Establishment of Cell Free Conversion System With Biotin-labelled Recombinant PrP^(sen) Expressed in E.coli

Objective To report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope. Methods A hamster PrP protein （HaPrP） was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrP^sen preparation from scrapie strain 263K. Results Protease-resistant bands were detected after four-day incubation. Conclusion The new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

Cell-Free大规模MIMO系统中基于传输时延的缓存策略研究

为了满足未来移动互联网中用户的超低时延和超高可靠需求,将无线缓存技术与无小区大规模多输入多输出系统相结合,设计了基于AP间协作缓存及区域流行度评估的缓存模型。推导出涉及AP分簇、协作缓存以及区域流行度的传输时延表达式,并将内容放置问题表述为最小化总内容传输时延问题。通过对优化问题的NP-hard和拟阵约束下次模单调性的证明,提出了基于贪婪算法的缓存部署优化策略。仿真结果表明,所提策略可有效降低系统内容传输时延和提升缓存命中率。

Diagnostic value of negative enrichment and immune fluorescence in situ hybridization for intraperitoneal free cancer cells of gastric cancer

Objective:To explore the intraperitoneal free cancer cell(IFCC)detection value of negative enrichment and immune fluorescence in situ hybridization(NEimFISH)on chromosomes(CEN)8/17.Methods:To verify the reliability of NEimFISH,29 gastric cancer tumors,their adjacent tissues and greater omental tissues were tested.Our study then included 105 gastric cancer patients for IFCC.We defined patients as IFCC-positive if a signal was detected,regardless of the detailed cancer cell numbers.A comparison of clinicopathological features was conducted among IFCC groups.We also compared the diagnosis value and peritoneal recurrence predictive value among different detection methods.The comparison of IFCC number was also conducted among different groups.Results:A cutoff of 2.5 positive cells could distinguish all benign tissue samples and 97%of malignant tissue samples in our study.Compared to intestinal gastric cancer,patients with diffuse gastric cancer tended to have more IFCCs(6 vs.4,P=0.002).The IFCC counts were often higher in the lymphovascular invasion positive group than negative group(3 vs.1,P=0.022).All IFCC samples that were considered positive using conventional cytology were also found to be positive using NEimFISH.When compared to conventional cytology and paraffin pathology,NEimFISH had a higher IFCC positive rate(68.9%)and higher one-year peritoneal recurrence predictive value with area under the curve(AUC)of 0.922.Conclusions:Gastric cancer could be effectively diagnosed by NEimFISH.The IFCC number found using NEimFISH on CEN8/17 is closely associated with Lauren type and vascular invasion of cancer.NEimFISH is a reliable detection modality with a higher positive detection rate,higher one-year peritoneal recurrence predictive value and quantitative features for IFCC of gastric cancer.

Effects of Chuanxiongqin hydrochloride on increasing the fluidity of brain cell membrane and scavenging free radicals in model rats with ischemia/reperfusion injury

BACKGROUND: The fluidity of cell membrane can be affected by various factors. Many experiments have confirmed that the ischemia/reperfusion of organic tissue can increase the contents of free radicals, which lead to high rigidity and low fluidity of cell membrane, and the conditions can be changed by Chuanxiongqin. OBJECTIVE: To observe the effect and mechanism of Chuanxiongqin hydrochloride on the fluidity of brain cell membrane in rat models of ischemia/reperfusion. DESIGN: A completely randomized controlled animal trial. SETTINGS: Institute of Brain Sciences; Department of Physiology, Medical College, Datong University. MATERIALS: Twenty male grade Ⅰ Wistar rats of 170-220 g were randomly divided into model group (n =10) and control group (n =10). Chuanxiongqin hydrochloride (molecular mass was 172.2) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (batch number: 0817-9803); Spin labelers: 5-doxyl-stearlic acid methylester (5DS), 16-doxyl-stearlic acid methylester (16DS), xanthine, xanthine oxidase (XOD) and 5,5-dimeth-1-pyrroline- N-oxide (DMPO) from Sigma Company; Bruker ESP 300 electron paramagnetic resonance (EPR) spectrometer by Bruker Company (Germany). METHODS: The experiments were carried out in the State Key Laboratory of Natural and Biomimetic Drugs, Peking University from June 2001 to July 2002. In the model group, rats were made into models of cerebral ischemia by 30-minute ligation and 2-hour reperfusion of common carotid arteries; The rats in the control group were not made into models. The order parameter (S) and rotational correlation time (τc) were detected with the ESR spectrometer by means of spin labeling. The greater the S and τc, the smaller the fluidity. Meanwhile, the clearance rate of free radicals was detected with ESR spin trapping. The measurement data were compared using the t test. MAIN OUTCOME MEASURES: The S, τc and clearance rates of O2 · and OH· free radicals were compared between the model group and control group. RESULTS: The S and τc in the model group [0.738 4±0.003 5; (8.472±0.027)×10-10 s/circle] were obviously different from those in the control group [0.683 9±0.008 3; (7.945±0.082)×10-10 s/circle, t =5.731, 5.918, P < 0.05], which suggested that ischemia/reperfusion injury decreased the fluidity of brain cell membrane. After adding Chuanxiongqin hydrochloride, there were no obvious differences between the model group [0.688 5±0.030 5; (7.886±0.341)×10-10 s/circle] and control group (P > 0.05), indicating that Chuanxiongqin hydrochloride could recover the fluidity of brain cell membrane after ischemia/reperfusion injury close to the level in the normal control group. Chuanxiongqin hydrochloride could directly scavenge the O2 · and OH· free radicals, and the maximal clearance rates were 83.92% and 44.99% respectively. CONCLUSION: Chuanxiongqin hydrochloride increases the fluidity of membrane of ischemia-injured brain cell by scavenging both O2 ·and OH· free radicals.

