Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.
Alleles ; Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA, Circular/metabolism* ; Embryo, Nonmammalian ; Gene Editing/methods* ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, Reporter ; Genetic Loci ; Genotyping Techniques ; Green Fluorescent Proteins/metabolism* ; Integrases/metabolism* ; Luminescent Proteins/metabolism* ; Mutagenesis, Insertional ; Single-Cell Analysis ; Zebrafish/metabolism*

Objective To explore the application value of fractional anisotropy(FA)values of magnetic resonance diffusion tensor imaging(DTI)parameters in rat rat glioma grading.Methods Sixty-seven female Wistar rats were divided into the experi-mental group(n=57)and control group(n=10)according to the random number table method.All the surviving rats were exam-ined by 3.0T DTI at 1-2 weeks(22 cases)and 3-4 weeks(35 cases)after inoculation,and the FA values of the tumor were ob-tained and compared with the pathological results.Results Among 57 tumor-loading rats,there were 18 low-grade gliomas and 39 high-grade gliomas.DTI showed that the FA value of high-grade gliomas was higher than that of the low-grade glioma,the differ-ence was statistically significant[(0.167 ± 0.035)vs.(0.147 ± 0.015),t=2.34,P<0.05].Conclusion The FA value of DTI pa-rameters can provide accurate,reliable and noninvasive imaging information for preoperative glioma grading.

Objective To evaluate preoperative oral carbohydrate in patients receiving LC day surgery.Methods 117 patients undergoing LC day surgery in PLA General Hospital from Oct 2016 to Mar 2017 were evenly divided into three groups.Patients in group A took oral carbohydrate preoperatively,group B by iv glucose,group C by overnight fasting.The preoperative thristy and the indicators of inflammation,heart rate,blood glucose,serum insulin,insulin resistance index postoperatively were collected and compared.Results No patients suffered aspiration.There were no complications above Clavien Ⅰ level.Differences in insulin resistance index,fasting serum insulin were statistically significant between group A and group B (Z =-5.60,-4.78,P ＜ 0.05).That of insulin resistance index,fasting serum insulin and procalcitonin between group A and group C were statistically significant (Z =-2.65,-2.49,-4.02,P ＜0.05).Procalcitonin between group B and group C were statistically different (Z =-2.183,P ＜ 0.05).The preoperative thristy between group A and group B,group C were statistically different (x2 =6.47,P ＜ 0.05).Conclusions Preoperative oral carbohydrate intake is safe and feasible with the benefits of improving the quality of life in patients operated on LC day surgery.

Objective To explore the safety and economic benefits of day surgery laparoscopic cholecystectomy (LC/DS).Methods Clinical data of 5771 patients undergoing LC/DS and 2450 undergoing conventional laparoscopic cholecystectomy (CLC) in PLA General Hospital from November 2009 to January 2017 were retrospectively analyzed. The informed consents of all patients were obtained and the local ethical committee approval was received. In LC/DS group, there were 2204 males and 3567 females with the average age of (51±13) years old. In CLC group, there were 992 males and 1458 females with the average age of (51±13) years old. The hospitalization time and total medical expenses of two groups were compared by Mann-Whitney test. The ratio of conversion to laparotomy, postoperative complication, postoperative 24-hour discharge rate, and 30-day readmission rate were compared by Chi-square test.Results The ratio of conversion to laparotomy and 30-day readmission rate was respectively 0.36％, 0.49％ in LC/DS group and 0.16％, 0.49％ in CLC group, and no significant difference were observed (χ2=2.83, 0.00; P>0.05). The postoperative 24-hour discharge rate in LC/DS Group was 83.34％, significantly higher than 49.22％in CLC group (χ2=1020.30, P<0.05). The postoperative complication rate in LC/DS group was 6.59％, significantly lower than 14.56％ in CLC group (χ2=130.19, P<0.05). The hospitalization time and total medical expenses in LC/DS group was respectively [3.0(1.0-33.0)] d and [0.98(0.56-4.59)] million RMB, significantly lower than [5.0(1.0-36.0)] d and [1.40(0.38-2.95)] million RMB in CLC group (Z=-17.33,-45.40; P<0.05).Conclusions LC/DS is safe and effective. It can reduce the incidence of postoperative complications, shorten the hospitalization time and reduce the hospitalization cost of patients.

