Previously, mouse bone marrow-derived stem cells （MSC） treated with the unspecific DNA methyltransferase inhibitor 5-azacytidine were reported to differentiate into cardiomyocytes. The aim of the present study was t...Previously, mouse bone marrow-derived stem cells （MSC） treated with the unspecific DNA methyltransferase inhibitor 5-azacytidine were reported to differentiate into cardiomyocytes. The aim of the present study was to investigate the efficiency of a similar differentiation strategy in human mononuclear cells obtained from healthy bone marrow donors. After 1-3 passages, cultures were exposed for 24 h to 5-azacytidine （3 μM） followed by 6 weeks of further culture. Drug treatment did not induce expression of myogenic marker MyoD or cardiac markers Nkx2.5 and GATA-4 and did not yield beating cells during follow-up. In patch clamp experiments, approximately 10-15% of treated and untreated cells exhibited L-type Ca^2＋ currents. Almost all cells showed outwardly rectifying K^＋ currents of rapid or slow activation kinetics. Mean current amplitude at ＋60 mV doubled after 6 weeks of treatment compared with time-matched controls. Membrane capacitance of treated cells was significantly larger than in controls 2 weeks after treatment and remained high after 6 weeks, Expression levels of mRNAs for the K^＋ channels Kv 1,1, Kv 1,5, Kv2,1, Kv4,3 and KCNMA 1 and for the Ca^2＋ channel Cav 1.2 were not affected by 5-azacytidine. Treatment with potassium channel blockers tetraethylammonium and clofilium at concentrations shown previously to inhibit rapid or slowly activating K^＋ currents of hMSC inhibited proliferation of these cells. Our results suggest that despite the absence of differentiation ofhMSC into cardiomyocytes, treatme.nt with 5-azacytidine caused profound changes in current density.展开更多
Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small ...Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )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
In this study,we investigated the effects of different concentrations of 5-azacytidine(AZA),a DNA methyltransferase in-hibitor,on the growth,antioxidant activities and germination of pellicle cysts of Scrippsiella acu...In this study,we investigated the effects of different concentrations of 5-azacytidine(AZA),a DNA methyltransferase in-hibitor,on the growth,antioxidant activities and germination of pellicle cysts of Scrippsiella acuminata.The purpose of this study is to understand the toxic effects of AZA on marine microalgae,and to demonstrate the effect of DNA methyltransferase inhibitors on the germination of pellicle cysts.Results showed that AZA inhibited the growth of S.acuminata significantly,and displaced a clear dose-dependent inhibition trend with the 96h EC50 of 146.77μmolL^(-1)(35.84mgL^(-1)).Pellicle cysts of S.acuminata were less sensitive to AZA than the vegetative cells,and the EC50 value of AZA to the germination of pellicle cysts of S.acuminata was 8.08mmolL^(-1)(1.97g L^(-1)).After exposed to AZA,the antioxidant activities in S.acuminata responded rapidly and significantly.Among them,soluble pro-tein and superoxide dismutase(SOD)were more sensitive to AZA,and significant promotions occurred after exposed to 10μmolL^(-1)AZA for 24h.Meanwhile,malondialdehyde(MDA)contents in algal cells did not change significantly after exposed to low concen-trations of AZA,but increased firstly and then decreased under high concentration of AZA.The glutathione(GSH)levels in S.acu-minata increased significantly under high concentrations of AZA,and remained unchanged at low concentrations of AZA.The results suggested that the enhanced protein level and SOD activity of S.acuminata eliminated reactive oxygen species(ROS)to a certain ex-tent,and thus protected algal cells against damages of ROS caused by AZA.展开更多
DNA methylation is widespread in plants and associated with plant development and defense mechanisms.However,the relationship between DNA methylation and plant secondary metabolism has rarely been reported.Here,when b...DNA methylation is widespread in plants and associated with plant development and defense mechanisms.However,the relationship between DNA methylation and plant secondary metabolism has rarely been reported.Here,when birch suspension cells were treated with 5-azacytidine(5-azaC),which blocks DNA methylation,triterpenoid accumulation was significantly promoted and antioxidant and defense enzymatic activity changed.For studying triterpenoid accumulation,0.1 mM azaC was optimal.A qRT-PCR assay revealed increased expression of genes encoding key triterpenoid biosynthetic enzymes.Evaluation of methylation polymorphisms at CCGG sites showed that the methylation level was lower in cells treated with 5-azaC.These results demonstrated that 5-azaC treatment led to an increase in the production of triterpenoids in cell cultures through a mechanism that involved in DNA methylation,which resulted in the induction of genes encoding the key enzymes.The study provides evidence of a relationship between DNA methylation and regulation of secondary metabolism.展开更多
