Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.
Animals ; Base Sequence ; Cloning, Organism ; Electrophoresis ; Endonucleases ; genetics ; metabolism ; Fetus ; Fibroblasts ; metabolism ; Gene Knockout Techniques ; Gene Targeting ; methods ; Goats ; Green Fluorescent Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Zinc Fingers

ObjectiveTo explore the clinical characteristics and radiological manifestations of immune reconstitution inflammatory syndrome(IRIS)in acquired immunodeficiency syndrome(AIDS)patients with tuberculosis(TB)during highly active antiretroviral therapy(HAART).MethodsThe clinical and radiological data in 4 AIDS patients with TB who presented IRIS were analyzed retrospectively.ResultsThe clinical presentations of IRIS in 4 patients included fever(4 cases),weakness and weight loss(3 cases),abdominal pain(2 cases),cough with sputum(1 case),dyspnea(1 case).Cervical and(or)supra-clavicular lymph node enlargement were seen in 3 patients,inguinal lymph node enlargement in 1 patient,abdominal lymph node enlargement in 1 patient,hilar or mediastinal lymph node enlargement in 2 patients,pulmonary parenchyma and liver were involved in 2 patients,the involvement of kidney,adrenal gland,mesentery,peritoneum,psoas,brain and cutis was respectively found in 1 patient.The clinical and radiological presentations of IRIS were temporary and self-limited,improvement can be seen with antituberculosis therapy and HAART. ConclusionsIt is possible to have IRIS during HAART in AIDS patients with TB.Imaging examinations play an important role in the early diagnosis,monitoting and evaluating the response to therapy of IRIS.

To study the changes of pupil diameter and its effects during phacoemulsification. METHODS: Block design, prospective study. 60 cataract patients (60 eyes) were selected, which divided into two groups according to the age, young age group (group A, 20 eyes of 20 patients) and older age group (group B, 40 eyes of 40 patients). Two groups were all given compound tropicamide eye drops for fully mydriasis before operation, computer software to process images during operation, and according to the cornea amplification ratio measure pupil diameter during the operation phase: before the corneal tunnel incision ( t1 ) , after injection of sodium hyalurantae ( t2 ) , before artificial lens implantation (t3), at the end of the surgery ( t4 ). Statistical analysis of data using SPSS18. 0 software.RESULTS: The pupil diameter measurements of t1-t4 were followed respectively:group A were 8. 36 ± 0. 65, 8. 97±0. 50, 8. 67±0. 63, 8. 72±0. 96mm; group B were 7. 73± 0. 58, 8. 23 ± 0. 59, 7. 89 ± 0. 16, 7. 70 ± 0. 63mm. Overall comparisons between the two groups, the pupil diameter measurements had differences during the operation phase ( F = 26. 696, P< 0. 05 ). Comparisons between the two groups at different time points: the pupil diameter of t1-t4 in group A was larger than that in group B, with statistically differences (P<0. 05). The pupil diameter of each groups had a tendency to change in different operation phrases, with no statistically differences (F=2. 617,P>0. 05). Comparisons in a group at different time points: the pupil diameter of t2 was larger than that in t1, with statistically differences ( P>0. 05), the pupil diameter of t3 and t4 were decreased in group B, with statistically differences (P<0. 05).CONCLUSlON: Compound tropicamide eye drops are given for mydriasis before operation, young patients with cataract and senile patients with cataract can achieve the effect of surgery for mydriasis, the pupil stability of phacoemulsification in senile patients with cataract are lower than that in young patients with cataract.

The kinetics of nicotine degradation by O.intermedium DN2 and its application in tobacco waste were investigated. The results showed that the optimum temperature of nicotine degradation by O.intermedium was 30 ℃, the pH value was 6.5 and a mount of inoculum was 5 %. Under above conditions, the kinetics of nicotine degradation of initial concentration 500 mg/L was studied. The results indicated that the degradation process of nicotine with no-induced strain DN2 followed inverse S-shaped curve, and degradation process of nicotine with induced cells of DN2 followed Eckenfelder mode. The half life of nicotine degradation was 17.43 h and 4.10 h, respectively. And the results also showed that tolerance of O.intermedium DN2 to nicotine was up to 5000 mg/L when 0.1 % of glucose was added. Nicotine (2 220 mg/L) in extract of tobacco wastes degraded about 95.22 % by strain DN2 in 60 h incubation, indicating that strain DN2 was of application value in treatment nicotine pollution.

