Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T(CT) or A→G(AG) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F_0 are mosaic, and breeding of F_1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing(GVBE) to produce point mutant F_0 porcine embryos by editing the maternal alleles during the GV to MⅡ transition. Injection of cytosine base editor 3(BE3) mRNA and X-linked Dmdspecific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MⅡ oocytes developed into blastocysts after parthenogenetic activation(PA) or in vitro fertilization(IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4%±2.1%. There 40%–78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only CT mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five(5/19) F_0 male embryos are homozygous and three of them were Dmd precise CT mutation. Nine(9/19) female IVF embryos had two different alleles including a WT and a CT mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F_0 embryos with maternal point mutated alleles in a single step.     

Alternate partial root-zone irrigation induced dry/wet cycles of soils stimulate N mineralization and improve N nutrition in tomatoes

Given the same amount of irrigation volume, applying alternate partial root-zone irrigation (PRI) has improved crop N nutrition as compared to deficit irrigation (DI), yet the mechanisms underlying this effect remain unknown. Therefore, the objective of this study was to investigate whether PRI induced soil dry/wet cycles facilitate soil organic N mineralization hereby contributing to the improvement of N nutrition in tomatoes. The plants were grown in split-root pots in a climate-controlled glasshouse and were subjected to PRI and DI treatments during early fruiting stage. ¹⁵N-labeled maize residues were incorporated into the soils. Results showed that PRI resulted in 25% higher net ¹⁵N mineralization than did DI, indicating that the enhanced mineralization of soil organic N alone could account for the 16% increase of N accumulation in the PRI than in the DI plants. The higher net N mineralization under PRI was coincided with an intensified soil microbial activity. In addition, even though soil chloroform fumigation labile carbon (CFL-C, as an index of microbial biomass) was similar for the two irrigation treatments, a significant increase of chloroform fumigation labile nitrogen (CFL-N) was found in the PRI wetting soil. Consequently, the C:N ratio of the chloroform fumigation labile pool was remarkably modified by the PRI treatment, which might indicate physiological changes of soil microbes or changes in labiality of soil organic C and N due to the dry/wet cycles of soils, altering conditions for net N mineralization. Moreover, in both soil compartments PRI caused significantly less extractable organic carbon (EOC) as compared with DI; whilst in the PRI wetting soil significantly higher extractable organic nitrogen (EON) was observed. A low EOC:EON ratio in the PRI wetting soil may indicate an increasing net mineralization of the organic N as a result of microbial metabolism. Conclusively, PRI induced greater microbial activity and higher microbial substrates availability are seemingly responsible for the enhanced net N mineralization and improved N nutrition in tomato plants.     

长白山不同演替阶段森林植物功能性状及其与地形因子间的关系

植物功能性状是近年来生态学研究的热点。不同种群功能性状的差异决定竞争优势,导致群落结构和性质发生改变,进而演替。以长白山森林演替过程中4个阶段的典型群落为研究对象,分析了长白山地区森林不同层次优势种的茎叶功能性状之间的关系、功能性状与地形因子的关系,并对不同演替阶段群落功能性状进行了比较。结果发现,比叶面积(SLA)与叶氮浓度(LNC)和茎磷浓度(SPC)正相关,与叶干物质质量(LDMC)负相关;叶厚度(LT)与LDMC负相关;LNC与LPC、SNC和LDMC呈正相关;茎氮浓度(SNC)与茎磷浓度(SPC)正相关;茎组织密度(STD)与LNC、SNC正相关。通过灰色关联度分析发现,海拔对SLA、LNC、LPC、STD、SPC影响最大;坡向对于LT、LDMC影响最大;坡位对SNC影响最大。各演替群落间的乔木层功能性状均有显著差异,灌木层的功能性状无显著差异,而草本层除了LDMC、LPC、SNC有显著差异外,其它指标均无显著差异;SLA、STD、LNC随演替的进行呈现明显增加趋势,LDMC、LPC随演替呈减少的趋势。LDMC和SLA是能体现群落演替差异的主要功能性状。     

MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis

Background

As an important factor affecting meat quality, intramuscular fat (IMF) content is a topic of worldwide concern. Emerging evidences indicate that microRNAs play important roles in adipocyte differentiation. However, miRNAome has neither been studied during porcine intramuscular preadipocyte differentiation, nor compared with subcutaneous preadipocytes. The objectives of this study were to identify porcine miRNAs involved in adipogenesis in primary preadipocytes, and to determine whether intramuscular and subcutaneous adipocytes differ in the expression and regulation of miRNAs.