Objective 5-azacytidine could induce the differentiation of stem cells into cardiomyocytes （CMs）. The aim of this study was to screen the optimal condition for 5-azacytidine inducing differentiation of human mesench...Objective 5-azacytidine could induce the differentiation of stem cells into cardiomyocytes （CMs）. The aim of this study was to screen the optimal condition for 5-azacytidine inducing differentiation of human mesenchumal stem cells （hMSCs） into CMs, and the effect of 5-azacytidine on adherence, cell vigor and chromosome karyotype of hMSCs. Methods hMSCs were isolated from human bone marrow and cultured in vitro. The phenotypes ofhMSCs were identified by flow cytometric analyses. MTT test was used to investigate the effect of different concentrations of 5-azacytidine on proliferation ofhMSCs. Four weeks after 5-azacytidine induction, semi-quantitative RT-PCR, transmission electron microscopy （TEM）, single-cell action potentials, detection of cardio-enzyme AST and LDH, cell adherence, cell viability and chromosome karyotype test were performed. Results The typical morphological features of hMSCs were fibroblast-like in shape, hMSCs expressed CD44 and CD105,and did not express CD34, CD45 and CD31. The optimal concentration of 5-azacytidine was 10μ mol/L. The shape of hMSCs treated with 5-Azacytidine changed from fusiform to polygon or astrocyte gradually, and passaged cells were evenly arranged as polarity structure. Indueed-hMSCs connected with neighbouring cells, fbrming myotube-like structures 4 weeks later. It was confirmed that induced hMSCs shaped myotubule-like structure and had some of micro-histologic structures of CMs by TEM. RT-PCR showed that induced hMSCs expressed cardiac specific product BNNP and early cardio-myogenesis specific transcription factor NKX2.5mRNA. Besides, induced-MSCs led to the weak action potential and secreted cardio-enzyme AST and LDH. There was no significant difference in cell adherence and viability before and after induction. Both hMSCs and induced-hNSCs kept stable normal diploid nucleus. Conclusion The optimal condition for inducing effect of 5-azacytidine is 10 la mol/L and 24-hour incubation; and under this condition, the adherence, vigor and chromosome karyotype ofhMSCs would not be affected （J Geriatr Cardio12009; 6：182-188）.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
文摘Previously, mouse bone marrow-derived stem cells （MSC） treated with the unspecific DNA methyltransferase inhibitor 5-azacytidine were reported to differentiate into cardiomyocytes. The aim of the present study was to investigate the efficiency of a similar differentiation strategy in human mononuclear cells obtained from healthy bone marrow donors. After 1-3 passages, cultures were exposed for 24 h to 5-azacytidine （3 μM） followed by 6 weeks of further culture. Drug treatment did not induce expression of myogenic marker MyoD or cardiac markers Nkx2.5 and GATA-4 and did not yield beating cells during follow-up. In patch clamp experiments, approximately 10-15% of treated and untreated cells exhibited L-type Ca^2＋ currents. Almost all cells showed outwardly rectifying K^＋ currents of rapid or slow activation kinetics. Mean current amplitude at ＋60 mV doubled after 6 weeks of treatment compared with time-matched controls. Membrane capacitance of treated cells was significantly larger than in controls 2 weeks after treatment and remained high after 6 weeks, Expression levels of mRNAs for the K^＋ channels Kv 1,1, Kv 1,5, Kv2,1, Kv4,3 and KCNMA 1 and for the Ca^2＋ channel Cav 1.2 were not affected by 5-azacytidine. Treatment with potassium channel blockers tetraethylammonium and clofilium at concentrations shown previously to inhibit rapid or slowly activating K^＋ currents of hMSC inhibited proliferation of these cells. Our results suggest that despite the absence of differentiation ofhMSC into cardiomyocytes, treatme.nt with 5-azacytidine caused profound changes in current density.
基金supported by research grants from State 863 high technology R&D Project of China(2002AA205051and 2003AA205160)the National Key Rroject for Basic Research of China(2001CB509906)
文摘Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )
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。
基金supported by the National Natural Science Foundation of China(No.42076141).
