From the Cover: PNAS Plus: Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified “burst” patterns—radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator.The ParA/MinD family of adenosine triphosphatases (ATPases) forms dynamic patterns on biological surfaces, such as the nucleoid or the inner membrane, to spatially organize a variety of processes including the segregation of plasmids, chromosomes, and organelles as well as positioning the cell division machinery (1, 2). These ATPases are commonly associated with a partner protein that stimulates the local release of the ATPase from its binding surface, resulting in the formation of a dynamic ATPase pattern that imparts positional information to the cell (3). Despite the ubiquity of these minimal self-organizing positioning systems and their importance in a wide variety of essential processes throughout the microbial world, the patterning mechanism is not fully understood.In Escherichia coli, the MinD ATPase forms a cell-pole to cell-pole oscillator on the membrane, as a standing wave with a node at the cell center, in response to its stimulator protein MinE (4–6). The final component of the Min system is the inhibitor of divisome assembly, MinC, which is a passenger protein on MinD and not required for oscillation (7, 8). Oscillations apparently result from the perpetual chase and release of MinD by MinE on the membrane, which produces a time-averaged concentration of MinC that is lowest at midcell (4, 8–11). The Min system therefore promotes symmetric cell division at midcell by inhibiting division near the poles (5).The patterning reaction occurs from a series of ATP-driven protein–protein and protein–membrane interactions. ATP promotes MinD dimerization and membrane binding via its membrane-targeting sequence (MTS) (12–15). It is currently thought that membrane-bound MinD at a cell pole recruits MinE dimers to the membrane in the form of an E-ring residing on the periphery of the MinD-bound zone (5, 6, 16). MinE also has an MTS essential for the spatial regulation of cell division (17). Structural studies suggest that, in solution, the MTS and the adjacent MinD–interaction domain of MinE are sequestered in the hydrophobic core of the dimer (18, 19). Thus, under physiological conditions, MinE does not interact with MinD in the absence of membrane and shows only a weak affinity for E. coli membrane in the absence of MinD (17). This transient interaction with membrane apparently helps unveil the adjacent MinD interaction interface, which is bound and stabilized by membrane-bound MinD (18, 19). MinD interaction with MinE leads to ATP hydrolysis and membrane release (16). After MinD release, MinE dimers remain bound to the membrane (17, 20–22). This “lingering” MinE species in vivo could remain on the membrane at the cell pole from which MinD just disassembled, thereby providing positional memory to the system by preventing MinD from rebinding the same pole and directing de novo MinD binding to the opposing pole.The role of lingering MinE in patterning via its membrane-binding activity has been debated. Previous in vitro reconstitutions suggest a role for MinE lingering on the membrane after releasing MinD (22, 23), but doubt remains as to whether this property is important for self-organization. For example, in vivo dynamics have been recapitulated by simulations based on different models either with (24) or without (25, 26) considering MinE membrane-binding activity. Here we address the molecular mechanism by directly studying the MinE membrane-binding requirement on patterning. We find that a MinE mutant lacking its MTS can still form patterns, but cannot form an E-ring or regulate the periodicity of the standing wave.The apparent simplicity of this binary system makes it attractive for modeling, and many mathematical models recapitulate qualitative aspects of Min patterns (11, 23, 25, 27). Each model is predicated on significantly different molecular mechanisms, but all recapitulate the in vivo dynamics quite well, which makes it difficult to discriminate among the considered mechanisms. To date, the underlying mechanistic basis for patterning has not been uniquely constrained by in vivo or in vitro experiments. Specifically, to achieve self-organized spatiotemporal patterning, at least one nonlinear reaction term is required, e.g., a higher-order concentration-dependent rate, in addition to at least one energy-coupled reaction step that is irreversible. In previous modeling exercises, this critical nonlinearity has been included in a variety of manners as ad hoc cooperativity parameters without direct experimental validation. Therefore, it remains critically important to experimentally identify the molecular mechanistic source of this nonlinearity.To experimentally dissect the mechanistic details of the system, we developed a cell-free imaging technique to visualize Min patterning on a supported lipid bilayer (SLB). Min patterning was first reconstituted in the form of spiraling wave trains of MinD chased by MinE on the bottom of an SLB-coated well (28). In our flowcell setup, several modes of Min patterning, including spirals, have been reconstituted (22, 29). However, in all of these previous reconstitutions, patterns are achieved with protein densities on the bilayer far in excess of those in vivo. Furthermore, the observed patterns lack standing wave dynamics with nodes where the time-averaged local MinD concentration is minimum, as observed at midcell in vivo. Recently, standing wave dynamics were reconstituted by isolating the reaction in small volumes (30, 31). However, the mechanistic basis for standing wave oscillation has not been experimentally addressed. We hypothesized that Min protein depletion from the cytoplasm as proposed earlier by Meinhardt and de Boer (11) is a critical, but experimentally unexplored, feature of the oscillation mechanism.With our flowcell setup, we find that, under limiting MinD conditions, MinD and MinE form a previously unidentified pattern that we term “bursts.” Bursts share several features with in vivo Min patterns such as the ability to spontaneously form standing wave patterns even at a membrane-surface area-to-volume ratio much lower than a bacterial cell. Our observations indicate that pole-to-pole oscillation of Min proteins is correlated with periodic depletion of MinD dimers from the cytoplasm during the formation of MinD polar zones on the membrane. Together, the data reported here support a patterning mechanism that is largely governed by the local MinD:MinE stoichiometry on the membrane, which acts as a toggle switch promoting MinE-stimulated MinD recruitment to the membrane when MinD is in excess or MinE-stimulated MinD release from the membrane when MinE is in excess. We propose that this toggle switch mechanism, coupled with cytoplasmic MinD depletion, provides the crucial nonlinear coupling terms that support standing wave oscillations of the Min system.     

