Design-phase indicators for LECMPAU airways.
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Barely three months into the new year and we are happy to announce a monumental milestone reached - 150 million downloads.
\n\nThis achievement solidifies IntechOpen’s place as a pioneer in Open Access publishing and the home to some of the most relevant scientific research available through Open Access.
\n\nWe are so proud to have worked with so many bright minds throughout the years who have helped us spread knowledge through the power of Open Access and we look forward to continuing to support some of the greatest thinkers of our day.
\n\nThank you for making IntechOpen your place of learning, sharing, and discovery, and here’s to 150 million more!
\n\n\n\n\n'}],latestNews:[{slug:"intechopen-signs-new-contract-with-cepiec-china-for-distribution-of-open-access-books-20210319",title:"IntechOpen Signs New Contract with CEPIEC, China for Distribution of Open Access Books"},{slug:"150-million-downloads-and-counting-20210316",title:"150 Million Downloads and Counting"},{slug:"intechopen-secures-indefinite-content-preservation-with-clockss-20210309",title:"IntechOpen Secures Indefinite Content Preservation with CLOCKSS"},{slug:"intechopen-expands-to-all-global-amazon-channels-with-full-catalog-of-books-20210308",title:"IntechOpen Expands to All Global Amazon Channels with Full Catalog of Books"},{slug:"stanford-university-identifies-top-2-scientists-over-1-000-are-intechopen-authors-and-editors-20210122",title:"Stanford University Identifies Top 2% Scientists, Over 1,000 are IntechOpen Authors and Editors"},{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"},{slug:"all-intechopen-books-available-on-perlego-20201215",title:"All IntechOpen Books Available on Perlego"}]},book:{item:{type:"book",id:"3847",leadTitle:null,fullTitle:"Oligomerization of Chemical and Biological Compounds",title:"Oligomerization of Chemical and Biological Compounds",subtitle:null,reviewType:"peer-reviewed",abstract:"Many thanks to the authors for high quality chapters and to the referees for helping improve the manuscripts. The book is interdisciplinary, it covers fields from organic chemistry to mathematics, and raises different aspects of oligomerization. It is a great source of information as every chapter introduces general knowledge and deep details. Mixing communities is to instigate novel ideas and hopefully help looking at oligomerization with new eyes.",isbn:null,printIsbn:"978-953-51-1617-2",pdfIsbn:"978-953-51-4232-4",doi:"10.5772/57075",price:139,priceEur:155,priceUsd:179,slug:"oligomerization-of-chemical-and-biological-compounds",numberOfPages:452,isOpenForSubmission:!1,isInWos:1,hash:"11969d8e08568e065d3f375ebea337a4",bookSignature:"Claire Lesieur",publishedDate:"June 18th 2014",coverURL:"https://cdn.intechopen.com/books/images_new/3847.jpg",numberOfDownloads:29938,numberOfWosCitations:49,numberOfCrossrefCitations:25,numberOfDimensionsCitations:49,hasAltmetrics:1,numberOfTotalCitations:123,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"May 14th 2013",dateEndSecondStepPublish:"June 4th 2013",dateEndThirdStepPublish:"September 8th 2013",dateEndFourthStepPublish:"December 7th 2013",dateEndFifthStepPublish:"March 19th 2014",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6,7,8,9",editedByType:"Edited by",kuFlag:!1,editors:[{id:"169626",title:"Dr.",name:"Claire",middleName:null,surname:"Lesieur",slug:"claire-lesieur",fullName:"Claire Lesieur",profilePictureURL:"https://mts.intechopen.com/storage/users/169626/images/system/169626.jpg",biography:"After getting a master's degree in physics and chemistry, Dr. Claire Lesieur switched to biochemistry and biophysics for her PhD. She worked on the pore-forming toxin aerolysin, her favorite example of protein fold plasticity: it starts as a soluble monomer and ends as a heptameric pore. Dr. Lesieur did a post doc on the oligomerization of the cholera toxin B into pentamers aimed at isolating assembly intermediates. The difficulty of producing such intermediates experimentally drove her to explore computational approaches. The idea was to identify the amino acids determinant for the oligomerization and to investigate how they conduct subunit association. Ultimately, one may hope that the molecular detail of the process will help in designing better inhibitors against pathological