AIM：To investigate the roles of integrins in choroidal neovascularization（CNV） and their associations with the stromal cell-derived factor-1（SDF-1）/CXCR4 axis.METHODS：CNV lesions were induced in mice using laser...AIM：To investigate the roles of integrins in choroidal neovascularization（CNV） and their associations with the stromal cell-derived factor-1（SDF-1）/CXCR4 axis.METHODS：CNV lesions were induced in mice using laser photocoagulation.After CNV induction,all animals were randomly assigned to：control,SDF-1,SDF-1＋age-related macular degeneration（AMD） 3100（CXCR4 inhibitor）,and SDF-1＋ATN161（integrin α5β1 inhibitor） groups;their effects on CNV progression were observed using hematoxylin eosin（HE） staining,fundus fluorescein angiography（FFA） grading and optical coherence tomography（OCT）,and their effects on CXCR4/integrin α5 expression were evaluated using Western blot and double immunofluorescence staining.Hypoxia-exposed endothelial cells（ECs） were used to simulate CNV in vitro,they were treated with SDF-1,combined with CXCR4 siRNA/AMD3100 or ATN161,and expression of integrin α5,cell migration and tube formation were analyzed.RESULTS：Integrin subunit α5 increased at 3^ rd and 7^ th day and decreased at 14 ^th day in CNV mice,with no significant change of β1-integrin.CXCR4 expression in CNV mice had persistent increase within 14 d after induction.SDF-1 treatment significantly promoted the CNV progression during 3-14 d.The mean CNV length in AMD3100 andATN161 group at day 7 was 270.13 and 264.23 μm in HE images,significantly lower than the mean length in SDF-1（345.70 μm） group.AMD3100 and ATN161 also significantly reduced thickness and leakage of CNV induced by SDF-1.Mean integrin α5 positive area in SDF-1 group reached 2.31×104 μm^2,significantly higher than control（1.25×104 μm^2）,which decreased to 1.78×104 μm^2 after AMD3100 treatment.About 61.36% of ECs in CNV lesions expressed α5 in SDF-1 group,which significantly decreased to 43.12% after AMD3100 treatment.In vitro,integrin α5 peaked by 6 folds after 6 h of hypoxia exposure and CXCR4 gradually increased by up to 2.3 folds after 24 h of hypoxia.Approximately 25.12% of ECs expressed integrin α5 after SDF-1 stimulation,which decreased to 7.2%-9.5% after si-CXCR4 or AMD3100 treatment.ATN161 exerted an inhibitory effect comparable to that of si-CXCR4 on EC migration and tube formation in the presence of SDF-1.CONCLUSION：SDF-1/CXCR4 signaling induces integrin α5β1 expression in ECs to promote CNV.展开更多
As a key regulator of immune response,CD40 L is usually associated with chronic disease-related inflammation,autoimmune diseases and malignant diseases.Receptor recognition of platelet CD40 L is the initial event that...As a key regulator of immune response,CD40 L is usually associated with chronic disease-related inflammation,autoimmune diseases and malignant diseases.Receptor recognition of platelet CD40 L is the initial event that mediates platelet aggregation and leukocyte immune response.Unlike soluble CD40 L,the interaction between transmembrane platelet CD40 L and its receptors occurs within the cell junction surface,usually,in a physiological and pathological high blood flow shear stress environment.This two-dimensional reaction kinetics should be a mechano-chemical coupling process.In addition to its classical receptor CD40,CD40 L also binds to receptorα5β1,CD40 L can bind to the resting state of integrinα5β1,but the mechanical regulation mechanism of integrinα5β1 activation under fluid shear stress remains unclear.We assume that the force can promote CD40 L-inducedα5β1 activation.To check this hypothesis,we performed flow chamber experiment to investigate interaction of CD40 L andα5β1.In experiments,the bottom of the flow chamber is functionalized by a suitable concentration of CD40 L,and the fiber spheres of 6μm diameter was coated withα5β1.The selection of CD40 L concentration was based on the observation that as many tether events ofα5β1-coated spheres as possible were observed rather than stable adhesion events of these spheres.Theα5β1-coated sphere suspension was poured over the CD40 L-coated substrates in the flow chamber under different shear rates.A high-speed camera was used to observe and record tether events of fiber spheres at a rate of 100 frames per second.According to our affinity state transition model for integrin,the data were analyzed to obtain the rate of integrin activation and its mechanical regulation characteristics.Our results demonstrated that the interaction betweenα5β1 and CD40 L is biphasic force-dependent,showing mechano-chemical regulation mechanism of'Catch-slip bond'transition.The affinity jumping model was well fitted with the data obtained from flow chamber experiment at various wall shear stresses.We