Objective To evaluate effect of Xugu-Huoxue decoction on the treatment of patients with femoral neck fracture after internal fixation with hollow screw fixation. Methods A total of 120 patients with femoral neck fracture were randomly divided into the treatment group and the control group according to the inclusion criteria, 60 patients in each group. The control group was treated with cannulated screw internal fixation; the observation group was treated with Xugu-Huoxue decoction at the second day after the operation. Both groups were assessed with the Harris hip function score at the end of 12 months after operation. The SF-36 scale was used to evaluate the quality of life of the patients. Results The total effect rate of the observation group was 88.3% (53/60) and the control group was 71.7% (43/60). The difference between the 2 groups was statistically significant (χ2=5.208, P=0.022). At 12 months after surgery, the pain (41.4 ± 7.3 vs. 32.2 ± 5.7, t=7.738), the joint function (45.7 ± 6.2 vs. 36.3 ± 7.2, t=7.701), joint mobility (5.0 ± 0.8 vs. 3.1 ± 0.8, t=13.115), and the total score of Harris (87.7 ± 4.6 vs. 65.4 ± 5.4, t=24.461) in the observation group were significantly higher than those in the control group (P<0.001). The physical function (83.1 ± 7.2 vs. 78.8 ±14.2, t=2.095), body pain (82.1 ± 9.9 vs. 67.7 ± 11.1, t=7.524), the overall health (76.6 ± 10.3 vs. 68.8 ± 14.4, t=3.401) and activity (81.1 ± 7.9 vs. 76.6 ± 11.2, t=2.549) in the observation group were higher than those in the control group (P<0.05 or P<0.01). Conclusions The Xugu-Huoxue decoction could improve the prognosis of patients with femoral neck fractures after cannulated screw fixation, promote the recovery of hip function and improve the quality of life.

Objective To investigate the value of multi-b value DWI combined with PSA evaluation on the effect of endocrinotherapy for prostate cancer (PCa), and to study the correlation between the signal intensity(SI), apparent diffusion coefficient(ADC) with PSA.Methods Forty patients with PCa diagnosed by pathology or biopsy were tested for serum PSA levels before and after endocrine therapy.All patients underwent DWI at b-value of 300,800 and 1 000 s/mm2 by using a GE Signa HDx 3.0T MRI scanner.The serum PSA,the value of SI and ADC at different b values before and after endocrine therapy,and their correlation with PSA were observed and measured, respectively.Results (1)The serum PSA before and after endocrine therapy were (35.63±20.91) ng/mL and (5.98±3.84) ng/mL, respectively.The difference was statistically significant (P<0.05).(2)On the condition of different b values, ADC values were (1.06±0.06)×10-3 mm2/s, (0.83±0.04)×10-3 mm2/s, (0.73±0.03)×10-3 mm2/s, respectively before endocrine therapy, and (1.33±0.09)×10-3 mm2/s, (1.16±0.09)×10-3 mm2/s, (1.10±0.08)×10-3 mm2/s, respectively after endocrine therapy.There were significant difference between the ADC values before endocrine therapy and those after endocrine therapy (P<0.05).SI were 809.09±79.06, 417.42±26.65 and 327.42±25.26 in 40 patients with Pca before the endocrine therapy, and 780.10±78.52,409.33±33.93 and 320.00±31.10 after endocrine therapy.The difference was not statistically significant (P>0.05).(3)Pearsoncorrelation analysis showed that ADC value was negatively correlated with serum PSA (r300=-0.58,P<0.01;r800=-0.60,P<0.01;r1 000=-0.66,P<0.01) before endocrine therapy.ADC value was also negatively correlated with serum PSA (r300=-0.55,P<0.01;r800=-0.52,P<0.01;r1 000=-0.61,P<0.01) after endocrine therapy.In addition,SI was negatively correlated with serum PSA (r300=-0.09,P<0.01;r800=-0.18,P<0.01;r1 000=-0.28,P<0.01) before the treatment, as well.Conclusion ADC value at different b values increased after endocrine therapy in Pca, and is negatively correlated with serum PSA.The correlation is the most significant at b value of 1 000 s/mm2.The combination of DWI and serum PSA could be used to monitor and evaluate the effect of endocrinotherapy for Pca, and better guide the clinical treatment of Pca.

