Phosphodiesterase 9 localization in cytoplasm and nucleus:the gateway to selective targeting in neuroprotection?

The umbrella term"neurodege ne rative disorders"(NDDs) refers to several conditions characterized by a progressive loss of structure and function of cells belonging to the nervous system.Such diseases affect more than 50million people worldwide.Neurodegenerative disorders are characterized by sundry factors and pathophysiological mechanisms that a re challenging to be fully profiled.Many of these rely on cell signaling pathways to preserve homeostasis,involving second messengers such as cyclic adenosine monophosphate (cAMP)and cyclic guanosine 3',5'-monophosphate(cGMP).Their ability to control the duration and amplitude of the signaling cascade is given by the presence of several common and uncommon effectors.Protein kinases A and G (PKA and PKG),phosphodiesterases (PDEs),and scaffold proteins are among them.

Ancient Zhenyuan, Gateway to East Guizhou

This ancient city has a history going back over 2,000 years.It has witnessed the fusion of multiple ethnic cultures including the Miao,Dong,and Han,and hosts a number of cultural heritage sites.THE old town of Zhenyuan is located in Zhenyuan County,Qiandongnan Miao and Dong Autonomous Prefecture in southwest China’s Guizhou Province.Covering an area of just around three square kilometers,it sits on the banks of the Wuyang River and is surrounded by mountains.Zhenyuan has been a county for more than 2,281 years and was the prefectural capital during the Yuan(1271-1368)and Qing(1644-1911)dynasties.It is a multicultural region,with Miao,Dong,and other ethnic minorities making up nearly half of the population.

Application Strategies of Shotcrete Anchor Support Technology in Highway Bridge and Tunnel Engineering

This article analyzes the application strategies of shotcrete anchor support technology using a highway bridge-tunnel construction project as an example.The article covers various strategies,including support plan formulation,mortar shotcrete anchor construction,grid steel frame construction,steel mesh construction,and concrete support construction.This analysis aims to provide a guideline for those interested in applying this technology and improving the quality and safety of highway bridges and tunnels construction.

Comprehensive utilization of solid waste resources: Development of wet shotcrete for mines

The development of solid waste resources as constituent materials for wet shotcrete has significant economic and environmental advantages. In this study, the concept of using tailings as aggregate and fly ash and slag powder as auxiliary cementitious material is proposed and experiments are carried out by response surface methodology(RSM). Multivariate nonlinear response models are constructed to investigate the effect of factors on the uniaxial compressive strength(UCS) of tailings wet shotcrete(TWSC). The UCS of TWSC is predicted and optimized by constructing Gaussian process regression(GPR) and genetic algorithm(GA). The UCS of TWSC is gradually enhanced with the increase of slag powder dosage and fineness modulus, and it is enhanced first and then decreased with the increase of fly ash dosage. The microstructure of TWSC has the highest gray value and the highest UCS when the fly ash dosage is about 120 kg·m^(-3). The GPR–GA model constructed in this study achieves high accuracy prediction and optimization of the UCS of TWSC under multi-factor conditions.

Development of Wet Shotcrete with Solid Waste as Aggregate:Strength Optimization and Mix Proportion Design

The super-fine particle size of tailings is its drawback as a recycled resource,which is reflected in the low strength of the new construction and industrial materials formed when it is mixed with cement and other cementitious materials.Therefore,it is crucial to study the effect of tailings particle size and cementitious material on the strength of tailings wet shotcrete(TWSC)and to investigate the optimal mix proportion.In this paper,a multivariate nonlinear response model was constructed by conducting central composite experiments to investigate the effect of different factors on the strength of TWSC.The strength prediction and mix proportion optimization of TWSC are carried out by machine learning techniques.The results show that the response model has R^(2)>0.94 and P<0.01,which indicates that the model has high reliability.Moreover,the strength of TWSC increases with the increase of tailings fineness modulus and decrease of water-binder ratio,while it also increases and then decreases with the increase of replacement rate of slag powder to cement(SRC rate).The extreme learning machine(ELM)constructed in this paper predicts the strength of TWSC with an accuracy of more than 98%and achieves rapid prediction under multi-factor conditions.It is worth mentioning that the ELM combined with the genetic algorithm(ELM-GA)collaboratively solved to obtain the mix proportion for C15 and C20 strength grades of TWSC and the maximum error is verified by experiments to be less than 2%.