oligomers.",institutionString:null,position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"1",institution:null}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"494",title:"Bioorganic Chemistry",slug:"organic-chemistry-bioorganic-chemistry"}],chapters:[{id:"46507",title:"End-capped Oligomers of Ethylene, Olefins and Dienes, by means of Coordinative Chain Transfer Polymerization using Rare Earth Catalysts",doi:"10.5772/58217",slug:"end-capped-oligomers-of-ethylene-olefins-and-dienes-by-means-of-coordinative-chain-transfer-polymeri",totalDownloads:1628,totalCrossrefCites:1,totalDimensionsCites:1,signatures:"Thomas Chenal and Marc Visseaux",downloadPdfUrl:"/chapter/pdf-download/46507",previewPdfUrl:"/chapter/pdf-preview/46507",authors:[{id:"169644",title:"Dr.",name:"Thomas",surname:"Chenal",slug:"thomas-chenal",fullName:"Thomas Chenal"},{id:"169881",title:"Dr.",name:"Marc",surname:"Visseaux",slug:"marc-visseaux",fullName:"Marc Visseaux"}],corrections:null},{id:"46348",title:"The Use of Ionic Liquids in the Oligomerization of Alkenes",doi:"10.5772/57478",slug:"the-use-of-ionic-liquids-in-the-oligomerization-of-alkenes",totalDownloads:2204,totalCrossrefCites:1,totalDimensionsCites:5,signatures:"Csaba Fehér, Eszter Kriván, Zoltán Eller, Jenő Hancsók and Rita\nSkoda-Földes",downloadPdfUrl:"/chapter/pdf-download/46348",previewPdfUrl:"/chapter/pdf-preview/46348",authors:[{id:"169646",title:"Dr.",name:"Csaba",surname:"Fehér",slug:"csaba-feher",fullName:"Csaba Fehér"},{id:"169882",title:"Dr.",name:"Eszter",surname:"Krivan",slug:"eszter-krivan",fullName:"Eszter Krivan"},{id:"169883",title:"Dr.",name:"Jeno",surname:"Hancsok",slug:"jeno-hancsok",fullName:"Jeno Hancsok"},{id:"169884",title:"Dr.",name:"Rita",surname:"Skoda-Foldes",slug:"rita-skoda-foldes",fullName:"Rita Skoda-Foldes"},{id:"170468",title:"MSc.",name:"Zoltán",surname:"Eller",slug:"zoltan-eller",fullName:"Zoltán Eller"}],corrections:null},{id:"46460",title:"Silk Fiber — Molecular Formation Mechanism, Structure- Property Relationship and Advanced Applications",doi:"10.5772/57611",slug:"silk-fiber-molecular-formation-mechanism-structure-property-relationship-and-advanced-applications",totalDownloads:6398,totalCrossrefCites:11,totalDimensionsCites:26,signatures:"Xinfang Liu and Ke-Qin Zhang",downloadPdfUrl:"/chapter/pdf-download/46460",previewPdfUrl:"/chapter/pdf-preview/46460",authors:[{id:"169635",title:"Dr.",name:"Ke-Qin",surname:"Zhang",slug:"ke-qin-zhang",fullName:"Ke-Qin Zhang"},{id:"169872",title:"Dr.",name:"Xinfang",surname:"Liu",slug:"xinfang-liu",fullName:"Xinfang Liu"}],corrections:null},{id:"46495",title:"Ethylene Oxide Homogeneous Heterobifunctional Acyclic Oligomers",doi:"10.5772/57610",slug:"ethylene-oxide-homogeneous-heterobifunctional-acyclic-oligomers",totalDownloads:1257,totalCrossrefCites:1,totalDimensionsCites:1,signatures:"Calin Jianu",downloadPdfUrl:"/chapter/pdf-download/46495",previewPdfUrl:"/chapter/pdf-preview/46495",authors:[{id:"169634",title:"Dr.",name:"Calin",surname:"Jianu",slug:"calin-jianu",fullName:"Calin Jianu"}],corrections:null},{id:"46402",title:"Higher Oligomeric Surfactants — From Fundamentals to Applications",doi:"10.5772/57655",slug:"higher-oligomeric-surfactants-from-fundamentals-to-applications",totalDownloads:1845,totalCrossrefCites:1,totalDimensionsCites:1,signatures:"D. 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Zvonko Pacanoski is an associated professor at the\nFaculty of Agricultural Sciences and Food, Institute for Plant\nProtection in Skopje, Republic of Macedonia. He graduated in\n1998 at the Faculty for Agriculture in Skopje. He had his master’s\nthesis in 2003 and dissertation in 2008 at the same faculty. He participated at the different training programs and courses in the Netherlands, Germany, Switzerland, and Greece, and in 2009, he specialized in Sustainable Agriculture and Rural\nDevelopment (SARD) in CIHAEM, Bari, Italy.",institutionString:null,position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"3",totalChapterViews:"0",totalEditedBooks:"1",institution:{name:"SS. 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The beginning of RPAS integration in non-segregated airspace is expected to be reached by the time frame 2025, according to European RPAS Steering Group [1]. This aim requires broad and structured analysis of the current situation as well as the potential solutions to be implemented. In this way, the development of a risk-based framework to ensure the safe integration of RPAS is crucial for its achievement.
