Modelling of a DNA packaging motor

During the assembly of many viruses, a powerful molecular motor packages the genome into a preassembled capsid. The Bacillus subtilis phage φ29 is an excellent model system to investigate the DNA packaging mechanism because of its highly efficient in vitro DNA packaging activity and the development of a single-molecule packaging assay. Here we make use of structural and biochemical experimental data to build a physical model of DNA packaging by the φ29 DNA packaging motor. Based on the model, various dynamic behaviours such as the packaging rate, pause frequency and slip frequency under different ATP concentrations, ADP concentrations, external loads as well as capsid fillings are studied by using Monte Carlo simulation. Good agreement is obtained between the simulated and available experimental results. Moreover, we make testable predictions that should guide future experiments related to motor function.

Mitochondria Dynamically Transplant into Cells in Vitro and in Mice and Rescue Aerobic Respiration of Mitochondrial DNA-Depleted Motor Neuron NSC-34

It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells’ mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson’s disease rats. To prepare for mitochondrial transplantation study in human neurodegenerative diseases, we select human fibroblasts as mitochondrial donor because that fibroblasts share many characteristics with mesenchymal stromal cells (MSCs). We isolate human primary fibroblasts and develop a mitochondrial DNA (mtDNA)-depleted mouse motor neuron NSC-34 cells (NSC-34 ρ° cells). Fibroblast and NSC-34 cell’s mitochondria are co-cultured with NSC-34 ρ° cells. Mitochondrial transplantation is observed by fluorescent microscopy. Gene expression is determined by polymerase chain reaction (PCR) and real time PCR (qPCR). Also, mitochondria are injected to mice bearing mammary adenocarcinoma 4T1 cells. We find results as following: 1) There are abundant mitochondria in fibroblasts (337 ± 80 mitochondria per fibroblast). 42.4% of viable mitochondria are obtained by using differential centrifugation. The isolated mitochondria actively transplant into NSC-34 ρ° cells after co-culture. 2) Fibroblasts transfer mitochondria to human mammary adenocarcinoma MCF-7 cells. 3) There is no expression of HLA-I antigen in fibroblast’s mitochondria indicating they can be used for allogeneic mitochondrial transplantation without HLA antigen match. 4) PCR and qPCR show that NSC-34 ρ° cells lose mitochondrially encoded cytochrome c oxidase I (MT-CO1) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and upregulate expression of glycolysis-associated genes hexokinase (HK2), glucose transporter 1 (SLC2A1) and lactate dehydrogenase A (LDHA). 5) Transplantation of NSC-34 mitochondria restores MT-CO1 and MT-ND1 and downregulates gene expression of HK2, SLC2A1 and LDHA. 6) Normal mammary epithelial mitochondria successfully enter to 4T1 cells in mice. Subcutaneous injection of mitochondria is safe for mice. In summary, mitochondrial transplantation replenishes mtDNA and rescues aerobic respiration of diseased cells with mitochondrial dysfunction. Human primary fibroblasts are potential mitochondrial donor for mitochondrial transplantation study in human neurodegenerative diseases.

机动车排放PM_(2.5)对EA.hy926细胞DNA甲基化效应研究

目的探讨机动车排放PM2.5对EA.hy926细胞(人脐静脉内皮细胞和人肺腺癌细胞株A549融合的细胞株)增殖功能、DNA甲基化及凋亡的影响。方法机动车排放PM2.5对EA.hy926细胞染毒24 h。采用MTS法测定机动车排放PM2.5对细胞增殖功能的影响;DNA甲基化定量检测试剂盒(比色法)测定机动车排放PM2.5对细胞DNA甲基化的影响;Annexin V-FITC/PI染色,流式细胞仪测定机动车排放PM2.5对细胞凋亡的影响。结果机动车排放PM2.5染毒24 h可抑制细胞增殖功能,当染毒质量浓度为200μg/m L时,与PBS对照组比较差异有统计学意义(P<0.05);随着染毒质量浓度的增加细胞DNA甲基化率(5-m C%)下降,在染毒质量浓度为50μg/m L和200μg/m L时,与PBS对照组比较差异有统计学意义(P<0.05);随染毒质量浓度升高细胞诱导凋亡率逐渐升高。结论机动车尾气排放PM2.5可以抑制EA.hy926细胞的增殖功能,改变EA.hy926细胞的DNA甲基化率,对EA.hy926细胞产生诱导凋亡作用。此外DNA甲基化可能是细胞增殖和凋亡的机制,定点基因的甲基化仍需要进一步研究。

