Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster.

Although the Cas9 protein from Streptococcus pyogenes (SpCas9) is the most widely used clustered regularly interspaced short palindromic repeats (CRISPR) variant in genome engineering experiments, it does have certain limitations. First, the stringent requirement for the protospacer adjacent motif (PAM) sequence limits the target DNA that can be manipulated using this method in insects. Second, its complementarity specifications are not very stringent, meaning that it can sometimes cause off-target effects at the target site. A recent study reported that an evolved SpCas9 variant, xCas9(3.7), with preference for various 5'-NG-3' PAM sequences not only has the broadest PAM compatibility but also has much greater DNA specificity and lower genome-wide off-target activity than SpCas9 in mammalian cells. Here we applied the CRISPR/xCas9 system to target the white gene in Drosophila melanogaster, testing the genome-editing efficiency of xCas9 at different PAM sites. On the GGG PAM site, xCas9 showed less activity than SpCas9. For the non-NGG PAM site TGA, xCas9 could produce DNA cleavage and indel-mediated disruption on the target gene. However, for other non-NGG PAM sites, xCas9 showed no activity. These findings show that the evolved Cas9 variant with broad PAM compatibility is functional in Drosophila to induce heritable gene alterations, increasing the targeting range for the applications of genome editing in insects.

The effect of borneol on the concentration of meropenem in rat brain and blood.

Meropenem is a carbapenem antibiotic with a wide spectrum of activity against both Gram-positive and Gram-negative bacteria. Because of its clinical efficacy, meropenem is an excellent choice for the treatment of serious infections in both adults and children. The knowledge of tissue concentrations of antibiotic in an infection site is valuable for the prediction of treatment outcome. The aim of the present study is to investigate the effect of borneol on the concentration of meropenem in rat brain and blood and to find the potential relationships of the combined use of medicine and traditional Chinese medicine. Analysis of meropenem in the dialysates was achieved using the microdialysis technique and HPLC. At 40 min after the administration of an intraperitoneal injection of meropenem, the concentration of meropenem in brain in borneol+meropenem group was 2.25 (0.35) µg ml(-1), which was significantly higher than that in meropenem group [1.20 (0.12) µg ml(-1); P < 0.01]. Within 80 min of drug administration, the AUCbrain/AUCblood (area under the curve, AUC) in the borneol+meropenem group was 1.2 times that of the meropenem group. Borneol can increase the concentration of meropenem in the cerebrospinal fluid, but has no influence on its blood concentration. This study represents a successful application of the microdialysis technique, which is an effective method for the study of pharmacokinetics of meropenem.

[Significance of double-oblique multiplanar reconstruction of the treatment decision-making in sacral fracture with sacral neurological damage].

OBJECTIVE: To study diagnostic and surgical significance of the multiplanar reconstruction (MPR) of spiral CT in sacral fracture with sacral neurological damage. METHODS: From April 2007 to April 2009, 10 cases of sacral fracture with sacral neurological damage in Shengjing Hospital of China Medical University were examined by double-oblique MPR of sacrum to show a total length of the sacral neural tube, and observe the course of sacral neural tube and the relationship between fracture and the neural tube. There were 7 males and 3 females, aged 30 - 55 years. The time from injury to hospitalization varied from 1 day to 1 months. The injury was caused by traffic accident in 6 cases, smash of heavy object in 3 cases and crush in 1 cases. All patients were examined with double-oblique MPR (coronal oblique 45 degrees and 30 degrees oblique-shaped bit lost)to display the course of sacral neural tube and the condition of clearly, which was confirmed by operation and clinical validation. RESULTS: The clinical manifestations and international standards for Neurological Classification of Spinal Cord Injury recommended by American Spinal Injury Association International Spinal Cord Society were the basis for clinical diagnose. Nerve injury diagnosed by clinical manifestation were S1 (6 cases), S2 (2 cases), S1 and S2 (2 cases). After double-oblique MPR in patients, we found 3 patients have fractures in sacral neural tube of S1, 1 patients in S2, and 2 patients both in S1 and S2, whom recovered average 12 month after operation (S1 and S2 nerve were pressed by fractures according to operating observation); And there were no fractures in sacral neural tube of other patients (3 cases in S1, 1 cases in S2), these were contund, whom were recovered average 13 month after expectant treatment. CONCLUSIONS: Double-oblique MPR of sacrum has important clinical significance in diagnosis of nature and location with sacral nerve injury, as well as surgical planning. It can be used as routine examination of patients with sacral fracture.

[Experimental expression of HIF-1α in rabbit adipose-derived mesenchymal stem cells by dynamic compression plus IGF-1 gene transfection].

OBJECTIVE: To investigate the effects on HIF-1α expression of rabbit adipose-derived mesenchymal stem cells (ADSCs) by dynamic compression plus IGF-1 gene transfection and explore the mechanism of promoting chondrogenesis by HIF-1α. METHODS: The ADSCs were harvested after the digestion of typeIcollagenase and transfection with pcDNA3.1-IGF-1. And then the cells were seeded onto chitosan/gelatin scaffolds with a density of 5 × 10(7) cells/ml and divided into groups: Group A (control), non-transfected ADSCs; Group B (IGF-1), hIGF-1 gene transfected ADSCs; Group C (loading), untransfected ADSCs with stimulation of compressive loading; Group D (loading + IGF-1), hIGF-1 gene transfected ADSCs and loading stimulation. The dynamic compression was carried out with a bio-reactor at a frequency of 0.1 Hz and a sinusoidal strain amplitude of 2% (2% at 0.1 Hz). The dynamic load was performed every 20 minutes, 4 hours daily. After 7 days, morphological observation was performed. The MTT assay was used to detect the cell proliferation. Meanwhile the CM-DiI cell-labeling solution was used to observe the distribution of cells. The total amount of GAG and the expression of related genes of IGF-1, HIF-1α, type II collagen (COL II) and Sox-9 were quantified. RESULTS: The best morphology was found in loading+ IGF-1 group. The results of proliferating capacity were: Group A < C < B < D (P < 0.01); Dil fluorescence showed that the cells were well-distributed in Group D. Meanwhile, the content of total GAG and the expression of related genes demonstrated: Group A < C < B < D (P < 0.01). And the combined effect was more significant than either alone. CONCLUSION: In three-dimensional culture conditions, dynamic compression plus IGF-1 transfection can significantly enhance the level of autocrine IGF-1. Both of two experimental intervention factors can significantly up-regulate the mRNA level of HIF-1α. And they exert a synergistic effect. HIF-1α plays an important role in promoting the chondrogenesis and the synthesis and secretion of extra-cellular matrix.

[Study on bone mesenchymal stem cells transfected by polyethylene glycol/bone morphogenetic protein-2].

OBJECTIVE: Polyethylene glycol/bone morphogenetic protein-2 (PEG/BMP-2) nanoparticles were transfected into Rabbit bone mesenchymal stem cells (rBMSCs) and the expression of BMP-2 was detected. METHODS: Dissociated rBMSCs were primarily cultured in vitro and BMP-2 gene was transfected into rBMSCs by PEG/BMP-2 nanoparticals and lipofectamine, respectively. The efficiency of transfection was detected by flow cytometry and the expression of BMP-2 was detected by Western Blot and real time RT-PCR. RESULTS: PEG/BMP-2 nanoparticals were successfully synthesized and transfected into rBMSCs. Compared with the lipofectamine transfection group, PEG/BMP-2 transfection group had higher efficiency and higher BMP-2 expression. CONCLUSION: PEG/BMP-2 nanoparticals transfected rBMSCs highly expressed BMP-2,which provided novel strategies for the treatment of bone defect.