Effects of primary glaucoma on sleep quality and daytime sleepiness of patients residing at an equatorial latitude

AIM: To investigate the efficacy and the safety of umbilical cord derived mesenchymal stem cell (UC-MSC) implantation in patients with retinitis pigmentosa (RP). METHODS: This prospective, single-center, phase 3 clinical study enrolled 124 eyes of 82 RP patients. The patients received 5 million UC-MSCs to the suprachoroidal area with a surgical procedure. Patients were evaluated on the 1st day, 1st and 6th month postoperatively. Best corrected visual acuity (BCVA), anterior segment and fundus examinations, color photography, optical coherence tomography (OCT), and visual ?eld (VF) tests were carried out at each visit. Fundus ?uorescein angiography (FFA) and multifocal electroretinography (mfERG) recordings were performed at the end of the 6th month. Ocular and systemic adverse events of the surgical procedure were also noted. RESULTS: All of the 82 patients completed the 6-month follow-up period. None of them had any serious systemic or ocular complications. There were statistically significant improvements in BCVA and VF during the study (all P<0.05). The amplitudes of the P1 waves in the central areas showed significant improvements in mfERG recordings. There were also significant increases in implicit times of P1 waves in the central areas. CONCLUSION: Suprachoroidal administration of UC-MSCs has beneficial effect on BCVA, VF, and mfERG measurements during the 6-month follow-up period. Cell mediated therapy based on the secretion of growth factors (GFs) seems to be an effective and safe option for degenerative retinal diseases.     

骨髓单个核细胞复合异种骨修复坏死股骨头

目的:观察骨髓单个核细胞复合异种骨对坏死股骨头的修复效果。方法:实验于2006-04/10在辽宁医学院附属第一医院骨科实验室完成。15只新西兰白兔采用杨述华等液氮冷冻法的改进方法建立双侧股骨头坏死模型,即术中只切开关节囊,不切断股骨头圆韧带,股骨头不脱位行液氮冷冻。手术后行自体对照,一侧髋关节为实验组,另一侧为对照组。实验组为髓芯减压后植入复合骨髓单个核细胞的异种骨,对照组髓芯减压后植入异种骨。术后2,4,8周两组分别行X射线检查、Masson染色检查及计算机图像分析。观察坏死股骨头修复情况。结果:纳入新西兰白兔15只,均进入结果分析。X射线及Masson染色检查示实验组股骨头坏死修复效果优于对照组。术后2,4,8周实验组新生骨小梁体积比分别高于对照组,差异有显著性意义[分别为(25.49±0.60)%,(24.44±0.76)%;(32.45±0.37)%,(31.08±0.38)%;(37.93±0.40)%,(36.30±0.74)%,t=2.901,4.936,6.627,P<0.05]。结论:骨髓单个核细胞复合异种骨对坏死股骨头有良好的修复效果。     

