Real-time virtual reference service based on applicable artificial intelligence technologies:The début of the robot Xiaotu at Tsinghua University Library

The adoption of applicable artificial intelligence technologies to library real-time virtual reference services is an innovative experimentation in one of the key areas of library services. Based on the open source software Artificial Linguistic Internet Computer Entity(A.L.I.C.E.) and a combined application of several other relevant supporting technologies for facilitating the use of the current existing library resources, Tsinghua University Library has recently developed a real-time smart talking robot, named Xiaotu,for the enhancement of its various service functions, such as reference services, book searching, Baidu Baike searching, self-directed learning, etc. The operation of Xiaotu is programmed into Renren website(a social networking website), which adds significantly an innovative feature to the modus operandi of the real-time virtual reference service at Tsinghua University Library.

Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole(Cynoglossus semilaevis)

Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization during Cynoglossus semilaevis early developmental process, eight candidate reference genes （ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S） were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the mature C. semilaevis. EFIA was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages （from oosphere to 90 days old） in this study. However, when divided the Ct value data of the above mentioned early developmental stages into two separate periods （embryo and post-hatching periods）, TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.

Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by Quantitative Real-Time PCR

The brown planthopper Nilaparvata lugens Stal （Homoptera： Delphacidae） can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but several N. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels in N. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes in N. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated from N. lugens, including two different treatments （resistant or susceptible rice） and three virulent N. lugens populations. Three software programs （BestKeeper, Normfinder and geNorm） were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes 18S, E-ACT, E-TUB and a-TUB as the most stable reference genes. BestKeeper identified ETIF1 as the optimal reference gene with the least overall variation, whereas 18S and a-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes 18S and a-TUB were the most suitable reference genes in N. lugens. These results will facilitate future transcript profiling studies on N. lugens populations that show variation in virulence levels on different rice varieties.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.

Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship（R^2=0.7763, P〈0.05）. This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.

Precise Point Positioning Technique with IGS Real-Time Service (RTS) for Maritime Applications

The maritime navigation accuracy requirements for radionavigation systems such as GPS are specified by the International Maritime Organization (IMO). Maritime navigation usually consists of three major phases identified as Ocean/Coastal/Port approach/Inland waterway, in port navigation and automatic docking with an accuracy requirement that ranges from 10 m to 0.1 m. With the advancement in autonomous GPS positioning techniques such as Precise Point Positioning (PPP) and with the advent of the new IGS-Real-Time-Service (RTS), it is necessary to assess the possibility of a wider role of the PPP-based positioning technique in maritime applications. This paper investigates the performance of an autonomous real-time PPP-positioning solution by using the IGS- RTS service for maritime applications that require an accurate positioning system. To examine the performance of the real-time IGS-RTS PPP-based technique for maritime applications, kinematic data from a dual frequency GPS receiver is investigated. It is shown that the real-time IGS-RTS PPP-based GPS positioning technique fulfills IMO requirements for maritime applications with an accuracy requirement ranges from 10 m for Ocean/Coastal/Port approach/Inland waterways navigation to 1.0 m for in port navigation but cannot fulfill the automatic docking application with an accuracy requirement of 0.10 m. To further investigate the real-time PPP-based GPS positioning technique, a comparison is made between the real-time IGS-RTS PPP-based positioning technique and the real-time PPP-based positioning by using the predicted part of the IGS Ultra-Rapid products and the real-time GPS positioning technique with the Wide Area Differential GPS service (WADGPS). It is shown that the IGS-RTS PPP-based positioning technique is superior to the IGS-Ultra-Rapid PPP-based and WADGPS-based positioning techniques.

Selection of Reference Genes in Saccharopolyspora Spinosa for Real-Time PCR

Reverse transcription quantitative PCR （RT-qPCR） combined with the published genome information of Saccharopolyspora spinosa can allow sophisticated studies about S. spinosa, including Studying the regulation of spinosyn biosynthesis, finding new target genes for engineering, and discovering and exploiting other macrolide secondary metabolites. Studies have demonstrated that appropriate internal control is needed to normalize target genes at transcription levels. However, many studies have shown that no single reference gene is universal for all strains under all experimental conditions. Thus, eight candidate reference genes of three different S. spinosa strains in two different cultures were studied to find suitable reference gene（sl. The number of amplification cycles of these candidate genes was calculated by BestKeeper, NormFinder and geNorm. The results indicated that the most suitable reference genes for normalization during the fermentation of S. spinosa were 16S rRNA and rbL13.