found that,CD40 L ligation-induced jumping ofα5β1 affinity state from low to medium(or high)one will occur within 0.5-1.0 second,resulting in prolonging of bond lifetimes.And,frequency distribution of the tether events number with tether lifetime under each force,exhibits obvious doublet peaks,one within 0.5-1 s and second within 1.5-2.5 s,indicating theα5β1 affinity state transform from low to high one.The probability distribution of the tether lifetime under different shear forces are not linear,and exists a turning point,which shows that the rate ofα5β1 dissociation from CD40 L is fast first,and then become slow,showing a force-induced conformation transformation of the integrinα5β1 from low affinity state to high affinity one.Our findings suggest that,the continuous force stimulation will quickly cause the affinity state change of integrinα5β1. The dissociation rate of theα5β1/CD40 L complex decreases first and then increases with wall shear stress,exhibiting a'Catch-slip bond'transformation of interaction betweenα5β1-CD40 L.This mechanical regulation mechanism exists in interaction of CD40 L not only toα5β1 at low affinity state but also to one at high affinity state.Our results should be useful in understanding the mechanical regulation mechanism of a5β1-CD40 L interaction-mediated cellular immune response and inflammatory processes.展开更多
针对实时行人检测场景存在遮挡、形态姿势不同的行人目标,YOLOv5模型对于这些目标检测有明显的漏检问题,提出一种像素差异度注意力机制(pixel difference attention,PDA),不同于传统的通道注意力机制用全局均值池化(global average pool...针对实时行人检测场景存在遮挡、形态姿势不同的行人目标,YOLOv5模型对于这些目标检测有明显的漏检问题,提出一种像素差异度注意力机制(pixel difference attention,PDA),不同于传统的通道注意力机制用全局均值池化(global average pooling,GAP)、全局最大值池化(global max pooling,GMP)来概括整张特征图的信息,全局池化将空间压缩成一个值来表征整个通道,造成了空间信息的流失,PDA将空间信息沿高和宽分别压缩,并将其分别与通道信息联系起来做注意力加权操作,同时提出一种新的通道描述指标表征通道信息,增强空间信息与通道信息的交互,使模型更容易关注到综合了空间和通道维度上的特征图的重要信息,在主干网络末端插入PDA后使模型平均精度(mean average precision,mAP)0.5提升了2.4个百分点,mAP0.5:0.95提升了4.4个百分点;针对实时检测场景的部署和检测速度要求模型拥有较少的参数量和计算量,因此提出了新的轻量化特征提取模块AC3代替原YOLOv5模型中的C3模块,该模块使插入PDA后的改进模型在精度仅仅损失0.2个百分点的情况下,参数量(parameters,Param.)减少了20%左右,浮点运算量(giga floating-point operations,GFLOPs)减少了30%左右。实验结果表明,最终的改进模型比YOLOv5s原模型在VOC行人数据集上mAP0.5提升了2.2个百分点,mAP0.5:0.95提升了3.1个百分点,且参数量减少了20%左右,浮点运算量减少了30%左右,在GTX1050上的检测速度(frames per second,FPS)提升了4。展开更多
背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供...背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。展开更多
基金Supported by the National Natural Science Foundation of China(No.81770936No.81570856+2 种基金No.81670863No.81500748No.81370020)
文摘AIM：To investigate the roles of integrins in choroidal neovascularization（CNV） and their associations with the stromal cell-derived factor-1（SDF-1）/CXCR4 axis.METHODS：CNV lesions were induced in mice using laser photocoagulation.After CNV induction,all animals were randomly assigned to：control,SDF-1,SDF-1＋age-related macular degeneration（AMD） 3100（CXCR4 inhibitor）,and SDF-1＋ATN161（integrin α5β1 inhibitor） groups;their effects on CNV progression were observed using hematoxylin eosin（HE） staining,fundus fluorescein angiography（FFA） grading and optical coherence tomography（OCT）,and their effects on CXCR4/integrin α5 expression were evaluated using Western blot and double immunofluorescence staining.Hypoxia-exposed endothelial cells（ECs） were used to simulate CNV in vitro,they were treated with SDF-1,combined with CXCR4 siRNA/AMD3100 or ATN161,and expression of integrin α5,cell migration and tube formation were analyzed.RESULTS：Integrin subunit α5 increased at 3^ rd and 7^ th day and decreased at 14 ^th day in CNV mice,with no significant change of β1-integrin.CXCR4 expression in CNV mice had persistent increase within 14 d after induction.SDF-1 treatment significantly promoted the CNV progression during 3-14 d.The mean CNV length in AMD3100 andATN161 group at day 7 was 270.13 and 264.23 μm in HE images,significantly lower than the mean length in SDF-1（345.70 μm） group.AMD3100 and ATN161 also significantly reduced thickness and leakage of CNV induced by SDF-1.Mean integrin α5 positive area in SDF-1 group reached 2.31×104 μm^2,significantly higher than control（1.25×104 μm^2）,which decreased to 1.78×104 μm^2 after AMD3100 treatment.About 61.36% of ECs in CNV lesions expressed α5 in SDF-1 group,which significantly decreased to 43.12% after AMD3100 treatment.In vitro,integrin α5 peaked by 6 folds after 6 h of hypoxia exposure and CXCR4 gradually increased by up to 2.3 folds after 24 h of hypoxia.Approximately 25.12% of ECs expressed integrin α5 after SDF-1 stimulation,which decreased to 7.2%-9.5% after si-CXCR4 or AMD3100 treatment.ATN161 exerted an inhibitory effect comparable to that of si-CXCR4 on EC migration and tube formation in the presence of SDF-1.CONCLUSION：SDF-1/CXCR4 signaling induces integrin α5β1 expression in ECs to promote CNV.