髋关节置换术后股骨假体周围骨密度的变化

目的:总结国内外全髋关节置换术后股骨假体周围骨密度变化的研究,为全髋关节置换术后假体周围骨溶解的早期发现提供依据。资料来源:检索Medline1980-01/2006-04和Embase1980-10/2006-08期间的相关文章,检索词"Total Hip Arthroplasty,bone loss",并限定文章语言种类为English。同时检索万方数据库1990-01/2001-04期间的相关文章,限定文章语言种类为中文,检索词"全髋置换术,假体周围,骨密度"。资料选择:纳入标准是与全髋置换术后假体周围骨密度相关的文章,以临床研究类文章为主,同时包括综述性文章。资料提炼:共收集相关文献56篇,按上述标准纳入30篇,其余文献均被排除。资料综合:全髋置换术股骨假体有两种固定方式:骨水泥固定型与非骨水泥固定型。两种类型虽然固定方式不同,但术后假体周围均存在骨量丢失,应用双能X线吸收测定法能够早期检测出假体周围骨量的丢失。结论:双能X线吸收测定法逐渐成为定量监测假体周围骨量丢失的重要手段,术后连续性检测假体周围骨密度对于早期发现假体松动有重要意义。     

主动脉超微结构和细胞间黏附分子1在慢性强迫游泳应激大鼠模型的表达

目的:观察主动脉超微结构改变和主动脉细胞间黏附分子1在慢性强迫游泳应激模型大鼠体内的表达特点,为应激增加心血管系统疾病危险性的研究机制提供新的依据。方法:实验于2005-07/10在中国医科大学生理实验室完成。①实验分组:将26只健康青年雄性Wistar大鼠随机分成实验组和对照组各13只。②实验方法:实验第1～21天,每天上午8:00将大鼠置于水温4℃,水深为30cm的水池中强迫其游泳,每次游5min,1次/d。对照组不给予任何刺激。③实验评估:实验第22天各组大鼠麻醉后断头处死,处死前再次测定行为并称质量。透射电镜观察大鼠主动脉超微结构改变;采用免疫组化ABC法检测主动脉细胞间黏附分子1表达情况。结果:26只大鼠均进入结果分析。①开场实验结果及体质量:经过21d应激,实验组大鼠体质量明显下降,水平穿越格数、直立次数、修饰次数减少,粪便粒数、中央格停留时间增加。②主动脉超微结构:实验组大鼠主动脉组织部分内皮细胞脱落,平滑肌细胞由收缩表型向合成表型转化,向内膜迁移,线粒体变性,对照组大鼠主动脉超微结构基本正常。③主动脉组织细胞间黏附分子1表达:实验组主动脉组织细胞间黏附分子1表达增强,显著高于对照组(11.35±5.41,22.85±5.71,t=5.06,P<0.01)。结论:慢性强迫游泳应激可以导致大鼠主动脉超微结构发生改变,主动脉细胞间黏附分子1的表达增强,慢性强迫游泳应激所致主动脉血管内皮细胞损伤可能和炎症反应有关。     

血管内皮生长因子联合骨髓单个核细胞自体移植治疗大鼠肢体缺血

目的:将血管内皮生长因子应用于骨髓单个核细胞自体移植治疗肢体缺血,探索一种简单、安全有效治疗肢体缺血的方法,同时初步分析血管内皮生长因子促进血管新生的机制和疗效。方法:实验于2005-03/2006-07在上海交通大学附属第六人民医院动物实验中心完成。将48只雄性Wistar大鼠采用随机单位组设计分为4组:缺血对照组、血管内皮生长因子治疗组、骨髓单个核细胞治疗组、转染血管内皮生长因子基因的骨髓单个核细胞治疗组,每组12只。结扎所有动物左后肢股动脉及分支,制备后肢缺血模型。以微量加样器自结扎处以下2cm开始,间隔0.2cm为一注射点,共注射于内收肌和腓肠肌5点,每注射点各注射60μL,缺血对照组大鼠注射300μL培养液,血管内皮生长因子治疗组大鼠注射含5μg基因pcDNA3.1-hVEGF165的DNA-脂质体复合物,骨髓单个核细胞治疗组大鼠注射3×106个骨髓单个核细胞,转染血管内皮生长因子基因的骨髓单个核细胞治疗组大鼠注射含3×106个血管内皮生长因子的骨髓单个核细胞。于移植后4周行动脉造影,采用RT-PCR检测血管内皮生长因子基因的体内表达,采用免疫组化法检测缺血区新生血管密度,评价血管新生情况。结果:48只大鼠均造模成功,并进入结果分析。①移植后4周动脉造影显示,缺血对照组大鼠股动脉及其分支均未显影,血管内皮生长因子治疗组和骨髓单个核细胞治疗组可见中等量新生血管,两组效果相似,转染血管内皮生长因子基因的骨髓单个核细胞治疗组可见有大量新生血管新生,丰富的侧枝循环建立。②大鼠毛细血管密度测定结果显示,血管内皮生长因子治疗组和骨髓单个核细胞治疗组均较缺血对照组新生血管数量增加,转染血管内皮生长因子基因的骨髓单个核细胞治疗组可进一步增强疗效[(10.0±0.8),(21.7±1.9),(20.4±3.3),(42.1±3.5)个/HP,P<0.05],血管内皮生长因子治疗组和骨髓单个核细胞治疗组两组之间差异无明显统计学意义(P>0.05)。③RT-PCR检测大鼠血管内皮生长因子基因的体内表达,转染血管内皮生长因子基因的骨髓单个核细胞治疗组和血管内皮生长因子治疗组均有扩增,转染血管内皮生长因子基因的骨髓单个核细胞治疗组人血管内皮生长因子mRNA相对含量表达高于血管内皮生长因子治疗组(0.191±0.044,0.094±0.032,P<0.05)。结论:本组实验结果说明采用骨髓单个核细胞作为基因载体细胞治疗大鼠肢体缺血,能使血管内皮生长因子获得稳定有效的表达,其效果优于直接基因注射。     

