Mechanosensitive ion channels are essential for sensing and converting mechanical forces into electrical or chemical signals.These channels are widely distributed across bacteria,animals,and plants.In Arabidopsis thal...Mechanosensitive ion channels are essential for sensing and converting mechanical forces into electrical or chemical signals.These channels are widely distributed across bacteria,animals,and plants.In Arabidopsis thaliana,the OSCA family has been identified as mechanically activated ion channels that respond to osmotic stress by allowing calcium ions to enter the cell.This influx increases the cytoplasmic calcium concentration,triggering osmotic stress-induced signal transduction cascades in plants.In this study,we determined the structures of OSCA2.2 and OSCA3.1 via cryoelectron microscopy(cryo-EM).Both proteins form homodimers consisting of 11 transmembrane helices(TM1–11).The ion conduction pathway is formed by TM4–8.Despite belonging to the same family,OSCA2.2 and OSCA3.1 exhibit notable structural variations.Structural analysis revealed that both OSCA2.2 and OSCA3.1 exhibit a closed conformation.We also conducted functional studies on OSCA proteins via electrophysiological experiments and confirmed the role of key amino acids in the process of ion permeation.展开更多
The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and func...The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest(GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes “scars/indels” at integration junctions.Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination(HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs.This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci(e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER^(T2)line, we fate-map spinal cord p3 neural progenitor cells,revealing that Nkx2.2^(+) cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.展开更多
基于ISCCP(International Satellite Cloud Climatology Project)和NCEP(National Centers for Environmental Prediction)资料分析了BCC_AGCM2.1(Beijing Climate Center_Atmospheric General Circulation Model 2.1)和BCC_AGCM2.2模...基于ISCCP(International Satellite Cloud Climatology Project)和NCEP(National Centers for Environmental Prediction)资料分析了BCC_AGCM2.1(Beijing Climate Center_Atmospheric General Circulation Model 2.1)和BCC_AGCM2.2模拟的云在东亚的垂直分布特点,并探讨了误差来源。两个模式大体上模拟出了总云量的分布形势,较好地模拟出了垂直方向上云量大值带随地形的变化特点,但模拟的总云量偏少。AGCM2.2模拟的云量整体上小于AGCM2.1,除复杂地形外AGCM2.2没有体现出高分辨率的优势。模式对中国东部环流场的模拟效果差导致模拟的云量偏少,尤其是AGCM2.2。模拟的对流层高层相对湿度明显偏大导致高层云量偏大。模式在近海面模拟的相对湿度偏小,四川盆地及周围地区冷季模拟的水汽含量偏少,因而模拟的云量偏少。模式云量对相对湿度的响应能力较好,模拟出了云量对垂直速度和稳定度的响应,但地区差异不明显。模式的云参数化方案中云与相对湿度的关系系数需要调整,应更利于云的生成。展开更多
通过实验室25 kg真空感应炉冶炼和锻轧研究了0.26%～0.95%Al含量对0.5 mm 2.2%Si无取向硅钢冷轧板组织、织构和磁性能的影响。试验结果表明,0.26%～0.81%Al含量时,随钢中Al含量的增加,成品退火钢板的晶粒尺寸增加同时铁损减少;当Al含量...通过实验室25 kg真空感应炉冶炼和锻轧研究了0.26%～0.95%Al含量对0.5 mm 2.2%Si无取向硅钢冷轧板组织、织构和磁性能的影响。试验结果表明,0.26%～0.81%Al含量时,随钢中Al含量的增加,成品退火钢板的晶粒尺寸增加同时铁损减少;当Al含量大于0.81%时,随Al含量增加,钢板的晶粒尺寸减小,同时铁损增加。Al含量对硅钢板磁感的影响没有明显的规律。2.2%Si无取向硅钢的合适Al含量为0.48%～0.81%。展开更多
基金supported by the National Natural Science Foundation of China(32322041,W2412029,32321001,32471279)USTC Research Funds of the Double First-Class Initiative(YD9100002004,YD9100002020)+2 种基金Fundamental Research Funds for the Central Universities(WK9100000031)Research Funds of Center for Advanced Interdisciplinary Science and Biomedicine of IHM(QYPY20230034)the Natural Science Foundation of Anhui Province(2408085JX005).
文摘Mechanosensitive ion channels are essential for sensing and converting mechanical forces into electrical or chemical signals.These channels are widely distributed across bacteria,animals,and plants.In Arabidopsis thaliana,the OSCA family has been identified as mechanically activated ion channels that respond to osmotic stress by allowing calcium ions to enter the cell.This influx increases the cytoplasmic calcium concentration,triggering osmotic stress-induced signal transduction cascades in plants.In this study,we determined the structures of OSCA2.2 and OSCA3.1 via cryoelectron microscopy(cryo-EM).Both proteins form homodimers consisting of 11 transmembrane helices(TM1–11).The ion conduction pathway is formed by TM4–8.Despite belonging to the same family,OSCA2.2 and OSCA3.1 exhibit notable structural variations.Structural analysis revealed that both OSCA2.2 and OSCA3.1 exhibit a closed conformation.We also conducted functional studies on OSCA proteins via electrophysiological experiments and confirmed the role of key amino acids in the process of ion permeation.
基金supported by the National Key R&D Program of China(2021YFA0805000,2023YFA1800600,2019YFE0106700)the National Natural Science Foundation of China(92268114,31970782,32070819)+1 种基金the High-level Hospital Construction Project of GuangdongProvincial People'sHospital(DFJHBF202103 and KJ012021012)BGI grant(BGIRSZ20210002).
文摘The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest(GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes “scars/indels” at integration junctions.Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination(HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs.This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci(e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER^(T2)line, we fate-map spinal cord p3 neural progenitor cells,revealing that Nkx2.2^(+) cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.
文摘基于ISCCP(International Satellite Cloud Climatology Project)和NCEP(National Centers for Environmental Prediction)资料分析了BCC_AGCM2.1(Beijing Climate Center_Atmospheric General Circulation Model 2.1)和BCC_AGCM2.2模拟的云在东亚的垂直分布特点,并探讨了误差来源。两个模式大体上模拟出了总云量的分布形势,较好地模拟出了垂直方向上云量大值带随地形的变化特点,但模拟的总云量偏少。AGCM2.2模拟的云量整体上小于AGCM2.1,除复杂地形外AGCM2.2没有体现出高分辨率的优势。模式对中国东部环流场的模拟效果差导致模拟的云量偏少,尤其是AGCM2.2。模拟的对流层高层相对湿度明显偏大导致高层云量偏大。模式在近海面模拟的相对湿度偏小,四川盆地及周围地区冷季模拟的水汽含量偏少,因而模拟的云量偏少。模式云量对相对湿度的响应能力较好,模拟出了云量对垂直速度和稳定度的响应,但地区差异不明显。模式的云参数化方案中云与相对湿度的关系系数需要调整,应更利于云的生成。
文摘通过实验室25 kg真空感应炉冶炼和锻轧研究了0.26%～0.95%Al含量对0.5 mm 2.2%Si无取向硅钢冷轧板组织、织构和磁性能的影响。试验结果表明,0.26%～0.81%Al含量时,随钢中Al含量的增加,成品退火钢板的晶粒尺寸增加同时铁损减少;当Al含量大于0.81%时,随Al含量增加,钢板的晶粒尺寸减小,同时铁损增加。Al含量对硅钢板磁感的影响没有明显的规律。2.2%Si无取向硅钢的合适Al含量为0.48%～0.81%。
