Receptor-mediated delivery of engineered nucleases for genome modification

Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.     

Association between variants of EXT2 and type 2 diabetes: a replication and meta-analysis

Recent publications have found an association between variants of exostosin 2 (EXT2) gene and the risk of type 2 diabetes in some population but not in others. In an attempt to address these inconsistencies, we investigated EXT2 variants in two independent cohorts, and conducted a literature-based meta-analysis. Through regression model, we assessed the relationship between the EXT2 single nucleotide polymorphisms (SNPs) (rs3740878, rs11037909 and rs1113132) and the risk of type 2 diabetes in Han Chinese population, including a total of 2,533 cases and 2,643 controls. We combined our data with that from previously published studies and performed a meta-analysis to evaluate the effect size of the gene. Consistent with some studies, we found marginal association for the rs3740878 (OR = 1.07, 95 % CI = 0.99, 1.16, p = 0.09), rs11037909 (OR = 1.07, 95 % CI = 0.99, 1.16, p = 0.08), and rs1113132 (OR = 1.08, 95 % CI = 1.00, 1.17, p = 0.06) in our 2 cohorts. Meta-analysis, comprising 9,224 type 2 diabetes and 10,484 controls, revealed that three SNPs (rs3740878, rs11037909 and rs1113132) in EXT2 were significantly associated with type 2 diabetes (ORs range from 1.06 to 1.07, p = 0.038, p = 0.008 and p = 0.005, respectively). Variation in the EXT2 locus appears to be associated with a small increase in the risk of type 2 diabetes. However, well-designed prospective studies with larger sample size and more ethnic groups are needed to further validate the results.     

Effect of GABA Receptor Agonists or Antagonists Injected Spinally on the Blood Glucose Level in Mice

The possible roles of gamma-amino butyric acid (GABA) receptors located in the spinal cord for the regulation of the blood glucose level were studied in ICR mice. We found in the present study that intrathecal (i.t.) injection with baclofen (a GABA_B receptor agonist; 1–10 μg/5 μl) or bicuculline (a GABA_A receptor antagonist; 1–10 μg/5 μl) caused an elevation of the blood glucose level in a dose-dependent manner. The hyperglycemic effect induced by baclofen was more pronounced than that induced by bicuculline. However, muscimol (a GABA_A receptor agonist; 1–5 μg/5 μl) or phaclofen (a GABA_B receptor antagonist; 5–10 μg/5 μl) administered i.t. did not affect the blood glucose level. Baclofen–induced elevation of the blood glucose was dose-dependently attenuated by phaclofen. Furthermore, i.t. pretreatment with pertussis toxin (PTX; 0.05 or 0.1 μg/5 μl) for 6 days dose-dependently reduced the hyperglycemic effect induced by baclofen. Our results suggest that GABA_B receptors located in the spinal cord play important roles for the elevation of the blood glucose level. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by baclofen. Furthermore, inactivation of GABA_A receptors located in the spinal cord appears to be responsible for tonic up-regulation of the blood glucose level.     

Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.     

Association of the variant rs2243421 of human DOC-2/DAB2 interactive protein gene (hDAB2IP) with gastric cancer in the Chinese Han population

Human DOC-2/DAB2 interactive protein (hDAB2IP) gene is a novel member of the Ras GTPase-activating family and has been demonstrated to be a tumor-suppressor gene that inhibits cell survival and proliferation and induces cell apoptosis. It was reported that the expression level of hDAB2IP in gastric cancer tissue was highly correlated with tumor progression, however, whether hDAB2IP genetic variants are associated with the risk of gastric cancer remains yet unknown. In this case–control study, we conducted a genetic analysis for hDAB2IP variants in 311 patients with gastric cancer and 425 controls from the Chinese Han population. We found that the SNP rs2243421 of hDAB2IP gene with the minor allele C significantly revealed strong association with decreased gastric cancer susceptibility (P = 0.007, adjusted odds ratio [OR] = 0.734, 95%CI = 0.586–0.919). Haplotypes rs2243421 and rs10985332 (HaploType: CC, P = 0.012, aOR = 0.760) and haplotypes rs2243421 and rs555996 (HaploType: CG, P = 0.034, aOR = 0.788) represented the decreased risk of gastric cancer, respectively. On the contrary, rs2243421 and rs555996 showed an elevated susceptibility (HaploType: TG, P = 0.010, aOR = 1.320). Our results for the first time provided new insight into susceptibility factors of hDAB2IP gene variants in carcinogenesis of gastric cancer.     

我国云南食用牛肝菌的DNA条形码研究

iFlora是结合传统分类学与DNA测序和信息技术,通过系列关键技术进行集成,构建便捷、准确识别物种和掌握相关数字化信息的新一代智能植物志。iFlora研发中首要和迫切的任务之一就是寻找适合于大多数植物和经济蘑菇的标准DNA条形码序列。为筛选适合大型经济蘑菇的DNA条形码,本研究以云南食用牛肝菌为例,选取野生食用菌市场上常见的、被当地人认为的4“种”牛肝菌为研究对象,利用核糖体大亚基（nrLSU）、翻译延长因子1-α（tefl-α）、RNA聚合酶II大亚基（rpbl）和RNA聚合酶II的第二个大亚基（rpb2）四个DNA序列,使用真核生物通用引物进行扩增、测序和测试。研究发现这4“种”样品实际上代表了12个独立的物种,进一步研究表明4个候选片段的扩增和测序成功率均为100％,且不存在种问和种内变异的重叠。4个片段的物种分辨率均较高,但与nrLSU相比,rpbl、tefl-α和rpb2具有更为明显的条形码间隔。鉴于rpbl比tefl-α和rpb2具有更高的种间变异和较低的种内变异,建议将rpbl作为牛肝菌属的核心条形码,tefl-α和rpb2可作为该属的辅助条形码。     

The application of super paramagnetic iron oxide-labeled mesenchymal stem cells in cell-based therapy

Mesenchymal stem cell (MSC)-based therapy has great potential for tissue regeneration. However, being able to monitor the in vivo behavior of implanted MSCs and understand the fate of these cells is necessary for further development of successful therapies and requires an effective, non-invasive and non-toxic technique for cell tracking. Super paramagnetic iron oxide (SPIO) is an idea label and tracer of MSCs. MRI can be used to follow SPIO-labeled MSCs and has been proposed as a gold standard for monitoring the in vivo biodistribution and migration of implanted SPIO-labeled MSCs. This review discusses the biological effects of SPIO labeling on MSCs and the therapeutic applications of local or systemic delivery of these labeled cells.