Objective To assess the accuracy of neuronavigation of 3D DSA and to evaluate the feasibility of 3D DSA neuronavigational neurosurgery through the coregistration of 3D DSA and MRI(A). Methods A Peg-Board Phantom was used in our study.The phantom consisted of 32 rods which were used for target localization;the height and the location of the rods were in normal distribution.For 3D DSA (Infinix NS/VC,Toshiba),the raw data was reconstructed to 3D images on the DSA workstation,and transferred to a online PC workstation where it was converted to standard 2D DICOM image data using WFU DICOM_Toolkit software.For MRI(A),the phantom was scanned with FSPGR sequence on the MRI scanner(GE SigMa VH/i 3.0 T),and the DICOM images were also transferred to the online PC workstation.Using the software 3D Slicer registration was performed on the PC workstation by using the location and shape of the rods in the phantom.The localization error of the rods was measured in image space as the Euclidean distance between targets defined in image space and those detected in the physical space. Paired t test was used to evaluate the difference between the accuracy of neuronaviagtion of 3D DSA and that of MRI(A).Results Through the coregistration of the rods in the phantom from different modality,all the images were better coregistrated.The mean localization error was(0.38?0.24)mm(3D DSA)and (0.31?0.12)mm[MRI(A)].There was no significant statistical difference between the accuracy of neuronavigation of 3D DSA and MRI(A)(t=-0.601,P=0.55).Conclusion 3D DSA images can be used in the neuronavigation system through the coregistration of 3D DSA and MRI(A).

To investigate the influence factors and effects of metal or dissolving microneedles on the formation and healing of skin microchannels, the pencil-shaped or conical stainless steel microneedles with different lengths, tip to tip space and base area, and the pencil-shaped dissolving microneedles with different tip to tip space were used. The microneedles were applied to the skin of epilatory mice and rats, and the effects of various microneedle parameters, application parameters, and animals on the microchannels were explored by the transepidermal water loss (TEWL) and methylene blue staining. Visually observe the skin local irritation caused by the microneedles. The animal experiments were approved by the Animal Experiments Welfare and Ethical Committee of Zhejiang University of Technology. The application time of the microneedle should be maintained at 30 s or more. When the insertion forces were 2, 4, 8 N, and the TEWL initial values of the formed microchannels were 12.9, 33.0, 40.4 g·m^-2·h^-1, respectively. When the length of metal microneedle were 400, 600, 800 μm, and the TEWL initial values of the formed microchannels were 37.1, 40.4, 49.5 g·m^-2·h^-1, respectively. When the tip to tip space of metal microneedle were 400, 600, 800 μm, and the TEWL initial values of the formed microchannels were 33.2, 40.4, 55.8 g·m^-2·h^-1, respectively. When the base area of metal microneedle were 0.16, 0.35, 0.62 cm², and the TEWL initial values of the formed microchannels were 35.1, 40.4, 67.1 g·m^-2·h^-1, respectively. The effects of conical and pencil-shaped microneedles are similar. When the tip to tip space of pencil shaped dissolving microneedle were 400, 600, 800 μm, and the TEWL initial values of the formed microchannels were 49.8, 60.5, 70.5 g·m^-2·h^-1, respectively. The TEWL baseline values of animal skins of different genders and series are different, but the tendency of microchannels formation and closure is similar. Visual inspection revealed that the slight erythema caused by the microneedles subsided within 24 h. Microneedles of different parameters have different effects on microchannels. The microchannels closed within 24 or 48 h, and the skin local irritation caused by microneedle was mild.

