查看更多>>摘要：Although single-particle cryogenic electron microscopy(cryo-EM)has been applied extensively for elu-cidating many crucial biological mechanisms at the molecular level,this technique still faces critical challenges,the major one of which is to prepare the high-quality cryo-EM specimen.Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation,novel supporting films including graphene-based two-dimensional materials have been explored in recent years.Here we report a ro-bust and simple method to fabricate EM grids coated with single-or few-layer reduced graphene oxide(RGO)membrane in large batch for high-resolution cryo-EM structural determination.The RGO mem-brane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide(GO)membrane.Moreover,we found that the RGO supporting film exhibited nice particle-absorption ability,thus avoiding the air-water interface problem.More importantly,we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa bio-molecules at near-atomic resolution,as exemplified by the study of RBD-ACE2 complex and other small protein molecules.We envision that the RGO membranes can be used as a robust graphene-based sup-porting film in cryo-EM specimen preparation.
查看更多>>摘要：Although single-particle cryogenic electron microscopy(cryo-EM)has been applied extensively for elu-cidating many crucial biological mechanisms at the molecular level,this technique still faces critical challenges,the major one of which is to prepare the high-quality cryo-EM specimen.Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation,novel supporting films including graphene-based two-dimensional materials have been explored in recent years.Here we report a ro-bust and simple method to fabricate EM grids coated with single-or few-layer reduced graphene oxide(RGO)membrane in large batch for high-resolution cryo-EM structural determination.The RGO mem-brane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide(GO)membrane.Moreover,we found that the RGO supporting film exhibited nice particle-absorption ability,thus avoiding the air-water interface problem.More importantly,we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa bio-molecules at near-atomic resolution,as exemplified by the study of RBD-ACE2 complex and other small protein molecules.We envision that the RGO membranes can be used as a robust graphene-based sup-porting film in cryo-EM specimen preparation.
查看更多>>摘要：Ischemic stroke results in cerebral tissue hypoxia and increased expression of hypoxia-inducible factor(HIF),which is critically implicated in ischemic brain injury.Understanding the mechanisms of HIF-1alpha regulation in the ischemic brain has been an important research focus.The generation of both nitric oxide(NO)and reactive oxygen species(ROS)is increased under hypoxic/ischemic conditions and each of them has been independently shown to regulate HIF-1alpha expression.In this study,we investigated the cross-effects of NO and ROS on the expression of HIF-1alpha in hypoxic astrocytes.Ex-posure of astrocytes to 2 h-hypoxia remarkably increased HIF-1alpha protein levels,which was accom-panied by increased NO and ROS production.Decreasing ROS with NAC,NADPH oxidase inhibitor DPI,or SOD mimetic MnTMPyP decreased hypoxia-induced HIF-1alpha protein accumulation and increased NO level in hypoxic astrocytes.The NO synthase(NOS)inhibitor L-NAME inhibited ROS generation,which led to a reduction in hypoxia-induced HIF-1alpha protein expression.Although NOS inhibitor or ROS scavengers alone reduced HIF-1alpha protein levels,the reduction was reversed when NOS inhib-itor and ROS scavenger present together.The NO scavenger PTIO increased hypoxia-induced HIF-1alpha protein expression and ROS production,while HIF-1alpha protein level was decreased in the presence of NO scavenger and ROS scavenger together.These results suggest that ROS,NO,and their interaction critically contribute to the regulation of hypoxia-induced HIF-1alpha protein accumulation under hypoxic condition.Furthermore,our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for balancing ROS-mediated hypoxic stress,as reflected by inhib-iting hypoxia-induced HIF-1alpha protein accumulation.
查看更多>>摘要：Ischemic stroke results in cerebral tissue hypoxia and increased expression of hypoxia-inducible factor(HIF),which is critically implicated in ischemic brain injury.Understanding the mechanisms of HIF-1alpha regulation in the ischemic brain has been an important research focus.The generation of both nitric oxide(NO)and reactive oxygen species(ROS)is increased under hypoxic/ischemic conditions and each of them has been independently shown to regulate HIF-1alpha expression.In this study,we investigated the cross-effects of NO and ROS on the expression of HIF-1alpha in hypoxic astrocytes.Ex-posure of astrocytes to 2 h-hypoxia remarkably increased HIF-1alpha protein levels,which was accom-panied by increased NO and ROS production.Decreasing ROS with NAC,NADPH oxidase inhibitor DPI,or SOD mimetic MnTMPyP decreased hypoxia-induced HIF-1alpha protein accumulation and increased NO level in hypoxic astrocytes.The NO synthase(NOS)inhibitor L-NAME inhibited ROS generation,which led to a reduction in hypoxia-induced HIF-1alpha protein expression.Although NOS inhibitor or ROS scavengers alone reduced HIF-1alpha protein levels,the reduction was reversed when NOS inhib-itor and ROS scavenger present together.The NO scavenger PTIO increased hypoxia-induced HIF-1alpha protein expression and ROS production,while HIF-1alpha protein level was decreased in the presence of NO scavenger and ROS scavenger together.These results suggest that ROS,NO,and their interaction critically contribute to the regulation of hypoxia-induced HIF-1alpha protein accumulation under hypoxic condition.Furthermore,our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for balancing ROS-mediated hypoxic stress,as reflected by inhib-iting hypoxia-induced HIF-1alpha protein accumulation.
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