A Duty Cycle-Based Gateway Selection Algorithm for LoRaWAN Downlink Communication

Long Range Wide Area Network(LoRaWAN)has been developed to meet the requirements for the enormous device-to-device communication of Inter-net of Things(IoT)networks,which consist of a large number of participating devices spread over large coverage areas with low data rates and low power con-sumption.It supports communications in both directions,uplink,and downlink directions.However,the downlink communication in the current LoRaWAN raises the bottleneck issue at gateways due to the used gateway selection algo-rithm.This paper proposes a novel gateway selection algorithm based on the duty cycle time-off values for the existing gateways,Duty Cycle Gateway Selection(DCGS),to direct acknowledgment packets as downlink traffic towards the most suitable gateway.Thus,the proposed system avoids subsequent retransmission of previously sent traffic that leads to excessive traffic overloading the network.The proposed system avoids exhausting a gateway duty cycle with downlink traffic by distributing the downlink traffic among available gateways based on the duty cycle time off.DCGS is evaluated using FloRa and INET frameworks in the well-known network simulator OMNeT++.The result shows the superior perfor-mance of the proposed approach over the existing Signal-to-Noise ratio(SNR)based selection mechanism.It clearly indicates that the DCGS maintains a better confirmed packet delivery rate while reducing number of retransmissions,colli-sions,and power consumption.

An Optimal Cluster Head and Gateway Node Selection with Fault Tolerance

In Mobile Ad Hoc Networks(MANET),Quality of Service(QoS)is an important factor that must be analysed for the showing the better performance.The Node Quality-based Clustering Algorithm using Fuzzy-Fruit Fly Optimiza-tion for Cluster Head and Gateway Selection(NQCAFFFOCHGS)has the best network performance because it uses the Improved Weighted Clustering Algo-rithm(IWCA)to cluster the network and the FFO algorithm,which uses fuzzy-based network metrics to select the best CH and entryway.However,the major drawback of the fuzzy system was to appropriately select the membership func-tions.Also,the network metrics related to the path or link connectivity were not considered to effectively choose the CH and gateway.When learning fuzzy sets,this algorithm employs a new Continuous Action-set Learning Automata(CALA)approach that correctly modifies and chooses the fuzzy membership functions.Despite the fact that it extends the network’s lifespan,it does not assist in the detection of defective nodes in the routing route.Because of this,a new Fault Tolerance(NQCAEFFFOCHGS-FT)mechanism based on the Distributed Connectivity Restoration(DCR)mechanism is proposed,which allows the net-work to self-heal as a consequence of the algorithm’s self-healing capacity.Because of the way this method is designed,node failures may be utilised to rebuild the network topology via the use of cascaded node moves.Founded on the fractional network information and topologic overhead related with each node,the DCR is suggested as an alternative to the DCR.When compared to the NQCAFFFOCHGS algorithm,the recreation results display that the proposed NQCAEFFFOCHGS-FT algorithm improves network performance in terms of end-to-end delay,energy consumption,Packet Loss Ratio(PLR),Normalized Routing Overhead(NRO),and Balanced Load Index(BLI).

Study on Mechanical Properties of High Fine Silty Basalt Fiber Shotcrete Based on Orthogonal Design

In order to improve the comprehensive utilization rate of highfines sand(HFS)produced by the mine,full solid waste shotcrete(HFS-BFRS)was prepared with HFS asfine aggregate in cooperation with basaltfiber(BF).The strength growth characteristics of HFS-BFRS were analyzed.And thefitting equation of compressive strength growth characteristics of HFS-BFRS under the synergistic effect of multiple factors was given.And based on the orthogonal experimental method,the effects on the compressive strength,splitting tensile strength andflex-ural strength of HFS-BFRS under the action of different levels of influencing factors were investigated.The effect of three factors on the mechanical properties of HFS-BFRS,3,and 28 d,respectively,was revealed by choosing the colloidal sand ratio(C/H),basaltfiber volume fraction(BF Vol)and naphthalene high-efficiency water reducing agent(FDN)as the design variables,combined with indoor tests and theoretical analysis.The results show that the sensitivity of the three factors on compressive strength andflexural strength is C/H>FDN>BF Vol,and split-ting tensile strength is BF Vol>FDN>C/H.Finally,thefitting ratio of HFS-BFRS was optimized by the factor index method,and the rationality was verified by thefield test.For thefluidity of HFS-BFRS,the slump can be improved by 139%under the action of 1.2%FDN,which guarantees the pump-ability of HFS-BFRS.