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.

THE RELATIONSHIP BETWEEN SEROSAL TYPES, PATHOLOGICAL CHARACTERISTICS AND FREE CANCER CELLS IN THE PERITONEAL CAVITY OF GASTRIC CANCER PATIENTS

The relationship between free cancer cells and the pathological characteristics of gastric cancer were studied. Of 100 cases of gastric cancer, free cancer cells in the peritoneal cavity were detected in 32 cases (32%). Free cancer cells were most often found in tendonoid (62.2%) and colour diffused (60.0%) serosal types. When the area of serosal invasion was over 20 cm, the positive free cancer cell rate was 56.6% and only 2.5% if below 20 cm. Free cancer cells were related to the depth of cancer infiltration, often found in the S2 and S3 invasion layers, as well as to the pathological characteristics of gastric cancer. Free cancer cells were often seen in infiltrated type cancer, histologically poorly differentiated or undifferentiated adenocarcinoma. and nest or diffused growth types. In patients without peritoneal metastasis (P0), the positive rate was 26.1%. This study proved that Chen's serosal classification is correct and useful in assessing whether the cancer cells have penetrated through the serosa or not during surgery. Different treatments should be used in the cases with different serosal types. In addition to surgery, destruction of free cancer cells should be considered in tendonoid and colour diffused serosal types, so as to prevent peritoneal metastasis.

Comparison of conventional and inverted structures in fullerene-free organic solar cells

A n-type small molecule DC-IDT2E with 4,4,9,9-tetrakis（4-hexylphenyl）-indaceno[1,2-b：5,6-bt]dithiophene as a central building block, furan as rr-bridges, and 1,1 -dicyanomethylene-3-indanone as end acceptor groups, was synthesized and used as an electron acceptor in solution-processed organic solar cells （OSCs）. DC-IDT2F exhibited good thermal stability, broad and strong absorption in 500-850 rim, a narrow bandgap of 1.54 eV, LUMO of-3.88 eV, HOMO of-5.44 eV and an electron mobility of 6.5 × 10-4 cm2/（V.s）. DC-IDT2F-based OSCs with conventional and inverted structures exhibited power conversion efficiencies of 2.26 and 3.08% respec- tively. The effect of vertical phase separation and morphology of the active layer on the device performance in the two structures was studied.

基于位置分配的去蜂窝massive MIMO系统导频分配功率控制算法

针对去蜂窝(cell free,CF)大规模多输入多输出(multiple-input multiple-output,MIMO)系统中存在严重的导频污染问题,提出了一种基于位置分配的贪婪导频分配功率控制算法(greedy pilot assignment based on location with pilot power control,GPABL with PPC).首先,遵循相邻用户不分配相同导频序列的原则进行贪婪导频分配(greedy pilot assignment,GPA);然后,在导频分配的基础上叠加了导频功率控制,选择合理的导频功率控制系数减小信道估计的均方误差.仿真结果表明,将两种方式结合起来进行导频优化,系统的吞吐能力有所提升.

系统免疫炎症指数对根治性放疗Ⅲ期肺鳞癌患者长期生存的预测价值

目的探讨系统免疫炎症指数(SII)对接受根治性放疗的Ⅲ期肺鳞癌患者长期生存的预测价值。方法回顾性收集接受根治性放疗的Ⅲ期肺鳞癌患者的临床资料。计算患者放疗前1周内的SII及相关炎症指标,同时应用X-Tile软件确定最佳截断值。分析患者的生存情况以及SII对患者总生存(OS)及无进展生存(PFS)的影响。结果共纳入了453例患者,低SII组336例(<1277.3),高SII组117例(≥1277.3)。高SII组的中位OS和中位PFS均较低SII组缩短(OS:20.8个月vs.31.0个月,Log-rankχ2=18.015,P<0.01;PFS:13.0个月vs.21.0个月,Log-rankχ2=15.062,P<0.01)。多因素Cox回归分析显示,高SII是患者OS(HR=1.628,95%CI:1.294~2.047,P<0.001)和PFS(HR=1.559,95%CI:1.240~1.961,P<0.001)的独立危险因素,其他的影响因素包括较晚的TNM分期、放疗疗效欠佳,HALP评分下降。结论SII可作为接受根治性放疗Ⅲ期肺鳞癌患者长期生存的评价指标,SII升高提示预后较差。