Results

miRNAomes in primary intramuscular and subcutaneous adipocytes during differentiation were first sequenced using the Solexa deep sequencing method. The sequences and relative expression levels of 224 known (98.2% in miRbase 18.0) and 280 potential porcine miRNAs were identified. Fifty-four of them changed in similar pattern between intramuscular vascular stem cells (IVSC) and subcutaneous vascular stem cells (SVSC) differentiation, such as miR-210, miR-10b and miR-99a. Expression levels of 10 miRNAs were reversely up-or down-regulated between IVSC and SVSC differentiation, 19 were up-or down-regulated only during IVSC differentiation and 55 only during SVSC differentiation. Additionally, 30 miRNAs showed fat-depot specific expression pattern (24 in cells of intramuscular origin and 6 in cells of subcutaneous origin). These adipogenesis-related miRNAs mainly functioned by targeting similar pathways in adipogenesis, obesity and syndrome.

Conclusion

Comparison of miRNAomes in IVSC and SVSC during differentiation revealed that many different miRNAs are involved in adipogenesis, and they regulate SVSC and IVSC differentiation through similar pathways. These miRNAs may serve as biomarkers or targets for enhancing IMF content, and uncovering their function in IMF development will be of great value in the near future.     

MicroRNA-344 inhibits 3T3-L1 cell differentiation via targeting GSK3β of Wnt/β-catenin signaling pathway

Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of complex events in which microRNAs might play an essential role. In this study, we found that the overexpression of microRNA-344 (miR-344) inhibits 3T3-L1 cell differentiation and decreases triglyceride accumulation after MDI stimulation. We demonstrated that miR-344 directly targets the 3′ UTR of GSK3β (Glycogen synthase kinase 3 beta). Knockdown of GSK3β with siRNA results in inhibiting 3T3-L1 differentiation, while its overexpression restores the effect of miR-344. In addition, miR-344 elevates the level of active β-catenin, which is the downstream effector of GSK3β in the Wnt/β-catenin signaling pathway. These data indicate that miR-344 inhibits adipocyte differentiation via targeting GSK3β and subsequently activating the Wnt/β-catenin signaling pathway.     

Association of CYP2A6*4 with Susceptibility of Lung Cancer: A Meta-Analysis

ObjectivesTo assess the association between the variant of Cytochrome P450 2A6 whole gene deletion (CYP2A6*4) polymorphism and risk of lung cancer.MethodsTwo investigators independently searched the PubMed, Elsevier, EMBASE, Web of Science, Wiley Online Library and Chinese National Knowledge Infrastructure (CNKI). Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) for CYP2A6*4 and lung cancer were calculated in a fixed-effects model (the Mantel-Haenszel method) and a random-effects model (the DerSimonian and Laird method) when appropriate.ResultsThis meta-analysis included seven eligible studies, which included 2524 lung cancer cases and 2258 controls (cancer–free). Overall, CYP2A6*4 was associated with the risk of lung cancer (allele*4 vs. allele non-*4, pooled OR  = 0.826, 95% CI  = 0.725−0.941, P-value  = 0.004). When stratifying for population, significant association was observed in Asian (additive model, pooled OR  = 0.794, 95% CI  = 0.694−0.909, P-value  = 0.001; dominant model, pooled OR  = 0.827, 95% CI  = 0.709−0.965, P-value  = 0.016; recessive model (pooled OR  = 0.444, 95% CI  = 0.293−0.675, P-value <0.0001). In the overall analysis, a comparably significant decrease in the frequency of *4/*4 genotype was detected between cases and controls in Asian while no *4/*4 genotype was detected in Caucasian in collected data.ConclusionThis meta-analysis suggests that the CYP2A6*4 polymorphism is associated with susceptibility of lung cancer in Asian. The whole gene deletion of CYP2A6 may decrease the risk of lung cancer in Asian samples.