Objective:To study the coronal bone structure matching of distal radius in normal population and some patients with postoperative distal radius fracture, and to explore the clinical significance of coronal bone structure reduction of distal radius fracture.Methods:CT scans of 80 asymptomatic wrists were performed. Mimics 20.0 and 3-Matic research software were used to measure the matching data of coronal bone structure of the distal radius. Total of 44 patients with distal radius fractures treated with open reduction and volar plate fixation were collected. According to the data coronal bone structure of the distal radius, the matching group was in the normal range, and the mismatching group was less than the normal range. X-ray films were used to evaluate fracture healing, humeral height, ulnar angle and palm tilt angle at 3 months and 12 months after operation. The clinical indexes of wrist pain, wrist function, grip strength and activity were recorded in 2 groups. The DASH score was used for evaluation, and statistical comparisons was made between the two groups of related indicators.Results:The coronal bone structure matching value of the distal radius in 80 normal adults was 45.0%±16.2%. All the 44 patients with distal radius fracture were followed up for an average of 16 months. The postoperative wound healing was good, and the bone healing standard was reached 3 months after the operation. 3 months after surgery, radius height, ulnar deviation angle and palmar inclination angle of the mismatched group were all smaller than those of the matched group, but the differences had no statistical significance. The pronation angle in the mismatched group (68.82°±11.62°) was lower than that in the matched group (76.91°±9.14°), and the difference was statistically significant ( t=2.567, P=0.014). The DASH score in the mismatched group (15.53±2.36) was higher than that in the matched group (13.62±2.52), and the difference was statistically significant ( t=2.591, P=0.013). 12 months after surgery, the VAS score of the matched group (2.08±2.95) was less than that of the mismatched group (2.95±1.24), and the difference was statistically significant ( t=2.348, P=0.024). There was no significant difference in wrist range of motion, grip strength and DASH score between the two groups. Conclusion:The coronal bone structure matching of distal radius is about 45.0% in normal population. Early wrist dysfunction, limited pronation, and wrist pain may occur when the postoperative matching degree of the distal radius fracture is not within the normal range.

Objective To evaluate the clinical efficacy of multiple stents placement in the management of hilar cholangiocarcinoma,especially in the complex cases of which the hepatic ducts are invaded.Methods Forty-five consecutive patients with hilar cholangiocarcinoma were treated with percutaneous transhepatic placement of two or three self-expandable metallic endoprostheses.The cause of hilar obstructions in these patients were all cholangiocarcinoma,including Bismuth classification type Ⅱ(n 12 ),Ⅲa(n 17),Ⅲb(n 10),and Ⅳ(n 6).Two or 3 stents were placed in the configuration of T,Y or X over the strictures.Results Stent placement with 2 or 3 endoprostheses was successful in all patients.All patients showed significant decrease in serum bilirubin level.The mortality rate within 30 days of stent placement was 2.2%(1/45).The mean survival and stent patency times were 215.3 d(26— 516 d)and 181.5 d(26—473 d),respectively.Conclusion Deploying of multiple metallic stents is an effective method to treat complex hilar cholangiocarcinoma,especially for the cases of which hepatic ducts are invaded:the henatic ducts should be drained as much as nossible.

Objective To summarize the experience of the therapy and diagnosis of thoracictuberculosis. Methods Diagnosis and operation of 163 cases of thoracictuberculosis were analyzed.Results 163 cases of thoracic- tuberculosis were treated with focuspurge upon two weeks' anti-tuberculosis treatment.153 cases were cured upon one operation.10 cases suffered incision delayed healing and there were no recurrence cases.Conclusion Thoraeictu- berculosis was treated with focuspurge upon two weeks anti-tuberculosis treatment before operation.Complete purge of focus and postoperative compression band and residual cavity filled with music flap were important measures to prevent incision delayed healing and recurrence.

Objective To study the characteristics of the transcranial electric stimulation(TES)-elicited motor evoked potentials(MEP)recorded from different points of the diaphragm,and to determine the optimal record- ing site in the diaphragm.Methods One EEG electrode was placed subcutaneously in the midline of the skull as stimulation electrode and another EEG electrode was inserted into hard palate submucously as cathode in 30 male rats.And single square wave electrical pulse was used for stimulation.The stimulation intensity was 15 mA,the du- ration of the pulse was 0.2 ms,and the stimulating interval was 200 ms.The concentric needle electrode were insert- ed into the following eight sites:the medial,intermediate,lateral crura of the diaphragm,the junction of the posterior axilla line and the inferior border of the eleventh rib,the junction of the anterior axilla line and the inferior border of the ninth rib,the junction of the intermediate line of the clavicle and the inferior border of the eighth rib,the junction of the para-sternal line and the inferior border of the seventh rib,the pars sternalis.The MEP was recorded from each point,respectively.The optimal sites of the diaphragm MEP were found and recorded.Results The MEPs were re- corded from each of the 8 sites of the diaphragm in all the rats after TES.There was no statistically significant differ- ence among the latencies of every site.But the amplitude varied from 6.08?0.46 mV in maximum along the inferior border of the ninth rib at the anterior line of the axilla to the minimum of O.91?0.03 mV at the pars sternalis. Conclusion It is safer to insert the needle along the inferior border of the ninth rib at the anterior line of the axilla, because there was certain distant from the folding part of the pleura and the amplitude of the recorded wave at this point is maximal.