Development of CRISPR technology and its application in bone and cartilage tissue engineering / 南方医科大学学报

The CRISPR/Cas9 system, consisting of Cas9 nuclease and single guide RNA (sgRNA), is an emerging gene editing technology that can perform gene reprogramming operations such as deletion, insertion, and point mutation on DNA sequences targeted by sgRNA. In addition, CRISPR/dCas9 (a mutant that loses Cas9 nuclease activity) still retains the ability of sgRNA to target DNA. The fusion of dCas9 protein with transcriptional activator (CRISPRa) can activate the expression of the target gene, and fusion transcriptional repressors (CRISPRi) can also be used to suppress target gene expression. Efficient delivery of the CRISPR/Cas9 system is one of the main problems limiting its wide clinical application. Viral vectors are widely used to efficiently deliver CRISPR/Cas9 elements, but non-viral vector research is more attractive in terms of safety, simplicity, and flexibility. In this review, we summarize the principles and research advances of CRISPR technology, including CRISPR/ Cas9 delivery vectors, delivery methods, and obstacles to the delivery, and review the progress of CRISPR-based research in bone and cartilage tissue engineering. Finally, the challenges and future applications of CRISPR technology in bone and cartilage tissue engineering are discussed.

Species Diversity and Seasonal Distribution of Culicoides spp. (Diptera: Ceratopogonidae) in Jeju-do, Republic of Korea

Biting midges belonging to the genus Culicoides (Diptera: Ceratopogonidae) were collected by Mosquito Magnet(R) and black light traps at 5 sites on Jeju-do, Republic of Korea (Korea), from May-November 2013 to determine species diversity and seasonal distribution. A total of 4,267 specimens were collected, of which 99.9% were female. The most common species was Culicoides tainanus (91.8%), followed by C. lungchiensis (7.2%) and C. punctatus (0.6%), while the remaining 4 species accounted for <0.5% of all Culicoides spp. that were collected. High numbers of C. tainanus were collected in May, followed by decreasing numbers through August, and then increasing numbers through November when surveillance was terminated. Peak numbers of C. lungchiensis were collected during September, with low numbers collected from May-August and October-November. The presence of C. lungchiensis in Korea was confirmed by morphological and molecular analyses.

Hepatitis C virus strain JFH1 down-regulates expression of growth arrest and DNA damage-inducible gene 45a in human hepatoma Huh7.5.1 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of hepatitis C virus (HCV) strain JFH1 on expression of the human gene, growth arrest and DNA damage-inducible gene 45 alpha (GADD45a), in infected hepatoma cells.</p><p><b>METHODS</b>HCV JFH1 RNA-containing supernatants were used to infect the human hepatoma cell line, Huh7.5.1; infection was confirmed by Western blot detection of the HCV-encoded non-structural 5A (NS5A) protein and core protein. Infection-induced changes in GADD45a mRNA and protein expressions were measured by real time PCR using SYBR Green and Western blotting, respectively. Significance of differences between the levels detected in JFH1-infected or uninfected Huh7.5.1 cells was analyzed by single factor analysis of variance testing.</p><p><b>RESULTS</b>The HCV infection system was successfully established, as evidenced by expression of NS5A protein and core protein. The GADD45a mRNA and protein levels were significantly down-regulated in JFH1-infected Huh7.5.1 cells, by 0.57+/-0.09 and 0.28+/-0.03, respectively, as compared to levels in uninfected Huh7.5.1 cells (F values were 75.407 and 560.04, respectively; P less than 0.01).</p><p><b>CONCLUSION</b>HCV inhibits the mRNA transcription and protein expression of host GADD45a, which may contribute to the pathogenesis of hepatocellular carcinoma caused by HCV infection.</p>

Efficiency comparison of preparing nano-scale microbubbles by oscillation and sonication / 中华超声影像学杂志

Objective To compare the efficiency of oscillation with sonication in preparing nano-scale microbubbles (NBs).Methods Nano-scale microbubbles were prepared using oscillation and sonication respectively,and then compared the NBs' size,size distribution,concentrations and time-consumption of the two methods.Results The sizes of nanobubbles prepared by sonication and oscillation were (373.88 ±18.43)nm and (360.74 ± 14.39)nm,respectively.There was no significant difference in size between the two methods (P =0.523).The polidispersity was larger in sonication before centrifugation,there was significant difference between the two methods (P ＜0.001).The concentration of nanobubbles prepared by oscillation was (1.48 ± 0.15) × 1010,which was higher than that by oscillation [(8.07 ± 0.62) × 108],there was significant difference between the two methods (P ＜ 0.001).The consuming time was shorter in oscillation,the difference was significant when compared with sonication (P ＜0.001).Conclusions Both two methods can successfully prepare NBs.Compared with sonication,oscillation can effectively produce NBs with smaller polidispersity,higher concentration and shorter time-consumption.

HCV NS5A protein down-regulates hepcidin gene expression and increases hepatic intracellular iron storage / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether the nonstructural protein 5A (NS5A) encoded by the hepatitis C virus RNA genome affects the expression of hepcidin gene.</p><p><b>METHODS</b>HCV NS5A expression plasmid (pCN5A) and pRc/CMV were transfected into QSG7701 cells individually, RT-PCR was employed to detect the HCV NS5A and hepcidin mRNA transcription. Western blot was used for detection of HCV NS5A and hepcidin proteins. Iron was stained to evaluate the intracellular iron level.</p><p><b>RESULTS</b>HCV NS5A plasmid was successfully transfected into QSG7701 cells, which was evidenced by HCV NS5A mRNA and protein from the transfected cells. The hepcidin mRNA relative quantification in untransfected cells, pRc/CMV transfected cells and pCNS5A transfected cells were 0.711+/-0.049, 0.718+/-0.052 and 0.264+/-0.030 respectively. The transcription of hepcidin mRNA decreased remarkably in the cells transfected with pCNS5A plasmid as compared to the untransfected cells and pRc/CMV transfected cells (P less than 0.01). The level of hepcidin protein expression was found also significantly lower in the pCN5A plasmid transfected cells as compared to the untransfected cells and pRc/CMV transfected cells. The intracellular iron staining was remarkably higher in the pcNS5A transfected cells than untransfected or pRc/CMV transfected cells.</p><p><b>CONCLUSIONS</b>HCV NS5A inhibits the transcription of hepcidin mRNA and expression of hepcidin protein, inducing hepatic intracellular iron storage.</p>