壳聚糖/肝素和壳聚糖/重组水蛭素共价包被镍钛合金片的体外生物相容性

目的:体外评价生物活性分子共价包被镍钛金属片后的生物相容性。方法:实验于2005-11/2006-10在南京医科大学心血管药理实验室完成。实验材料:镍钛合金片(镍含量55.3%～55.6%,钛含量43.5%～44.2%,厚度0.05cm,激光切割为0.5cm×0.5cm);壳聚糖(脱乙酰度90%,Mr200000);肝素、重组水蛭素。实验分组:①实验分4组:未包被组、壳聚糖组、壳聚糖/肝素组、壳聚糖/重组水蛭素组。将壳聚糖、肝素(1g/L)和重组水蛭素(20mg/L)用共价化学交联法包被于镍钛金属片单面。②溶血实验:将各组样品加0.2mL稀释血液和10mL生理盐水。阳性对照和阴性对照分别用10mL蒸馏水和10mL生理盐水各加0.2mL稀释血,与样品同样操作。③人全血动态接触实验:各组镍钛合金片分别放入10mL新鲜的人全血后行扫描电镜检查,并检测血常规(红细胞计数、白细胞计数、血小板计数)及凝血3项(部分凝血活酶活化时间、凝血酶原时间、凝血酶时间)。④内皮细胞种植、培养:人脐静脉内皮细胞培养、传代后,植入每孔已放入1片镍钛金属片的24孔板中,倒置显微镜下观察人脐静脉内皮细胞生长情况及细胞形态。⑤免疫荧光标记:Fn免疫荧光染色(红色激发波长510nm)观察细胞生长和黏附情况,Ki67免疫荧光标记(绿色激发波长510nm)观察细胞增殖情况。实验评估:①各组血液样本溶血率。②各组人全血接触实验后血液样本血细胞计数和凝血活性。③扫描电镜下各组人全血接触实验后镍钛金属片表面形态。④倒置显微镜下人脐静脉内皮细胞形态。⑤荧光显微镜下镍钛金属片表面人脐静脉内皮细胞生长、黏附情况。⑥荧光显微镜下镍钛金属片表面人脐静脉内皮细胞增殖情况。结果:①溶血率:各组镍钛金属片溶血率均小于1.7%。②血细胞计数和凝血活性:各组接触血液后红细胞、白细胞和血小板计数与未接触血液无差别;壳聚糖/重组水蛭素组与壳聚糖/肝素组部分凝血活酶活化时间、凝血酶原时间和凝血酶时间值较未包被组和壳聚糖组明显延长,差异有显著性意义(P<0.001);壳聚糖/重组水蛭素组部分凝血活酶活化时间和凝血酶时间值较壳聚糖/肝素组延长,差异有显著性意义(P<0.005)。③扫描电镜下各组人全血接触实验后镍钛金属片表面形态:镍钛合金片表面有纤维蛋白黏附、血小板黏附和聚集,顺序依次为壳聚糖组>未包被组>壳聚糖/肝素组>壳聚糖/重组水蛭素组。④倒置显微镜下人脐静脉内皮细胞形态:与人脐静脉内皮细胞72h孵育,见各组镍钛金属片边缘细胞生长、移行良好,无细胞变形。⑤荧光显微镜下镍钛金属片表面人脐静脉内皮细胞生长、黏附情况:利用Fn免疫荧光标记人脐静脉内皮细胞,镍钛金属片表面的人脐静脉内皮细胞黏附和生长顺序如下:壳聚糖组>未包被组>壳聚糖/重组水蛭素组>壳聚糖/肝素组。⑥荧光显微镜下镍钛金属片表面人脐静脉内皮细胞增殖情况:利用Ki67免疫荧光标记人脐静脉内皮细胞,镍钛金属片表面的人脐静脉内皮细胞增殖顺序如下:壳聚糖组=未包被组>壳聚糖/重组水蛭素组>壳聚糖/肝素组。结论:壳聚糖/肝素与壳聚糖/重组水蛭素包被镍钛金属后具有良好抗凝血活性,但壳聚糖/重组水蛭素与壳聚糖/肝素相比更有利于人脐静脉内皮细胞的生长。     