基金supported by the National Natural Science Foundation of China ( 116272109, 11432006)
文摘As a key regulator of immune response,CD40 L is usually associated with chronic disease-related inflammation,autoimmune diseases and malignant diseases.Receptor recognition of platelet CD40 L is the initial event that mediates platelet aggregation and leukocyte immune response.Unlike soluble CD40 L,the interaction between transmembrane platelet CD40 L and its receptors occurs within the cell junction surface,usually,in a physiological and pathological high blood flow shear stress environment.This two-dimensional reaction kinetics should be a mechano-chemical coupling process.In addition to its classical receptor CD40,CD40 L also binds to receptorα5β1,CD40 L can bind to the resting state of integrinα5β1,but the mechanical regulation mechanism of integrinα5β1 activation under fluid shear stress remains unclear.We assume that the force can promote CD40 L-inducedα5β1 activation.To check this hypothesis,we performed flow chamber experiment to investigate interaction of CD40 L andα5β1.In experiments,the bottom of the flow chamber is functionalized by a suitable concentration of CD40 L,and the fiber spheres of 6μm diameter was coated withα5β1.The selection of CD40 L concentration was based on the observation that as many tether events ofα5β1-coated spheres as possible were observed rather than stable adhesion events of these spheres.Theα5β1-coated sphere suspension was poured over the CD40 L-coated substrates in the flow chamber under different shear rates.A high-speed camera was used to observe and record tether events of fiber spheres at a rate of 100 frames per second.According to our affinity state transition model for integrin,the data were analyzed to obtain the rate of integrin activation and its mechanical regulation characteristics.Our results demonstrated that the interaction betweenα5β1 and CD40 L is biphasic force-dependent,showing mechano-chemical regulation mechanism of'Catch-slip bond'transition.The affinity jumping model was well fitted with the data obtained from flow chamber experiment at various wall shear stresses.We found that,CD40 L ligation-induced jumping ofα5β1 affinity state from low to medium(or high)one will occur within 0.5-1.0 second,resulting in prolonging of bond lifetimes.And,frequency distribution of the tether events number with tether lifetime under each force,exhibits obvious doublet peaks,one within 0.5-1 s and second within 1.5-2.5 s,indicating theα5β1 affinity state transform from low to high one.The probability distribution of the tether lifetime under different shear forces are not linear,and exists a turning point,which shows that the rate ofα5β1 dissociation from CD40 L is fast first,and then become slow,showing a force-induced conformation transformation of the integrinα5β1 from low affinity state to high affinity one.Our findings suggest that,the continuous force stimulation will quickly cause the affinity state change of integrinα5β1. The dissociation rate of theα5β1/CD40 L complex decreases first and then increases with wall shear stress,exhibiting a'Catch-slip bond'transformation of interaction betweenα5β1-CD40 L.This mechanical regulation mechanism exists in interaction of CD40 L not only toα5β1 at low affinity state but also to one at high affinity state.Our results should be useful in understanding the mechanical regulation mechanism of a5β1-CD40 L interaction-mediated cellular immune response and inflammatory processes.
文摘针对实时行人检测场景存在遮挡、形态姿势不同的行人目标,YOLOv5模型对于这些目标检测有明显的漏检问题,提出一种像素差异度注意力机制(pixel difference attention,PDA),不同于传统的通道注意力机制用全局均值池化(global average pooling,GAP)、全局最大值池化(global max pooling,GMP)来概括整张特征图的信息,全局池化将空间压缩成一个值来表征整个通道,造成了空间信息的流失,PDA将空间信息沿高和宽分别压缩,并将其分别与通道信息联系起来做注意力加权操作,同时提出一种新的通道描述指标表征通道信息,增强空间信息与通道信息的交互,使模型更容易关注到综合了空间和通道维度上的特征图的重要信息,在主干网络末端插入PDA后使模型平均精度(mean average precision,mAP)0.5提升了2.4个百分点,mAP0.5:0.95提升了4.4个百分点;针对实时检测场景的部署和检测速度要求模型拥有较少的参数量和计算量,因此提出了新的轻量化特征提取模块AC3代替原YOLOv5模型中的C3模块,该模块使插入PDA后的改进模型在精度仅仅损失0.2个百分点的情况下,参数量(parameters,Param.)减少了20%左右,浮点运算量(giga floating-point operations,GFLOPs)减少了30%左右。实验结果表明,最终的改进模型比YOLOv5s原模型在VOC行人数据集上mAP0.5提升了2.2个百分点,mAP0.5:0.95提升了3.1个百分点,且参数量减少了20%左右,浮点运算量减少了30%左右,在GTX1050上的检测速度(frames per second,FPS)提升了4。
