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Background: Targeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired. Results: We generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17-0.45%) that was used to knock in an approximately 5-kb long DNA fragment. Conclusion: We developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application. [ABSTRACT FROM AUTHOR]

Background: To evaluate the efficacy and safety of half-dose photodynamic therapy (PDT combined with ranibizumab for polypoidal choroidal vasculopathy (PCV). PCV is commonly treated with a combination of anti-vascular endothelial growth factor and standard-dose photodynamic therapy (PDT). Choroidal ischemia and visual loss can be resulted from the standard-dose PDT. Half-dose PDT has proved to produce similar results and safety profile in treating central serous chorioretinopathy. Half-dose PDT may offer an alternative for PCV cases where the damage to choroidal vasculature maybe less. Here, we report the efficacy of treating PCV cases with combination of ranibizumab and half-dose PDT. Methods: In this prospective, non-comparative, interventional case series, 19 treatment-naive eyes were treated with combined half-dose PDT and ranibizumab. All subjects were followed up for 12 months with measurement of best-corrected visual acuity (BCVA), central foveal thickness (CFT) by optical coherence tomography. Indocyanine green angiogram (ICG) was performed every 3-monthly, and subjects assessed in terms of polyp regression rates, changes in vision and central foveal thickness, need to repeat half-dose PDT. Subgroup analysis was performed based on ICG features. Results: Themean logMAR BCVA improved from 0.64 at baseline to 0.41 at 12 months. The mean CFT improved from 459.6mum at baseline to 384.2mum at 12 months. The difference between baseline BCVA and CFT and that at 12 months were statistically significant (both P = 0.03). Polyp regression rate after one half-dose PDT was 42.1 %. This was 61.5 % in the polyp-only group, while that in the branching-vascular-network (BVN) group was 0 % (P = <0.01). Conclusion: Half-dose PDT combined with intravitreal ranibizumab was able to induce high polyp regression rate in PCV cases that had one single polyp. [ABSTRACT FROM AUTHOR]

Background: To evaluate the efficacy and safety of half-dose photodynamic therapy (PDT combined with ranibizumab for polypoidal choroidal vasculopathy (PCV). PCV is commonly treated with a combination of anti-vascular endothelial growth factor and standard-dose photodynamic therapy (PDT). Choroidal ischemia and visual loss can be resulted from the standard-dose PDT. Half-dose PDT has proved to produce similar results and safety profile in treating central serous chorioretinopathy. Half-dose PDT may offer an alternative for PCV cases where the damage to choroidal vasculature maybe less. Here, we report the efficacy of treating PCV cases with combination of ranibizumab and half-dose PDT. Methods: In this prospective, non-comparative, interventional case series, 19 treatment-naive eyes were treated with combined half-dose PDT and ranibizumab. All subjects were followed up for 12 months with measurement of best-corrected visual acuity (BCVA), central foveal thickness (CFT) by optical coherence tomography. Indocyanine green angiogram (ICG) was performed every 3-monthly, and subjects assessed in terms of polyp regression rates, changes in vision and central foveal thickness, need to repeat half-dose PDT. Subgroup analysis was performed based on ICG features. Results: Themean logMAR BCVA improved from 0.64 at baseline to 0.41 at 12 months. The mean CFT improved from 459.6mum at baseline to 384.2mum at 12 months. The difference between baseline BCVA and CFT and that at 12 months were statistically significant (both P = 0.03). Polyp regression rate after one half-dose PDT was 42.1 %. This was 61.5 % in the polyp-only group, while that in the branching-vascular-network (BVN) group was 0 % (P = <0.01). Conclusion: Half-dose PDT combined with intravitreal ranibizumab was able to induce high polyp regression rate in PCV cases that had one single polyp. [ABSTRACT FROM AUTHOR]