大鼠海马神经细胞体外缺糖缺氧模型制备方法的改进

目的:改进培养大鼠海马神经细胞体外缺糖缺氧模型制备方法,并通过兴奋性氨基酸N-甲基-D-天冬氨酸受体拮抗剂进行验证。方法:实验于2004-09/2005-06在南方医科大学基础医学院神经生物学教研室进行。实验材料:出生1d内清洁级SD大鼠,由南方医科大学实验动物中心提供(合格证号为粤证监字2004B023号)。N-甲基-D-天冬氨酸受体拮抗剂5-甲基二氢丙环庚烯亚胺马来酸(MK-801)和D-2-氨基-5-磷酰基戊酸(d-APV)购自Sigma公司。实验方法:取新生1dSD大鼠海马组织作神经细胞分散细胞原代培养,培养到13d时进行氧/葡萄糖剥夺模型的制备:将neurobasal培养基更换为不含葡萄糖的BSSo培养基,连续充以50mL/LCO2 950mL/LN2(体积比)混合气体。缺氧30,45,60,90min后取出细胞,更换为正常neurobasal培养基,恢复正常条件继续培养。将神经细胞随机分为正常对照组、单纯缺氧组、无糖缺氧组、MK-801组和d-APV组。将10μmol/LMK-801和500μmol/Ld-APV在通50mL/LCO2 950mL/LN2混合气前加入到BSSo培养基,缺氧结束后随BSSo培养基一起去掉。复氧24h后采用MTT比色法测神经细胞成活率及Hoechst荧光染料法测神经细胞凋亡率。结果:①随着氧/葡萄糖剥夺时间延长,神经细胞存活率下降。氧/葡萄糖剥夺30,45,60,90min再复氧24h,神经细胞存活率分别为(81.48±3.84)%、(63.14±3.14)%、(41.73±2.97)%和(16.78±2.12)%。②N-甲基-D-天冬氨酸受体拮抗剂MK-801(10μmol/L)和d-APV(500μmol/L)均能明显增加神经细胞存活率(P<0.05),并且两组细胞存活率与正常对照组比较,差异无统计学意义(P>0.05)。结论:实验制备的海马神经细胞短时间氧/葡萄糖剥夺模型可对神经细胞造成迟发性死亡,兴奋性氨基酸N-甲基-D-天冬氨酸受体拮抗剂可保护氧/葡萄糖剥夺诱导的神经细胞损伤,说明本实验建立的缺氧模型有效并简便可靠,并且缩短了缺氧时间。     

Effects of knockout of the receptor for advanced glycation end‐products on bone mineral density and synovitis in mice with intra‐articular fractures

Our group employed the mouse closed intra‐articular fracture (IAF) model to test the hypothesis that the innate immune system plays a role in initiating synovitis and post‐traumatic osteoarthritis (PTOA) in fractured joints. A transgenic strategy featuring knockout of the receptor for advanced glycation end‐products (RAGE ^?/?) was pursued. The 42 and 84 mJ impacts used to create fractures were in the range previously reported to cause PTOA at 60 days post‐fracture. MicroCT (μCT) was used to assess fracture patterns and epiphyseal and metaphyseal bone loss at 30 and 60 days post‐fracture. Cartilage degeneration, synovitis, and matrix metalloproteinase (MMP‐3, ‐13) expression were evaluated by histologic analyses. In wild‐type mice, μCT imaging showed that 84 mJ impacts led to significant bone loss at 30 days (p < 0.05), but recovered to normal at 60 days. Bone losses did not occur in RAGE^?/? mice. Synovitis was significantly elevated in 84 mJ impact wild‐type mice at both endpoints (30 day, p = 0.001; 60 day, p = 0.05), whereas in RAGE^?/? mice synovitis was elevated only at 30 days (p = 0.02). Mankin scores were slightly elevated in both mouse strains at 30 days, but not at 60 days. Immunohistochemistry revealed significant fracture‐related increases in MMP‐3 and ?13 expression at 30 days (p < 0.05), with no significant difference between genotypes. These findings indicated that while RAGE ^?/? accelerated recovery from fracture and diminished synovitis, arthritic changes were temporary and too modest to detect an effect on the pathogenesis of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2439–2449, 2018.