Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors.Safe harbor loci in the animal genome enable consistent o...Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors.Safe harbor loci in the animal genome enable consistent overexpression of foreign genes,without side effects.However,relatively few safe harbor loci are available in pigs,a fact which has impeded the development of multi-transgenic pig research.We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain(COL1A1)gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system.After the knock-in of a 2A peptide-green fluorescence protein(2A-GFP)transgene in the last codon of COL1A1 in multiple porcine cells,including porcine kidney epithelial(PK15),porcine embryonic fibroblast(PEF)and porcine intestinal epithelial(IPI-2I)cells,quantitative PCR(qPCR),Western blotting,RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus.The qPCR results showed that the GFP knock-in had no effect(P=0.29,P=0.66 and P=0.20 for PK15,PEF and IPI-2I cells,respectively)on the mRNA expression of COL1A1 gene.Similarly,no significant differences(P=0.64,P=0.48 and P=0.80 for PK15,PEF and IPI-2I cells,respectively)were found between the GFP knock-in and wild type cells by Western blotting.RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation(P<2.2e–16)with that of the wild type cells,indicating that the GFP knock-in did not alter the global expression of endogenous genes.Furthermore,the CCK8 assay showed that the GFP knock-in events had no adverse effects(P_(24)h=0.31,P_(48)h=0.96,P_(72)h=0.24,P_(96)h=0.17,and P_(120)h=0.38)on cell proliferation of PK15 cells.These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment.展开更多
为了探明生物质炭对华北平原土壤氨挥发的影响,以该区域4种典型土壤(水稻土、砂姜黑土、褐土、潮土)为研究对象进行微区试验,设置了对照(CK)、单施化肥(NPK)、单施生物质炭(BC)、化肥配施生物质炭(BC+NPK)4个处理,于冬小麦生育前期观测...为了探明生物质炭对华北平原土壤氨挥发的影响,以该区域4种典型土壤(水稻土、砂姜黑土、褐土、潮土)为研究对象进行微区试验,设置了对照(CK)、单施化肥(NPK)、单施生物质炭(BC)、化肥配施生物质炭(BC+NPK)4个处理,于冬小麦生育前期观测土壤氨挥发损失,分析土壤矿质氮含量、土壤pH和温度对土壤氨挥发的影响。结果表明,4种土壤单施化肥处理氨挥发累积损失分别为2.70、3.14、2.90、4.00 kg N·hm^-2,占施氮量的比例(氨挥发损失率)为3.3%、3.8%、3.5%、4.9%。与单施化肥相比,化肥配施生物质炭可以降低砂姜黑土(15.3%)和潮土(14.8%)的氨挥发损失,但增加了水稻土(3.0%)和褐土(6.9%)氨挥发。添加生物质炭显著提升土壤pH值和土壤温度,相关性分析表明,土壤pH值是决定生物质炭对土壤氨挥发增减的关键因素。综上所述,在华北平原砂姜黑土和潮土施用生物质炭可以有效降低小麦生育前期土壤氨挥发。展开更多
目的构建新型冠状病毒肺炎(COVID-19)确诊患者隔离病区的环境表面清洁与消毒流程,为隔离病区护理人员开展清洁与消毒工作提供参考,从而保障隔离病区的物表清洁与消毒质量。方法通过检索全球COVID-19防控相关的循证实践指南,以AGREE Ⅱ(A...目的构建新型冠状病毒肺炎(COVID-19)确诊患者隔离病区的环境表面清洁与消毒流程,为隔离病区护理人员开展清洁与消毒工作提供参考,从而保障隔离病区的物表清洁与消毒质量。方法通过检索全球COVID-19防控相关的循证实践指南,以AGREE Ⅱ(Appraisal of Guidelines for Research and Evaluation Ⅱ)评价工具进行严格评价及指南的筛选,通过开展德尔菲专家咨询及调查利益相关者即清洁与消毒岗护士对流程落实的认同度及观点反馈,最终通过小组会议形成隔离病区环境表面清洁与消毒流程。结果研究共纳入12篇指南,通过2轮德尔菲专家咨询结合对清洁与消毒岗护士的调查结果,形成6大步骤40条具体内容及备注(评估计划与组织、人员准备与培训、物资准备与提供、实施清洁与消毒、医废处理与整理、监控反馈与改进)的COVID-19确诊患者隔离病区物表清洁与消毒流程。结论基于循证实践指南构建的COVID-19确诊患者隔离病区环境表面清洁与消毒流程可为疫情中防止医务人员和患者的交叉感染、降低院内传播风险提供护理管理策略,为同质化落实清洁与消毒措施、保证隔离病区的清洁与消毒质量提供工作依据。展开更多
在公路交通中,针对复杂环境下交通标志识别率不高的问题,提出了一种基于K-means对图像聚类,切割图像感兴趣区域(Regions of Interest, ROI),并利用方向梯度直方图特征(Histogram of Oriented Gradient, HOG)与卷积运算,特征加权(CNN-Squ...在公路交通中,针对复杂环境下交通标志识别率不高的问题,提出了一种基于K-means对图像聚类,切割图像感兴趣区域(Regions of Interest, ROI),并利用方向梯度直方图特征(Histogram of Oriented Gradient, HOG)与卷积运算,特征加权(CNN-Squeeze)相结合的交通标志识别方法.首先,采用K-means对交通标志图像进行三角形、圆形图像二聚类,并利用制作的切割模板切割ROI并提取HOG特征;然后,利用卷积神经网络(Convolutional NeuralNetwork,CNN)对HOG特征进行过滤、降维,并通过Squeeze网络对过滤后的二次特征进行重要性标定;最后,训练该网络模型并实现对交通标志的识别.仿真结果表明,与BP网络、SVM及CNN对比,本文方法在保证训练时间的同时,识别精度达到98.58%.展开更多
基金supported by the Major Scientific Research Tasks for Scientific and Technological Innovation Projects of the Chinese Academy of Agricultural Sciences(CAAS-ZDRW202006)the National Transgenic Breeding Project(2018ZX08010-10B)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(ASTIP-IAS05).