Background: A growing number of studies reported the connection between the level of serum ferritin (SFL) and non-alcoholic fatty liver disease (NAFLD). However, such connection was still disputable. The aim of our meta-analysis was to estimate SFL between the groups as below: patients with NAFLD against control group; non-alcoholic steatohepatitis (NASH) patients against control group; non-alcoholic fatty liver (NAFL) patients against a control group and NASH patients vs NAFL patients., Methods: We screened the studies in PubMed, EMBASE, the Cochrane Database and the Cochrane Central register controlled trials from the beginning to July 10, 2016 to find the studies indicated the connection between SFL and NAFLD (NAFL and/or NASH). Fourteen published studies which evaluate the SFL in NAFLD patients were selected., Results: Higher SFL was noticed in NAFLD patients against control group (standardized mean difference [SMD] 1.01; 95% CI 0.89, 1.13), NASH patients against control group (SMD 1.21; 95% CI 1.00, 1.42), NAFL patients against control group (SMD 0.51; 95% CI 0.24, 0.79) and NASH patients against NAFL patients (SMD 0.63; 95% CI 0.52, 0.75). These results remained unaltered actually after the elimination of studies which were focused on paediatric or adolescent populations. Higher SFL was presented in NAFLD patients against the control group (SMD 1.08; 95% CI 0.95, 1.20) in adults and NASH patients against NAFL patients in adults (SMD 0.74; 95% CI 0.62, 0.87). The connection between SFL and NASH against NAFL group in paediatric or adolescent populations was observed inconsistently (SMD 0.10; 95% CI -0.18, 0.38)., Conclusions: The level of SFL was elevated in patients with NAFLD (NAFL and/or NASH) compared with the controls. Compared with NAFL, The level of SFL was increased in NASH. The result remained unaltered actually after the elimination of studies focused on paediatric or adolescent populations.

Background: Recently genome-wide association studies identified that NCAN rs2228603 polymorphism was associated with non-alcoholic fatty liver disease (NAFLD) mainly in subjects of European ancestry. While no research have been conducted to demonstrate the relationship between NCAN rs2228603 and NAFLD in Chinese Han adults. The aim of this study was to investigate whether NCAN rs2228603 is associated with NAFLD in Chinese population., Methods: Gene NCAN rs2228603 was genotyped in 182 patients with NAFLD and 195 healthy controls. The expression of NCAN was tested according to polymerase chain reaction analysis (PCR) and serum lipids were performed by biology techniques., Results: No significant difference was found in genotype and allele frequencies of NCAN rs2228603 between the NAFLD group and the controls (P > 0.05). Subjects with the NCAN rs2228603 CT genotype showed a higher level of alkaline phosphatase (AKP) (P = 0.017) and a higher high-density lipoprotein (HDL) (P < 0.05)., Conclusions: Our study for the first time identified that the gene NCAN rs2228603 is not a risk factor for the incidence of NAFLD in Chinese population. Also we found the dual and opposite role of T variant in protecting liver with a higher level of HDL and conferring risk for liver damage with a higher level of AKP., Trial Registration: Chinese Clinical Trial Register.gov Identifier: ChiCTR-ROC-15006447 .

Background: Cardiovascular disease (CAD) responsible and nonalcoholic fatty liver disease (NAFLD) are both metabolic diseases, and they are mostly influenced by genetic factors. The aim of our study is to evaluate the relationship between angiotensin II type-1 receptor (AGTR1) gene rs3772622 polymorphisms and the risk of developing coronary artery disease (CAD) in Chinese patients with NAFLD., Methods: Genotype for AGTR1 rs3772622 in 574 NAFLD patients with CAD or 589 NAFLD patients without CAD, 332 CAD patients exclude NAFLD and 338 health control subjects were determined by sequencing and polymerase chain reaction analysis. Relevant statistical methods were employed to analyze the genotypes, alleles and the clinical date. Inter-group differences and associations were assessed statistically using t-tests and Chi square and logistic analyses. The relative risk of AGTR1 rs3772622 for NAFLD was estimated by logistic regression analysis., Results: No significant difference in genotype and allele frequency of AGTR1 rs3772622 was found between the NAFLD without CAD population and the controls (P > 0.05). However, makeable difference was found when compared the CAD in patients with NAFLD and CAD free NAFLD patients (P < 0.001 OR = 2.09). Similarly, significant difference was found in AGTR1 rs3772622 genotype distribution between the groups of CAD patients and control (P = 0.046 OR = 1.71)., Conclusions: AGTR1 rs3772622 gene polymorphism was not associated with the risk of NAFLD, but could increase the risk of NAFLD patients suffering from CAD in the Chinese Han population. Deeply mechanisms underlying the association between AGTR1 rs3772622 gene polymorphism and the risk of CAD in NAFLD patients need more research.

Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease which represents a wide spectrum of hepatic damage. Several studies have reported that NAFLD is a strong independent risk factor for coronary artery disease (CAD). And patients with NAFLD are at higher risk and suggested undergoperiodic cardiovascular risk assessment. Cardiovascular disease (CVD) is responsible for the main cause of death in patients with NAFLD, and is mostly influenced by genetic factors. Both NAFLD and CAD are heterogeneous disease. Common pathways involved in the pathogenesis of NAFLD and CAD includes insulin resistance (IR), atherogenic dyslipidemia, subclinical inflammation, oxidative stress, etc. Genomic characteristics of these two diseases have been widely studied, further research about the association of these two diseases draws attention. The gene polymorphisms of adiponectin-encoding gene (ADIPOQ), leptin receptor (LEPR), apolipoprotein C3 (APOC3), peroxisome proliferator-activated receptors (PPAR), sterol regulatory elementbinding proteins (SREBP), transmembrane 6 superfamily member 2 (TM6SF2), microsomal triglyceride transfer protein (MTTP), tumor necrosis factors-alpha (TNF-α) and manganese superoxide dismutase (MnSOD) have been reported to be related to NAFLD and CAD. In this review, we aimed to provide an overview of recent insights into the genetic basis of NAFLD and CAD.

Background: HLA-DRB1 allele polymorphisms have been reported to be associated with hepatocellular carcinoma susceptibility, but the results of these previous studies have been inconsistent. The purpose of the present study was to explore whether specific HLA-DRB1 alleles (DRB1*07, DRB1*12, DRB1*15) confer susceptibility to hepatocellular carcinoma., Methods: Case-control studies on HLA-DRB1 alleles association with HCC were searched up to January 2010 through a systematic review of the literature. The odds ratios (ORs) of HLA-DRB1 allele distributions in patients with hepatocellular carcinoma were analyzed against healthy controls. The meta-analysis software REVMAN 5.0 was applied for investigating heterogeneity among individual studies and for summarizing all the studies. Meta-analysis was performed using fixed-effect or random-effect methods, depending on absence or presence of significant heterogeneity., Results: Eight case-control studies were included in the final analysis. Among the 3 HLA-DRB1 alleles studied, DRB1*07 and DRB1*12 were significantly associated with the risk of HCC in the whole populations (OR = 1.65, 95% CI: 1.08-2.51, P = 0.02 and OR = 1.59, 95% CI: 1.09-2.32, P = 0.02, respectively). No significant association was established for DRB1*15 allele with HCC in the whole populations. Subgroup analysis by ethnicity showed that DRB1*07, DRB1*12 and DRB1*15 alleles significantly increased the risk of hepatocellular carcinoma in Asians (OR = 2.10, 95% CI: 1.06-4.14, P = 0.03; OR = 1.73, 95% CI: 1.17-2.57, P = 0.006 and OR = 2.88, 95%CI: 1.77-4.69, P <0.001, respectively)., Conclusion: These results support the hypothesis that specific HLA-DRB1 alleles might influence the susceptibility of hepatocellular carcinoma. Large, multi-ethnic confirmatory and well designed studies are needed to determine the host genetic determinants of hepatocellular carcinoma.