BACKGROUND: Cells in contact with nanomaterials can induce oxidative stress, allergic reactions, and then produce cytotoxicity and genotoxicity. Therefore, studies on nano toxicology have attracted more and more attention.OBJECTIVE: To comparatively evaluate the cytocompatibility of Pluronic (P85, F127, F87) tri-block copolymer nanoparticles modified with folic acid (FA) and polylactic acid (PLA).METHODS: Pluronic (P85, F127, F87) tri-block copolymer nanoparticles were modified with FA and PLA to synthesize a variety of amphiphilic block copolymers, including PLA-P85-PLA, FA-P85-PLA, PLA-F127-PLA, FA-F127-PLA,PLA-F87-PLA and FA-F87-PLA. The cytotoxicity of these synthesized nanoparticles was analyzed by cell morphology,cell metabolic activity and cell membrane effects in HepG-2 cells.RESULTS AND CONCLUSION: The relative growth rate of HepG-2 cells had no significant differences under 24-hour induction of various concentrations (5, 10, 20, 50, 100 mg/L) of unmodified P85, F127, and F87 nanoparticles (P > 0.05).The growth and proliferation of cells under the low concentrations (5, 10, 20, 50 mg/L) were enhanced. P85 NPs and F87 NPs could significantly inhibit cell viability at dose of 400 mg/L. In contrast, there were no significant differences towards P85, F127 and F87 nanoparticles (5, 10, 20, 50, 100, 200, 400 mg/L) modified with FA and PLA when compared with the control group (P > 0.05). These findings indicate that the modification of FA and PLA can improve the cytocompatibility of Pluronic (P85, F127, F87) tri-block copolymers, and therefore, PLA-Pluronic-PLA and FA-Pluronic-PLA nanoparticles are both good candidates for drug vectors.

Objective To observe the therapeutic effect between less invasive stabilization system (LISS) plate and anatomical plate internal fixation for the treatment of tibial plateau fractures. Methods The clinical data of 58 patients with tibial plateau fractures were retrospectively analyzed. The patients were divided into LISS plate group and anatomical plate group according to the internal fixation method with 29 cases each. The operative time, blood loss, incision length, fracture healing time, postoperative weight-bearing time, postoperative complications and therapeutic effect (according to Rasmussen knee joint function score scale) were compared between 2 groups. Results The operative time, incision length, blood loss and incidence of postoperative complications in LISS plate group were significantly lower than those in anatomical plate group:(68.5 ± 7.1) min vs. (92.3 ± 9.4) min, (5.8 ± 1.4) cm vs. (8.6 ± 2.1) cm, (208.5 ± 27.8) ml vs. (329.7 ± 25.2) ml and 17.2%(5/29) vs. 41.4%(12/29), and the excellent rate was significantly higher than that in anatomical plate group: 86.2% 25/29) vs. 62.1%(18/29), and there were statistical differences (P<0.05);there were no statistical differences in fracture healing time and postoperative weight-bearing time between 2 groups (P>0.05). Conclusions Both of LISS plate and anatomical plate internal fixation for the treatment of tibial plateau fractures have good clinical efficacy, but the LISS plate has advantages of shorter operation time, less tissue trauma, less blood loss, quicker fracture healing and less postoperative adverse reaction.