文摘Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors.Safe harbor loci in the animal genome enable consistent overexpression of foreign genes,without side effects.However,relatively few safe harbor loci are available in pigs,a fact which has impeded the development of multi-transgenic pig research.We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain(COL1A1)gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system.After the knock-in of a 2A peptide-green fluorescence protein(2A-GFP)transgene in the last codon of COL1A1 in multiple porcine cells,including porcine kidney epithelial(PK15),porcine embryonic fibroblast(PEF)and porcine intestinal epithelial(IPI-2I)cells,quantitative PCR(qPCR),Western blotting,RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus.The qPCR results showed that the GFP knock-in had no effect(P=0.29,P=0.66 and P=0.20 for PK15,PEF and IPI-2I cells,respectively)on the mRNA expression of COL1A1 gene.Similarly,no significant differences(P=0.64,P=0.48 and P=0.80 for PK15,PEF and IPI-2I cells,respectively)were found between the GFP knock-in and wild type cells by Western blotting.RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation(P<2.2e–16)with that of the wild type cells,indicating that the GFP knock-in did not alter the global expression of endogenous genes.Furthermore,the CCK8 assay showed that the GFP knock-in events had no adverse effects(P_(24)h=0.31,P_(48)h=0.96,P_(72)h=0.24,P_(96)h=0.17,and P_(120)h=0.38)on cell proliferation of PK15 cells.These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment.
文摘为了探明生物质炭对华北平原土壤氨挥发的影响,以该区域4种典型土壤(水稻土、砂姜黑土、褐土、潮土)为研究对象进行微区试验,设置了对照(CK)、单施化肥(NPK)、单施生物质炭(BC)、化肥配施生物质炭(BC+NPK)4个处理,于冬小麦生育前期观测土壤氨挥发损失,分析土壤矿质氮含量、土壤pH和温度对土壤氨挥发的影响。结果表明,4种土壤单施化肥处理氨挥发累积损失分别为2.70、3.14、2.90、4.00 kg N·hm^-2,占施氮量的比例(氨挥发损失率)为3.3%、3.8%、3.5%、4.9%。与单施化肥相比,化肥配施生物质炭可以降低砂姜黑土(15.3%)和潮土(14.8%)的氨挥发损失,但增加了水稻土(3.0%)和褐土(6.9%)氨挥发。添加生物质炭显著提升土壤pH值和土壤温度,相关性分析表明,土壤pH值是决定生物质炭对土壤氨挥发增减的关键因素。综上所述,在华北平原砂姜黑土和潮土施用生物质炭可以有效降低小麦生育前期土壤氨挥发。
文摘目的构建新型冠状病毒肺炎(COVID-19)确诊患者隔离病区的环境表面清洁与消毒流程,为隔离病区护理人员开展清洁与消毒工作提供参考,从而保障隔离病区的物表清洁与消毒质量。方法通过检索全球COVID-19防控相关的循证实践指南,以AGREE Ⅱ(Appraisal of Guidelines for Research and Evaluation Ⅱ)评价工具进行严格评价及指南的筛选,通过开展德尔菲专家咨询及调查利益相关者即清洁与消毒岗护士对流程落实的认同度及观点反馈,最终通过小组会议形成隔离病区环境表面清洁与消毒流程。结果研究共纳入12篇指南,通过2轮德尔菲专家咨询结合对清洁与消毒岗护士的调查结果,形成6大步骤40条具体内容及备注(评估计划与组织、人员准备与培训、物资准备与提供、实施清洁与消毒、医废处理与整理、监控反馈与改进)的COVID-19确诊患者隔离病区物表清洁与消毒流程。结论基于循证实践指南构建的COVID-19确诊患者隔离病区环境表面清洁与消毒流程可为疫情中防止医务人员和患者的交叉感染、降低院内传播风险提供护理管理策略,为同质化落实清洁与消毒措施、保证隔离病区的清洁与消毒质量提供工作依据。
文摘在公路交通中,针对复杂环境下交通标志识别率不高的问题,提出了一种基于K-means对图像聚类,切割图像感兴趣区域(Regions of Interest, ROI),并利用方向梯度直方图特征(Histogram of Oriented Gradient, HOG)与卷积运算,特征加权(CNN-Squeeze)相结合的交通标志识别方法.首先,采用K-means对交通标志图像进行三角形、圆形图像二聚类,并利用制作的切割模板切割ROI并提取HOG特征;然后,利用卷积神经网络(Convolutional NeuralNetwork,CNN)对HOG特征进行过滤、降维,并通过Squeeze网络对过滤后的二次特征进行重要性标定;最后,训练该网络模型并实现对交通标志的识别.仿真结果表明,与BP网络、SVM及CNN对比,本文方法在保证训练时间的同时,识别精度达到98.58%.