Packet Scheduling Algorithm for Real-Time Services in Broadband WMAN

Packet scheduling algorithm is the key technology to guarantee Quality of Service (QoS) and balance the fairness between users in broadband Wireless Metropolitan Area Network (WMAN). Based on the research of Proportional Fairness (PF) algorithm and Modified Largest Weighted Delay First (M-LWDF) algorithm, a new packet scheduling algorithm for real-time services in broadband WMAN, called Enhanced M-LWDF (EM-LWDF), was proposed. The algorithm phases in new information to measure the load of service queues and updates the state parameters in real-time way, which remarkably improves system performance.Simulation results show that comparing with M-LWDF algorithm, the proposed algorithm is advantageous in performances of queuing delay and fairness while guaranteeing system throughput.

Models to Simulate Effective Coverage of Fire Station Based on Real-Time Travel Times

In recent years,frequent fire disasters have led to enormous damage in China.Effective firefighting rescues can minimize the losses caused by fires.During the rescue processes,the travel time of fire trucks can be severely affected by traffic conditions,changing the effective coverage of fire stations.However,it is still challenging to determine the effective coverage of fire stations considering dynamic traffic conditions.This paper addresses this issue by combining the traveling time calculationmodelwith the effective coverage simulationmodel.In addition,it proposes a new index of total effective coverage area(TECA)based on the time-weighted average of the effective coverage area(ECA)to evaluate the urban fire services.It also selects China as the case study to validate the feasibility of the models,a fire station(FS-JX)in Changsha.FS-JX station and its surrounding 9,117 fire risk points are selected as the fire service supply and demand points,respectively.A total of 196 simulation scenarios throughout a consecutiveweek are analyzed.Eventually,1,933,815 sets of valid sample data are obtained.The results showed that the TECA of FS-JX is 3.27 km^(2),which is far below the standard requirement of 7.00 km^(2) due to the traffic conditions.The visualization results showed that three rivers around FS-JX interrupt the continuity of its effective coverage.The proposed method can provide data support to optimize the locations of fire stations by accurately and dynamically determining the effective coverage of fire stations.

Bolzano Traffic: An Example of Open ITS Deployment for Advanced Traveller Information Services

This paper aims to present the experience gathered in the Italian alpine city of Bolzano within the project“Bolzano Traffic”whose goal is the introduction of an experimental open ITS platform for local service providers,fostering the diffusion of advanced traveller information services and the future deployment of cooperative mobility systems in the region.Several end-users applications targeted to the needs of different user groups have been developed in collaboration with local companies and research centers;a partnership with the EU Co-Cities project has been activated as well.The implemented services rely on real-time travel and traffic information collected by urban traffic monitoring systems or published by local stakeholders(e.g.public transportation operators).An active involvement of end-users,who have recently started testing these demo applications for free,is actually on-going.

Selection of Reference Genes in Transcription Analysis of Gene Expression of the Mandarin Fish, Siniperca chuasti

At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, β-actin, and 18S rRNA in six tissues and five developmental stages of the Mandarin fish Siniperca chuatsi were assayed with quantitative real-time PCR （qPCR）. Differences in expression levels were analyzed using geNorm program. The results demonstrate that β-actin is the most stable gene at developmental stages and GAPDH is the most stable in different tissues. While 18S rRNA expression during development is differentially regulated, which indicates it is suitable as an internal control for gene expression normalization at the developmental level. Overall, the data suggest that the two most stable housekeeping genes are enough to accurately calibrate gene expression in S. chuatsi. The significance of this study provided convincing references and methodology for housekeeping gene selection and normalization in gene expression analysis with regular PCR or qPCR.

基于Web Services的业务提供参考模型

在WebServcies应用于网络能力开放接口和终端的技术基础上,提出一个基于WebServices的业务提供参考模型,该模型结合了网络能力开放接口和WebServices服务组合等技术,从业务开发、业务环境、系统部署这3个视图描述了基于WebServices的业务提供,对开放融合网络下的业务提供给出了框架性的参考.

Application of Real-time Fluorescent Quantitative PCR in Plant

Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues （intestine, respiratory tree, and muscle） of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin （TUBB） gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 （RPSI 8）, TUBB, and NADH dehydrogenase （NADH）; for respiratory tree, it was β-actin （ACTB）, TUBB, and succinate dehydrogenase cytochrome B small subunit （SDHC）; and for muscle it was α-tubulin （TUBA） and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 （NDUFA13）. These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.

Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

An integrated DBP for streams with （m, k）-firm real-time guarantee

(m, k)-firm real-time or weakly hard real-time (WHRT) guarantee is becoming attractive as it closes the gap between hard and soft (or probabilistic) real-time guarantee, and enables finer granularity of real-time QoS through adjusting m and k. For multiple streams with (m, k)-firm constraint sharing a single server, an on-line priority assignment policy based on the most recent k-length history of each stream called distance based priority (DBP) has been proposed to assign priority.In case of priority equality among these head-of-queue instances, Earliest Deadline First (EDF) is used. Under the context of WHRT schedule theory, DBP is the most popular, gets much attention and has many applications due to its straightforward priority assignment policy and easy implementation. However, DBP combined with EDF cannot always provide good performance, mainly because the initial DBP does not underline the rich information on deadline met/missed distribution,specially streams in various failure states which will travel different distances to restore success states. Considering how to effectively restore the success state of each individual stream from a failure state, an integrated DBP utilizing deadline met/missed distribution is proposed in this paper. Simulation results validated the performance improvement of this pro-posal.

AN INTELLIGENT METHOD FOR REAL-TIME DETECTION OF DDOS ATTACK BASED ON FUZZY LOGIC

The paper puts forward a variance-time plots method based on slide-window mechanism tocalculate the Hurst parameter to detect Distribute Denial of Service(DDoS)attack in real time.Basedon fuzzy logic technology that can adjust itself dynamically under the fuzzy rules,an intelligent DDoSjudgment mechanism is designed.This new method calculates the Hurst parameter quickly and detectsDDoS attack in real time.Through comparing the detecting technologies based on statistics andfeature-packet respectively under different experiments,it is found that the new method can identifythe change of the Hurst parameter resulting from DDoS attack traffic with different intensities,andintelligently judge DDoS attack self-adaptively in real time.

Selection of Reference Genes for Expression Analysis of Kumamoto and Portuguese Oysters and Their Hybrid

Quantitative real-time polymerase chain reaction(q RT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts(expression analysis). It is also employed for studying heterosis, hybridization breeding and hybrid tolerability of oysters, an ecologically and economically important taxonomic group. For these studies, selection of a suitable set of housekeeping genes as references is crucial for correct interpretation of q RT-PCR data. To identify suitable reference genes for oysters during low temperature and low salinity stresses, we analyzed twelve genes from the gill tissue of Crassostrea sikamea(SS), Crassostrea angulata(AA) and their hybrid(SA), which included three ribosomal genes, 28 S ribosomal protein S5(RPS5), ribosomal protein L35(RPL35), and 60 S ribosomal protein L29(RPL29); three structural genes, tubulin gamma(TUBγ), annexin A6 and A7(AA6 and AA7); three metabolic pathway genes, ornithine decarboxylase(OD), glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and glutathione S-transferase P1(GSP); two transcription factors, elongation factor 1 alpha and beta(EF1α and EF1β); and one protein synthesis gene(ubiquitin(UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, ge Norm, Norm Finder and Best Keeper, were used to evaluate the expression stability of these candidate genes. Best Keeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α was stable under low temperature stress in hybrid(SA) oyster; the expression of RPS5 and GAPDH was stable under low salinity stress, and the expression of RPS5, UBQ, GAPDH was stable under low temperature stress in SS oyster; the expression of RPS5, GAPDH, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH and EF1β was stable under low temperature stress in AA oyster. Furthermore, to evaluate their suitability, the reference genes were used to quantify six target genes. In conclusion, we have successfully developed primers appropriate for the expression analysis in SS, SA and AA.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

AIM:To evaluate the suitability of reference genes in gastric tissue samples and cell lines.METHODS:The suitability of genes ACTB,B2M,GAPDH,RPL29,and 18S rRNA was assessed in21 matched pairs of neoplastic and adjacent nonneoplastic gastric tissues from patients with gastric adenocarcinoma,27 normal gastric tissues from patients without cancer,and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR).The ranking of the best single and combination of reference genes was determined by NormFinder,geNorm,BestKeeper,and DataAssist.In addition,GenEx software was used to determine the optimal number of reference genes.To validate the results,the mRNA expression of a target gene,DNMT1,was quantified using the different reference gene combinations suggested by the various software packages for normalization.RESULTS:ACTB was the best reference gene for all gastric tissues,cell lines and all gastric tissues plus cell lines.GAPDH+B2M or ACTB+B2M was the best combination of reference genes for all the gastric tissues.On the other hand,ACTB+B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines.According to the GenEx software,2 or 3 genes were the optimal number of references genes for all the gastric tissues.The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes.The level of expression of DNMT1 in neoplastic,adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH+B2M(P=0.32),ACTB+B2M(P=0.61),or GAPDH+B2M+ACTB(P=0.44).CONCLUSION:GAPDH+B2M or ACTB+B2M is the best combination of reference gene for all the gastric tissues,and ACTB+B2M is the best combination for the cell lines tested.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.