Widening the Gateway--China aims to foster internationally competitive manufacturing within an open environment

A mong the eight major steps China announced at the Third Belt and Road Forum for International Cooperation to support high-quality Belt and Road cooperation,the cancelation of all restrictions on foreign investment access in the manufacturing sector is the most striking measure,or at least one of the most eye-catching.

Gateway技术构建酵母双杂分析用辣椒cDNA文库

构建cDNA文库是利用酵母双杂交技术开展特定cDNA分离的重要基础工作。利用Gateway技术,在成功提取辣椒总RNA并分离mRNA的基础上,合成了3种读码框的双链cDNA,通过BP反应构建其相应的入门文库,再通过LR反应将cDNA迅速且定向地重组到酵母双杂分析用目的载体pDESTTM22上,构建成高质量目的文库。经检测入门文库和目的文库滴度均达到(4.0～5.0)×106cfu/mL,文库总容量为(2.4～3.0)×107cfu,平均插入片段大小为1250bp,其质量完全满足于进一步利用酵母双杂体系分析目的基因互作蛋白cDNA。

利用Gateway克隆技术大规模克隆拟南芥转录因子

随着越来越多基因组全序列的测定完成,基因组的研究进入了功能研究阶段。功能基因组的研究需要把大量基因连入不同载体,传统的酶切连接构建载体的方法不能满足这种大规模克隆的需要。Gateway技术是一种高效的大规模克隆系统,而且对载体和宿主没有依赖性。本文利用Gateway大规模克隆技术将16个拟南芥转录因子的ORF克隆入植物表达载pPTV和酵母表达载体pYTV中,酵母融合表达实验和West-ern-blot检测证明了该克隆途径的可行性,并且得到的His-Tag融合蛋白,为拟南芥转录因子的大规模蛋白质组学研究奠定了良好的基础,同时基因序列的聚类分析为将来进一步的功能研究提供了有利信息。

基于Gateway技术的低成本植物双分子荧光互补分析系统

创制基于Gateway重组技术的双分子荧光互补(bimolecular fluorescence complementation,BiFC)载体,同时创制低成本低克隆背景的Gateway入门载体。利用入门载体,采用Gateway技术快速将目标基因重组到BiFC目标载体,并采用农杆菌注射侵染烟草的方法观察荧光互作结果。构建的Gateway入门载体,由于带有ccdB基因,非线性化的载体无法在ccdB敏感菌株中生长,因此消除了载体本身的背景。同时由XcmⅠ酶切后产生的T突出,可以很好地与PCR产物结合,连接效率和普通商业化T载体一致。此外,由于载体可由实验室自行制备且采用了TA克隆,创制入门克隆的成本大大降低。借助该研究改造的BiFC目标载体,目的基因能快速地重组到目的载体用于基因互作鉴定。利用本研究构建的Gateway系统,能够以较低成本快速的进行基因克隆和互作研究。

使用与Gateway技术兼容的T载体获得入门克隆

与Gateway技术兼容的农杆菌双元载体系统已开始应用于植物功能基因组的研究 ,但应用这些载体系统的一个瓶颈问题 ,是如何简单、经济和高效地将PCR产物或其他来源的目的DNA片段构建到入门载体上获得入门克隆 .为此 ,将传统的TA克隆技术与Gateway重组克隆技术进行整合 ,构建了与Gateway技术兼容的两种TA克隆载体 ,用于在克隆PCR产物或其他来源的目的DNA片段的同时获得入门克隆 .利用兼容Gateway技术的TA克隆载体有效地解决了上述瓶颈问题 .

Gateway技术构建结缕草低温和干旱诱导cDNA文库及其质量鉴定

为研究结缕草(Zoysia japonica)胁迫响应信号转导途径中的关键基因,揭示结缕草抗逆分子机制,建立结缕草功能基因组学研究基础平台,以经低温、干旱处理的结缕草为材料,采用Gateway技术构建了首个结缕草低温和干旱诱导的标准cDNA文库。文库质量分析表明,未扩增的原始文库滴度1.76×106 pfu.mL-1,库容7.04×106 pfu,插入片段平均大小大于1kb,重组率90%。文库质量优良,可能包含大量新基因,不仅能为结缕草功能基因组分析提供必要资源,也可为后期进行高通量EST测序、发掘新抗逆相关基因、制作基因芯片等研究奠定基础。

