; IRC was also increased significantly; the relative content of VEGF was decreased by 21.2% and 21.7% respectively; MVD was increased by 62.5% and 75.0% respectively; the relative expression of HIF-1alpha was decreased by 27.2% and 29.0% respectively (all P<0.01 or P<0.05). There were no significant differences in the said indexes between group AE and AR. It is concluded that the blockade of RAS may ameliorate islets function of diabetic rats by increasing the MVD in islets.

To investigate the effects of rennin angiotensin system blockade on the microvessel density in islets of diabetic rats and its relationship with islet function,diabetes model was created by feeding of high-caloric laboratory chow plus intraperitoneal injection of a small dose of streptozotocin (30 mg/kg). After 8 weeks intervention with perindopril (AE,n=10) or valsartan (AR,n=10),the islet function of the animals was evaluated by intravenous insulin release test (IVIRT). The pancreases were immunohistochemically stained to analyze the content of insulin and vascular endothelial growth factor (VEGF) in the islets. The microvessel density (MVD) of islets was detected by counting CD34 positive cells. The hypoxia inducible factor (HIF)-1α mRNA expression in the islets was detected by RT-PCR. Compared with normal control group (NC,n=10),the area under the curve for insulin from 0 to 30 min (AUCI0-30) of diabetes group (DM,n=8) was decreased by 66.3%;the insulin relative concentration (IRC) of βcell was decreased significantly;the relative content of VEGF was increased obviously [(-4.21±0.13) vs (-4.06±0.29)];MVD in islets was decreased by 71.4%;the relative expression of HIF-1α mRNA was increased by 1.19 times (all P＜0.01). Compared with DM group,the AUCI0-30 of AE and AR group was increased by 44.6% and 34.9% respectively;IRC was also increased significantly;the relative content of VEGF was decreased by 21.2% and 21.7% respectively;MVD was increased by 62.5% and 75.0% respectively;the relative expression of HIF-1α was decreased by 27.2% and 29.0% respectively (all P＜0.01 or P＜0.05). There were no significant differences in the said indexes between group AE and AR. It is concluded that the blockade of RAS may ameliorate islets function of diabetic rats by increasing the MVD in islets.