佐利替尼一线治疗EGFR突变NSCLC伴中枢神经系统转移2例报告

目的探讨佐利替尼一线治疗具有原发表皮生长因子受体(EGFR)突变的非小细胞肺癌(NSCLC)伴中枢神经系统转移患者的疗效。方法2例患者均为一线使用佐利替尼,通过实体瘤的疗效评价标准RECIST v1.1和神经系统肿瘤脑转移RANO-BM标准评估肿瘤治疗反应。结果病例1基线伴多发脑转移,EGFR exon 19del突变,佐利替尼治疗51.4个月后仍维持肺部病灶部分缓解(PR)、颅内病灶完全缓解(CR)。病例2基线单个脑转移病灶,EGFR exon 19del突变,佐利替尼治疗期间达到肺部病灶PR、颅内病灶CR,13.7个月后肺部疾病进展(PD),新发单个脑转移病灶,综合评效PD。病例1出现3级不良反应,为皮肤干燥,其余主要为皮疹、肝功能异常、腹泻,不良反应总体可控。结论佐利替尼对EGFR突变NSCLC伴中枢神经系统转移患者有较好的颅内及颅外病灶的控制效果,与EVEREST研究一致,可作为一线初始治疗的选择。

室内自由小区VLC协作的用户接入和功率动态调整方法

针对室内自由小区VLC网络照明和吞吐量联合优化困难的问题,该文提出VLC协作用户接入和功率动态调整(VCUA-DPA)方法。在用户接入阶段,设计考虑网络的负载均衡、用户速率需求和协作小区数目限制的用户和VLC协作的用户接入算法;在功率分配阶段,设计联合优化照明均匀度和系统吞吐量的功率动态调整分配算法。仿真结果表明,所提VCUA-DPA在明显提升系统吞吐量的同时能优化室内照明均匀度。

Emerging role of non-invasive and liquid biopsy biomarkers in pancreatic cancer

A global increase in the incidence of pancreatic cancer(PanCa)presents a major concern and health burden.The traditional tissue-based diagnostic techniques provided a major way forward for molecular diagnostics;however,they face limitations based on diagnosis-associated difficulties and concerns surrounding tissue availability in the clinical setting.Late disease development with asymptomatic behavior is a drawback in the case of existing diagnostic procedures.The capability of cell free markers in discriminating PanCa from autoimmune pancreatitis and chronic pancreatitis along with other precancerous lesions can be a boon to clinicians.Early-stage diagnosis of PanCa can be achieved only if these biomarkers specifically discriminate the non-carcinogenic disease stage from malignancy with respect to tumor stages.In this review,we comprehensively described the non-invasive disease detection approaches and why these approaches are gaining popularity for their early-stage diagnostic capability and associated clinical feasibility.

不同无血清培养体系对人脐带间充质干细胞生物学功能的影响

目的 探讨3种不同无血清培养体系对人脐带间充质干细胞(hUC-MSC)体外培养的生物学功能的影响。方法 取3株脐带组织,各分3组实验组,经贴壁法分别置于3组不同无血清培养体系(A组:纯化学成分合成的无血清培养体系,B组:血小板裂解物+基础培养基的无血清培养体系,C组:纤维蛋白原包被+含重组人蛋白质的无血清培养体系)中培养至第3代,比较其细胞增殖能力、细胞表型、三系分化能力以及免疫调节功能。结果 3组实验组细胞均呈长而扁平的梭形,贴壁生长,大小均一。其增殖曲线趋势相似,A组和B组P2、P3代次的增殖能力优于C组(P均<0.05)。3组实验组细胞均符合国际干细胞协会对hUC-MSC细胞表面标志物鉴定的要求,A组和B组P3代次的CD29和CD90的表达量均高于C组(P均<0.05),并且3组实验组均具有三系分化能力。3组实验组中P3代次的T淋巴细胞增殖率、Th1和Th17细胞亚群分泌的细胞因子比例均低于对照组,调节性T细胞(CD4+CD25+FOXP3+)比例均高于对照组,而3组实验组之间两两比较差异无统计学意义(P均> 0.05)。结论 3种不同无血清培养体系的hUC-MSC均能保持其基本的生物学特性和免疫调节活性,纯化学成分合成的无血清培养体系和血小板裂解物+基础培养基的无血清培养体系的增殖能力和细胞表面标志物表达均优于纤维蛋白原包被+含重组人蛋白质的无血清培养体系。