文摘In this study,we investigated the effects of different concentrations of 5-azacytidine(AZA),a DNA methyltransferase in-hibitor,on the growth,antioxidant activities and germination of pellicle cysts of Scrippsiella acuminata.The purpose of this study is to understand the toxic effects of AZA on marine microalgae,and to demonstrate the effect of DNA methyltransferase inhibitors on the germination of pellicle cysts.Results showed that AZA inhibited the growth of S.acuminata significantly,and displaced a clear dose-dependent inhibition trend with the 96h EC50 of 146.77μmolL^(-1)(35.84mgL^(-1)).Pellicle cysts of S.acuminata were less sensitive to AZA than the vegetative cells,and the EC50 value of AZA to the germination of pellicle cysts of S.acuminata was 8.08mmolL^(-1)(1.97g L^(-1)).After exposed to AZA,the antioxidant activities in S.acuminata responded rapidly and significantly.Among them,soluble pro-tein and superoxide dismutase(SOD)were more sensitive to AZA,and significant promotions occurred after exposed to 10μmolL^(-1)AZA for 24h.Meanwhile,malondialdehyde(MDA)contents in algal cells did not change significantly after exposed to low concen-trations of AZA,but increased firstly and then decreased under high concentration of AZA.The glutathione(GSH)levels in S.acu-minata increased significantly under high concentrations of AZA,and remained unchanged at low concentrations of AZA.The results suggested that the enhanced protein level and SOD activity of S.acuminata eliminated reactive oxygen species(ROS)to a certain ex-tent,and thus protected algal cells against damages of ROS caused by AZA.
基金The work was supported by The Fundamental Research Funds for the Central Universities(2572017EA05)the National Natural Science Foundation of China(31870588).
文摘DNA methylation is widespread in plants and associated with plant development and defense mechanisms.However,the relationship between DNA methylation and plant secondary metabolism has rarely been reported.Here,when birch suspension cells were treated with 5-azacytidine(5-azaC),which blocks DNA methylation,triterpenoid accumulation was significantly promoted and antioxidant and defense enzymatic activity changed.For studying triterpenoid accumulation,0.1 mM azaC was optimal.A qRT-PCR assay revealed increased expression of genes encoding key triterpenoid biosynthetic enzymes.Evaluation of methylation polymorphisms at CCGG sites showed that the methylation level was lower in cells treated with 5-azaC.These results demonstrated that 5-azaC treatment led to an increase in the production of triterpenoids in cell cultures through a mechanism that involved in DNA methylation,which resulted in the induction of genes encoding the key enzymes.The study provides evidence of a relationship between DNA methylation and regulation of secondary metabolism.
文摘Objective 5-azacytidine could induce the differentiation of stem cells into cardiomyocytes （CMs）. The aim of this study was to screen the optimal condition for 5-azacytidine inducing differentiation of human mesenchumal stem cells （hMSCs） into CMs, and the effect of 5-azacytidine on adherence, cell vigor and chromosome karyotype of hMSCs. Methods hMSCs were isolated from human bone marrow and cultured in vitro. The phenotypes ofhMSCs were identified by flow cytometric analyses. MTT test was used to investigate the effect of different concentrations of 5-azacytidine on proliferation ofhMSCs. Four weeks after 5-azacytidine induction, semi-quantitative RT-PCR, transmission electron microscopy （TEM）, single-cell action potentials, detection of cardio-enzyme AST and LDH, cell adherence, cell viability and chromosome karyotype test were performed. Results The typical morphological features of hMSCs were fibroblast-like in shape, hMSCs expressed CD44 and CD105,and did not express CD34, CD45 and CD31. The optimal concentration of 5-azacytidine was 10μ mol/L. The shape of hMSCs treated with 5-Azacytidine changed from fusiform to polygon or astrocyte gradually, and passaged cells were evenly arranged as polarity structure. Indueed-hMSCs connected with neighbouring cells, fbrming myotube-like structures 4 weeks later. It was confirmed that induced hMSCs shaped myotubule-like structure and had some of micro-histologic structures of CMs by TEM. RT-PCR showed that induced hMSCs expressed cardiac specific product BNNP and early cardio-myogenesis specific transcription factor NKX2.5mRNA. Besides, induced-MSCs led to the weak action potential and secreted cardio-enzyme AST and LDH. There was no significant difference in cell adherence and viability before and after induction. Both hMSCs and induced-hNSCs kept stable normal diploid nucleus. Conclusion The optimal condition for inducing effect of 5-azacytidine is 10 la mol/L and 24-hour incubation; and under this condition, the adherence, vigor and chromosome karyotype ofhMSCs would not be affected （J Geriatr Cardio12009; 6：182-188）.