利用机油提高琼脂糖凝胶中DNA片段的回收率

目的:发展一种简单快速经济的回收琼脂糖凝胶中DNA的方法。方法:将切的琼脂糖凝胶胶块放入嵌套Eppendorf管中捣碎,加入50μL机油,室温下12 000r/min离心5min,取大Eppendorf管中收集的液体跑胶检验,凝胶上包括代表100bp～2000bp不同大小DNA分子回收率的分子量标准DL2000。结果:DNA回收率可由加机油前的约35%提高为加机油后的45%-90%,回收效率的波动主要取决于DNA片段的大小、切下的含有DNA的胶块的大小及操作者的熟练程度。结论:该方法快速、简便、经济,具有良好的重复性与特异性,比许多国产的试剂盒更可靠。

DNA纳米机器及其核酸传感应用

DNA纳米机器是由系列功能核酸序列组成的一类分子机器,由剪切作用和链置换反应驱动,可以在纳米轨道上实现物质的运输。DNA纳米机器易于构建与表征,其结构可以根据需要自由设计且变化可控,因而受到科研工作者的广泛关注。文章概述了DNA纳米机器近几十年来的发展与应用,重点探讨了DNA walker、DNA tweezer、DNA motor三种纳米机器以及它们在核酸传感器中的应用。

Nuclear TAR DNA-binding protein 43 A new target for amyotrophic lateral sclerosis treatment

Abnormal TAR DNA-binding protein 43 （TDP-43） inclusion bodies can be detected in the degen- erative neurons of amyotrophic lateral sclerosis. In this study, we induced chronic oxidative stress injury by applying malonate to cultured mouse cortical motor neurons. In the later stages of the malonate insult, TDP-43 expression reduced in the nuclei and transferred to the cytoplasm. This was accompanied by neuronal death, mimicking the pathological changes in TDP-43 that are seen in patients with amyotrophic lateral sclerosis. Interestingly, in the early stages of the response to malonate treatment, nuclear TDP-43 expression increased, and neurons remained relatively intact, without inclusion bodies or fragmentation. Therefore, we hypothesized that the increase of nuclear TDP-43 expression might be a pro-survival factor against oxidative stress injury. This hypothesis was confirmed by an in vitro transgenic experiment, in which overexpression of wild type mouse TDP-43 in cultured cortical motor neurons significantly reduced malonate-induced neuronal death. Our findings suggest that the loss of function of TDP-43 is an important cause of neuronal degen- eration, and upregulation of nuclear TDP-43 expression might be neuroprotective in amyotrophic lateral sclerosis.

发作性运动诱发性运动障碍患者PRRT2基因突变的研究

目的:探讨发作性运动诱发性运动障碍(Paroxysmal kinesigenic dyskinesia,PKD)个体与富含脯氨酸的跨膜蛋白2(Proline-rich transmembrane protein2,PRRT2)基因突变的相关性。方法:收集由佳木斯中心医院提供的14例散发病例的外周血3mL,同时取健康人30例对照。DNA提取试剂盒提取外周血DNA。PCR扩增PRRT2基因外显子2的全序列,PCR产物分别送上海生工测序,测序结果用Blast与Genebank中的序列(NM—145239.3)进行比对,寻找突变位点。结果:14例个体中发现4例错义突变,30例健康对照中均无基因突变。4例基因突变分别为c.846-847 insC(p.P217fsX)(5/14)、c.906G>A(p.Glys237Arg)(1/14)、c.609C>G(p.Pro138Ala)(5/14)和c.882G>A(p.Arg229 Lys)(9/14),其中(c.882G>A(p.Arg229 Lys)为新发现的突变。结论:PRRT2基因突变是PKD的主要致病基因,本研究发现了一个新的点突变,扩大了PKD发病的基因谱,为阵发性运动障碍疾病的诊断提供新的检测突变点。