OBJECTIVE: To investigate the value of liver perfusion imaging of 256-slice CT in evaluating the compensated and decompensated cirrhosis. 
 METHODS: A total of 20 patients with liver cirrhosis, who were confirmed by liver biopsy, clinical symptoms and imaging, were selected from December 2012 to June 2014. According to the results of liver biopsy and the Child-Pugh classification, the patients were divided into a compensated cirrhosis group (n=8) and a decompensated cirrhosis group (n=12). Eleven cases without liver and spleen diseases were served as a control group. All subjects were under the 256-CT liver perfusion (256-CTP). The data of CTP [hepatic arterial perfusion (HAP), portal venous perfusion (PVP), total liver perfusion (TLP), hepatic perfusion index (HPI)] were obtained according to liver perfusion type, and the data of CTP [liver perfusion (LP), peak enhanced (PE), time to peak (TTP), blood volume (BV)] were obtained according to general perfusion type. Spearman rank correlation was used to analyze the correlation of liver cirrhosis with perfusion parameters. The receiver operating characteristic (ROC) curve was used to predict liver cirrhosis, and the maximized Youden index was served as the optimal cutoff value, then the area under curve, sensitivity and specificity were calculated.
 RESULTS: The PVP, TLP and PE values in the control group, the compensated cirrhosis group and the decompensated cirrhosis group were (76.63±37.26), (38.78±16.13) and (36.14±
15.31) mL/(100 mL·min); (98.48±43.58), (55.63±14.47) and (54.41±20.81) mL/(100 mL·min); 
(55.62±18.25), (44.11±5.79) and (41.08±7.74) HU, respectively, showing a gradual downward trend and a significant difference among the 3 groups (all P <0.05). HPI values were (19.50±6.08)%, (31.81±16.48)% and (34.47±16.04)%; TTP values were (37.32±8.59), (47.06±14.61), (59.86±20.87) s, respectively, showing a gradual upward trend and significant difference among the 3 groups ( all P<0.05). There was no significant difference in the HAP, LP and BV among the 3 groups (all P>0.05). PVP, TLP, PE and LP were negatively correlated with the process of liver cirrhosis (r=-0.592, -0.567, -0.409, -0.569, all P<0.05), but HPI and TTP were positively correlated with the process of liver cirrhosis (r=0.434 and 0.538, both P<0.05). 
 CONCLUSION 256-CTP could provide useful information for the assessment of liver cirrhosis by measuring a plurality of perfusion parameters. The hepatic microvascular changes in patients with liver cirrhosis could be quantitatively assessed by perfusion CT. TTP shows high efficiency in prediction of liver cirrhosis and decompensated liver cirrhosis.
Case-Control Studies ; Humans ; Liver Cirrhosis ; diagnosis ; Perfusion Imaging ; Sensitivity and Specificity ; Tomography, X-Ray Computed

Objective To investigate the imaging features of alar ligament and its extent, and provide the basis forclinical treatment.Methods 3.0 T superconducting MRI was used to scan the alar ligament with high resolution PDWI sequence (Proton density weighted imaging, PDWI)in 109 patients of emergency admissions due to head and neck trauma. Based on imaging features, ligamentous injury was classified into three degrees(Ⅰ to Ⅲ degrees).Patients with Ⅰ degree ligamentous injury were treated conservatively, andⅡtoⅢdegree injury patients were treated with surgery, then follow-up was performed with MRI for the recovery of ligaments and clinical evaluation for symptoms (6 months follow-up period). Results High-resolution PDWI showed 78 patients with no ligament injury.On follow-up, patients recovered well (atlantoaxial joint motor function and clinical symptoms). Thirty one patients had alar ligament injury in varying degrees, of which 18 patients had grade Ⅰ injury, nine patients had degree Ⅱinjury, and four patients had degreeⅢinjury .All gradeⅠinjury patients received conservative treatment. Follow-up of patients showed good recovery, MR revealed the lesions shrank in varying degrees or disappear.
Six gradeⅡinjury patients had surgical treatment, and three received conservative treatment. On follow-up, seven patients had a good recovery, two patients underwent surgical treatment within 3 months after injury and recovered well.Three gradeⅢpatients treated by surgery, and all with good recovery postoperative, and a patient died of respiratory failure. Conclusions High resolution PDWI is an effective tool to evaluate the extent of the alar ligament injury. Grade Ⅰ ligamentous injury patients treated conservatively can achieve good results, GradeⅡandⅢligamentous injury patients should receive surgical treatment early.