单子叶植物RNA干扰和过表达Gateway载体的构建

基因枪和农杆菌介导的遗传转化是目前常用的两种单子叶植物遗传转化方法。载体的发展和改良是提高植物遗传转化效率的重要基础,RNA干扰载体和过表达载体是目前通过遗传转化研究植物基因功能的主要工具。Gateway克隆技术是一种基于lambda噬菌体特异位点重组特性的通用克隆技术,该技术可以将大批目的基因方便、快捷地连接到受体载体上。本文利用Gateway技术结合传统酶切、连接方法,构建了适用于单子叶植物基因枪和农杆菌转化的RNA干扰Gateway载体pAHC-PSK-RNAi、pClean-G185-RNAi和过表达Gateway载体pAHC-PSK-OE和pClean-G185-OE,为利用基因枪和农杆菌介导的遗传转化,在小麦和水稻等单子叶植物中进行规模化基因功能研究奠定了基础。

Gateway~技术构建交链孢菌JH505 cDNA文库

Gateway○R技术构建cDNA文库,利用λ噬菌体的位点特异性重组,避免使用限制性内酶切割cDNA,能够解决常规方法构建cDNA文库的技术缺陷。首次应用Gateway○R技术构建交链孢菌cDNA文库,经检测cDNA入门文库的滴度达到1×107cfumL,文库总容量为9×107cfu,平均插入片段为1510bp。通过LR重组把入门文库转换为表达文库,表达文库的滴度为1.58×106cfu/mL,文库总容量为6.32×106cfu,平均插入片段大小为1680bp。表达文库的构建为进一步克隆植物激活蛋白基因打下了基础。

应用Gateway技术构建水稻OsDAD1基因的RNA干涉载体

近年来,RNA干涉广泛应用于植物基因功能研究。Gateway技术是通过采用多重载体高效构建目标基因及任何DNA片段克隆和表达载体的研究平台,该技术以λ噬菌体的位点特异性重组体系为基础,两端具有重组位点的DNA片段或目标基因可以非常容易地被重组克隆到含同源重组位点的载体上。这种以重组为基础的克隆体系目前已被广泛应用于分子生物学中来分析基因的功能。本文应用Gateway技术体系替代传统载体构建方法,构建了水稻基因OsDAD1的RNA干涉载体,从而证明了Gateway技术的可行性。

利用Gateway技术构建黄瓜HPL基因的RNA干扰载体

为探讨黄瓜脂氢过氧化物裂解酶基因(HPL)下调表达对黄瓜果实挥发性醛类物质合成的影响,设计1对特异引物,以黄瓜pDONR201-HPL质粒为模板,通过PCR扩增出323bp的片段。利用基于同源重组原理的Gateway技术,将扩增的片段通过BP反应连接到入门载体pDONR201中。对扩增出的323bp的片段进行测序,测序结果显示该序列与原序列同源性为100%。在此基础上,通过LR反应将目的片段插入过表达的植物RNA干涉载体pHellsgate8,构建黄瓜HPL基因的RNAi表达载体pHellsgate8-HPLi,分别经限制性内切酶XhoⅠ、XbaⅠ酶切后获得323bp的片段,与预期结果一致。采用热击法将pHellsgate8-HPLi转化农杆菌GV3101,经菌液PCR扩增获得323bp的目的条带,表明RNAi表达载体pHellsgate8-HPLi已成功转入农杆菌中。

Gateway技术快速构建百合双价抗病毒RNAi表达载体

为获得适合转化百合的双价RNAi表达载体,以感病百合叶片的总RNA为模板,分别扩增400bp左右的LSV和LMoVCP基因。通过重叠延伸PCR技术获得长约800bp的LSV-LMoV融合基因。利用Gateway技术,通过BP反应及LR反应将LSV-LMoV融合基因克隆到双元载体pH7GWIWG2(Ⅱ),成功构建了含2种病毒基因的RNAi表达载体pH7GWIWG2(Ⅱ)-LSV-LMoV。将构建好的双价表达载体导入农杆菌EHA105,PCR和测序表明所构建的载体及获得的工程菌与预期的完全一致。

乳酸杆菌组成型启动子探测载体pgateway-612的构建

为筛选适宜乳酸菌(LAB)宿主表达载体的启动子,本研究以大肠杆菌-LAB穿梭载体pPG612为基本骨架,利用GatewayTM克隆技术操作构建了LAB组成型启动子探测载体,并利用该载体筛选了短乳杆菌的S层启动子。我们利用底物X-gluC与报告基因gusA编码的蛋白酶之间的特异性显色反应筛选阳性菌落,超声破碎后的菌体蛋白经SDS-PAGE和western blot分析表明该启动子在宿主菌中具有组成型启动子活性。本研究为进一步利用表达载体及筛选其他LAB启动子奠定基础。