无小区大规模MIMO系统中基于图着色的导频分配与功率控制算法

无小区大规模MIMO(Cell-Free Massive MIMO,CF-mMIMO)系统中大量的无线接入点在同一时间频率资源中服务所有的用户,具有较高的网络覆盖率和频谱效率,是下一代无线通信的前沿技术之一。然而,正交导频数量的限制以及用户信道质量的差异造成了较严重的用户间干扰。为了优化这一干扰问题,本文提出了无小区大规模MIMO系统中基于图着色的导频分配与功率控制算法。利用基于图着色的导频分配算法降低导频污染,提高了信道估计精度。在此基础上,通过最大-最小功率控制算法降低多用户同频干扰,满足了系统中用户的混合服务质量需求。数值仿真结果表明:所提出的基于图着色的导频分配与功率控制算法可以显著提高系统的95%用户频谱效率以及最小用户速率,较好抑制了CF-mMIMO系统中的用户间干扰。

Cell-free systems in the new age of synthetic biology

The advent of synthetic biology has ushered in new applications of cell-free transcription-translation systems. These cell-free systems are reconstituted using cellular proteins, and are amenable to modular control of their composition. Here, we discuss the historical advance- ment of cell-free systems, as well as their new applications in the rapid design of synthetic genetic circuits and components, directed evolution ofbiomolecules, diagnosis of infectious diseases, and synthesis of vaccines. Finally, we present our vision on the future direction of cell-free synthetic biology.

Construction of a cell-free system using extracts from apoptotic plant cells

To study the mechanism of plant apoptosis, a cell-free system has been developed using the extracts prepared from tobacco cells that become committed to apoptosis induced by heat shock treatment. Tobacco nuclei have displayed such characteristics of apoptosis as nucleus condensation and formation of apoptotic bodies during incubation in this system. Besides, these morphological changes are accompanied by DNA fragmentation, which is detected

NUCLEAR RECONSTITUTION IN THE Xenopus laevis EGG EXTRACTS CELL-FREE SYSTEM

Ⅰ. INTRODUCTIONNuclear reconstitution (nuclear assembly )was reported by several laboratories in recent years and regarded as an important advance in cell biology. In vitro nuclear assembly system provides a good model to study the organization and function of nucleus, chromatin, nuclear envelope and nuclear matrix. Although it is too early to evaluate its significance in theory and in bioengineering, there is no doubt that the prospect in this field is inspiring.

Nicotiana ovule extracts induce nuclear reconstitution of demembranated Xenopus sperm in cell-free system

Nicotiana tabaccum ovule extracts induced nuclear reconstitution of demembranated Xenopus leavis sperm in a cell-free system. Demembranated Xenopus sperm began to swell after 15 min of incubation with Nicotiana ovule extracts. Accompanying the process of incubation, Xenopus sperm decondensed and their shapes changed gradually from long and ellipse to round. The completely decondensed chromatin was surrounded with membrane structure, which was a mixture envelope of a double membrane and a single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion to re-constituted nuclei and DNA electrophoresis. Nicotiana ovule extracts supplied one more experimental model and system. The new system could promote powerfully the research on mechanisms of cell division and cell cycle regulation.

Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Genetic code expansion,which enables the site-specific incorporation of unnatural amino acids into proteins,has emerged as a new and powerful tool for protein engineering.Currently,it is mainly utilized inside living cells for a myriad of applications.However,the utilization of this technology in a cell-free,reconstituted platform has several advantages over living systems.The typical limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization.Herein,we describe a simplified method for the preparation of this system from Escherichia coli cells,which is specifically adapted for the expression of the components needed for cell-free genetic code expansion.Besides,we propose and demonstrate a modular approach to its utilization.By this approach,it is possible to prepare and store different extracts,harboring various translational components,and mix and match them as needed for more than four years retaining its high efficiency.We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein.Finally,we demonstrate the advantage of cell-free systems over living cells for the incorporation ofδ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair,which could not be achieved in a living cell.

Atomic force microscopy observation on nuclear reassembly in a cell-free system

Cell-free system is interesting and useful for studying nuclear assembly in mitosis. Atomic force micro- scopy (AFM), which is a simple way for imaging fixed reas-semble nuclei with high resolution, has not been used in the cell-free system. In this paper, we put forward an air-drying sample preparation for AFM. Using AFM, we observed nu-clear reassembly process within 100 nm resolution in a cell-free system. As a result, we found that the images were artifact-free, and with higher resolution compared with fluo-rescent optical microscope images. Furthermore, the mor-phology of membrane vesicles was obtained clearly, and a dynamic change of morphology during the vesicles?ap-proaching to nuclear envelope was also observed, which is enlightened to understand the mechanism of nuclear enve-lope assembly.