焦炉逸散物和汽车尾气有机提取物对人肝癌细胞的遗传毒性作用

目的：探讨焦炉逸散物和汽车尾气有机提取物对人肝癌HepG2细胞的遗传毒性作用。方法：不同浓度的焦炉逸散物有机提取物（0.2、1.1、5.5、28、138和688μg/L）和汽车尾气有机提取物（0.006、0.06、0.6、6.2、63和626.3 ng/L）染毒HepG2细胞24 h后,采用单细胞凝胶电泳实验和双微核实验分别测定细胞存活率、Olive尾矩、尾部DNA百分含量、尾长、微核率、核质桥率和核芽率。结果：焦炉逸散物和汽车尾气有机提取物染毒后,细胞Olive尾矩、尾部DNA含量和尾长均随着染毒剂量的增加而逐渐增加,与对照组相比,除0.006 ng/L汽车尾气有机提取物组的Olive尾矩外,差异均具有统计学意义（P〈0.05）。焦炉逸散物有机提取物染毒后,Hep G2细胞的微核率和核质桥率明显高于对照组（P〈0.05）,且均在5.5μg/L时达到最高值,分别为对照组的3.6和2.5倍。核芽率随着焦炉逸散物有机提取物浓度的增加而逐渐增加,且在最高染毒浓度时为对照组的2.3倍。与对照组相比,0.6~626.3 ng/L汽车尾气有机提取物染毒后细胞的微核率和核质桥率均显著增加（P〈0.01）,63和626.3 ng/L汽车尾气有机提取物作用后,细胞核芽率显著增加（P〈0.05）。结论：焦炉逸散物和汽车尾气有机提取物均可诱导HepG2细胞内的DNA和染色体损伤,单细胞凝胶电泳实验可作为低浓度环境复合污染物遗传毒性的快速评估方法。

Enzyme-free and multiplexed micro RNA detection using micro RNA-initiated DNA molecular motor

In this work,we have developed a sensitive,simple,and enzyme-free assay for detection of micro RNAs(mi RNAs)by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains.In the presence of mi RNA target,it can hybridize with one of the stem-loop DNA to open the stem and to produce a mi RNA/DNA hybrid and a single strand(ss)DNA,the ss DNA will in turn hybridize with another stem-loop DNA and finally form a double strand(ds)DNA to release the mi RNA.One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence.The formation of ds DNA can produced specific fluorescence signal for mi RNA detection.The released mi RNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor,which results in great fluorescence amplification.With the efficient signal amplification,as low as 1 pmol/L mi RNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained.Moreover,by designing different stem-loop DNAs specific to different mi RNA targets and labeling them with different fluorophores,multiplexed mi RNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum(SFS)technique.

应用基因组测序技术诊断缺失型脊肌萎缩症

目的评价基因组测序技术在诊断缺失型脊肌萎缩症（spinal muscular atrophy, SMA）中的应用。方法应用聚合酶链反应扩增100例SMA临床确诊患儿和110名对照样本的运动神经元存活基因（spinal muscular atrophy,SMN），基因组测序技术通过识别扩增片段中SMN1和SMN2的4个差异位点用来诊断SMN1的纯合缺失，并以多重连接探针扩增进行验证。结果100例SMA样本中基因组测序显示差异位点仅有SMN2基因特有碱基峰，多重连接探针扩增检测示SMN1与SMN2拷贝数为0：2或0：3，表明SMN1基因纯合缺失。对照组中5例样本的差异位点仅为SMN1基因特有碱基峰，SMN1与SMN2拷贝数为2：0，为SMN2纯合缺失。105例样本的差异位点均为杂合峰，SMN1与SMN2拷贝数为2：2，未显示SMN1和SMN2的缺失。所有测序结果与MLPA检测结果一致。结论基因组测序技术在进行缺失型脊肌萎缩症的分子诊断中具有特异、准确、实用的特点。

Smart Engineering of a Self-Powered and Integrated Nanocomposite for Intracellular MicroRNA Imaging

Over the past 2 years,many DNA motors have been synthesized and run in living cells,but there are still challenges in designing integrated DNA motors self-powered to enable autonomous intracellular walking without auxiliary additives.Herein,we report a smart strategy based on a DNA motor–MnO2 nanocomposite,which successfully meets these requirements of intracellular analysis and enables sensitive imaging of specific microRNAs(miRNAs)in living cells.

运动能力遗传学研究中的双生子类型诊断方法的发展

运动能力遗传学研究是当前解决与运动有关的性状的先天遗传与后天训练之争的关键。这一问题的解决，势必为科学选材和科学训练带来一次革命。双生子分析法是人类遗传学研究的经典方法。采用此法进行运动能力的遗传学研究，首先要解决的一个问题是双生子类型的准确诊断。诊断方法很多，各有利弊。从其发展来看，是由外部容貌观察到组织、细胞及分子水平的研究，即从现象到本质的发展。深入本质的探讨最终会为科学选材及科学训练提供可靠的保障。