利用Cas9TX实现非病毒TRAC定点整合制备T细胞

【目的】Cas9TX是Cas9的变体,能够显著降低基因编辑过程中染色体易位,大幅提升基因编辑的安全性。研究Cas9TX替换Cas9实现基因定点敲入的可行性。【方法】首先,利用His标签蛋白纯化技术制备Cas9TX蛋白,并在细胞水平上通过绘制EC50和核酸酶裂解动力学曲线等进行功能验证。其次,以RNP和dsDNA混合物电转的方式编辑体外激活的T细胞,流式检测定点敲入的效率。最后,探讨了供体模板进行稳定性修饰提升定点敲入效率的可行性。【结果】制备的Cas9TX在T细胞TRAC基因的A、R和S靶点的敲除效率分别为71.8%、81.0%和79.9%,具有与Cas9相当的基因敲除效率。在TRAC 1号外显子分别设计的dsDNA供体模板(2A-GFP编码序列)和ssDNA供体模板(+GTC bp),发现Cas9TX RNP定点敲入两种模板的效率均显著低于Cas9RNP。通过DNA修饰制备防TREX2核酸外切酶降解的供体模板,不能提升Cas9TXRNP定点敲入的效率。【结论】Cas9TX RNP在TRAC三个靶点的基因敲除方面可以替代Cas9RNP使用,但定点整合外源基因的效率约为Cas9RNP的一半。为Cas9TX应用于基因定点敲入提供了重要参考。

Detecting chimerism contributes to diagnosis of graft versus host disease after orthotopic liver transplantation

To the Editor:Graft versus host disease(GVHD)occurs when the immunocompetent cells from the donor mount an immune response against the tissues of the host[1].It is commonly seen in the patients who received hematopoietic stem cell transplantation(HSCT).GVHD is a rare but lethal complication in orthotopic liver

Crystal structures and decomposing of B–P compounds under pressure

We have systematically studied the structures, electronic properties, and lattice dynamics of B–P compounds at high pressures. BP and B_6 P are found to be thermodynamically stable below 100 GPa, and other stoichiometries are decomposable under pressure. The predicted structures of F-43 m BP and R-3 m B_6 P are in good agreement with the experimental results by comparing the powder diffraction file(PDF) standard cards with our simulated x-ray diffractions. The bonding properties of BP and B_6 P have also been analyzed by electronic localization functions, charge density difference, and Bader charge analysis. Our results show that BP and B_6 P decompose into B and P under high pressure, which is proven to be dominated by the volumes of them. Furthermore, the infrared and Raman spectra of F-43 m and R-3 m are investigated at selected pressures and will provide useful information for future experimental studies about B–P compounds.

Severe and delayed immune-mediated hemolysis post-liver transplantation

To the Editor:Hemolysis,which is caused by a variety of immune and non-immune mechanisms,is a well-recognized complication of solid organ transplantation.;Hemolysis post-liver transplantation can be induced by drug,infection,autoimmune disorders,blood-group incompatible

Charge state modulation on boron site by carbon and nitrogen localized bonding microenvironment for two-electron electrocatalytic H_(2)O_(2)production

Design of electrochemical active boron(B)site at solid materials to understand the relationships between the localized structure,charge state at the B site and electrocatalytic activity plays a crucial role in boosting the green electrochemical synthesis of hydrogen peroxide(H_(2)O_(2))via two-electron oxygen reduction(2eORR)pathway.Herein,we demonstrate a carbon(C)and nitrogen(N)localized bonding microenvironment to modulate the charge state of B site at the boron-carbon nitride solid(BCNs)to realize the efficient selective electrocatalytic H_(2)O_(2)production.The localized chemical structure of N-B-N,N-B-C and C-B-C bonds at B site can be regulated through solid-state reaction between boron nitride(BN)and porous carbon(C)at variable temperatures.The optimized BCN-1100 achieves an outstanding H_(2)O_(2)selectivity of 89%and electron transfer number of 2.2(at 0.55 V vs.RHE),with the production of 10.55mmol/L during 2.5 h and the catalytic stability duration for 15000 cycles.Further first-principles calculations identified the dependency of localized bonding microenvironment on the OOH~*adsorption energies and relevant charge states at the boron site.The localized structure of B site with BNC_(2)-Gr configuration is predicted to be the highest 2eORR activity.

New Cage-Like Cerium Trihydride Stabilized at Ambient Conditions

Metal hydrides,generally formed by high pressure combined with high-temperature conditions,have attracted substantial interest due to their promising high-energy density and high-temperature superconductivity.Although the synthesis and properties of these hydrides have been extensively studied,an important challenge is the lack of an efficient method to retain high-pressure phases.Herein,we have successfully quenched a high-pressure phase to ambient conditions with the precise control of different compression pathways.A newβ-CeH_(3) phase(space group Pm3n)isostructural toβ-UH_(3) phase was synthesized by the reaction of Ce and H2 above 33.0 GPa in a laser-heated diamond anvil cell.Theβ-CeH_(3)phase has a cage-like framework structure that can be retained at ambient conditions.The electrical resistance as a function of temperature determines its metallic properties,but no superconductivity is observed in the current temperature range.Thermodynamic and dynamic calculations further prove the stability ofβ-CeH_(3) at ambient pressure.This work demonstrates that different synthesized paths will drive the formation of different products.