RPAS operation in upper airspace does not require higher technological developments, but it demands detailed analysis about the safety of their integration with conventional aircraft. European Aviation Safety Agency (EASA) and Federal Aviation Administration (FAA) require that the integration of RPAS must not imply a diminish on current safety levels [2, 3]. This requirement means that further research is required to accomplish this goal. A new framework will be compulsory in the future to take the operational features of RPAS into account. One of the goals of this framework is to allow setting out the safety of the RPAS operation jointly with conventional aircraft [4, 5, 6].
Could RPAS fly safely in non-segregated aircraft? The complexity of the answer does not fall into a yes or not issue, because it must be yes, but instead we must focus on how. Currently, conventional aircraft fly according to prefixed routes that are modelled according to air traffic flow patterns, although there are several airspaces based on free-route [7]. Then, RPAS must adapt to the current airway network and current air traffic patterns. One of the main concerns is that RPAS operational patterns can differ from conventional aircraft ones [8, 9]. Although RPAS could be assumed to be modelled as slow conventional aircraft, there are uncertainties about communications, navigation and surveillance issues that must be analysed in advance [10].
Due to this lack of operational and technical knowledge about RPAS operation, regulators and Airspace Navigation Service Providers (ANSPs) seek to introduce RPAS based on a minimum interaction with conventional aircraft [11, 12]. The problem arises when both airspace users operate jointly in the same scenario where the interaction between them cannot be avoided. The first solution to his problem is the segregation of specific air traffic volumes for the different airspace users. However, this segregation should only focus on specific flight levels (FLs) or airways, as airspace cannot be completely segregated in different air traffic volumes for PRAS and conventional aircraft. One of the expected outcomes of this work is to appraise airways or FLs segregation for RPAS.
The most complex assessments about RPAS integration focus on three research areas. The first deals with the global problem of risk management. Clothier et al. [13] developed a framework for structuring the safety case of the RPAS operation. Moreover, various regulators assessed the primary difficulties that must be solved before RPAS operation [14, 15]. The second research area analyses the risk imposed by the single flight for one RPAS in terms of the number of casualties. Several authors developed different risk models to calculate what kind of populated areas are riskier for on-ground pedestrians [16, 17, 18]. The third research area involves the development of collision/conflict-risk models for the integration of RPAS. There are several studies about RPAS collision avoidance [9, 19, 20] (similar to conventional aircraft situations) but few of them focus on conflict risk [21, 22]. Conflict risk is a prior indicator of collision risk. However, none of those studies responds either how the RPAS integration should be or where RPAS could fly in non-segregated airspace.
With the goal of responding to the above research questions, it is required to assess the safety level of the airspace and to develop one specific methodology. Manual 9689 of International Civil Aviation Organisation (ICAO) [23] sets out that airspace planning requires a thorough analysis of every factor that could affect safety. In [24, 25], authors claimed the need for airspace design fulfilling levels of safety under different operational features. Different models were developed to evaluate the collision risk based on airspace geometry [26, 27]. A step further, Netjasov [28] developed a conflict-risk model to assess the level of safety, including air traffic flows. However, there is not a unique methodology that allows analysing the airspace risk-state for the integration of RPAS.
Therefore, the main goal of this research is to develop a risk-based framework to provide geographical and temporary restrictions for the safe integration of RPAS. The risk-based framework is split into two different temporal horizons: design and operation. The risk-based framework evaluates the state of the scenario regarding different risk-based indicators. The risk-based indicators relies on geometrical and operational features of airspace. The risk-based indicators sort airways and crossing points to detect airways (or flight levels): (1) where RPAS can operate because their integration is safe, and (2) when should be planned the operation of RPAS depending on a particular schedule of conventional aircraft. A further aim is to set out the pillars of a future decision-making process for ANSPs.
The rest of the article is structured as follows. Section 2 presents the structure of the risk-based framework and defines the different types of variables and indicators that must be considered. The risk-based indicators constitute the main outputs of the methodology that permit to assess the viability of the RPAS integration. It also describes the methodology for the design phase and the operational phase. Section 3 presents the case study and the application to one Spanish airspace volume and discusses the results. Lastly, Section 4 summarises the main contributions and further works.
The risk-based framework aims to analyse the safe integration of RPAS in non-segregated airspace. In non-segregated airspace, both conventional aircraft and RPAS must operate together. The problem arises when RPAS operate with different technical and operational features than conventional aircraft. Then, the integration of RPAS focuses on reducing their impact on conventional aircraft; in other words, RPAS must adapt themselves to current operations reducing their impact on current aviation. The risk-based framework is split into two phases depending on the operational information available:
design phase: this phase aims to appraise the impact of RPAS in non-segregated airspace for strategical phase. It can be applied both for design purposes and for analysing the operation of one particular scenario. This phase works with basic information of an airspace volume: airway structure and air traffic flow; and
operational phase: this phase addresses a temporal horizon where 1-hour schedule of conventional aircraft is evaluated. The goal is to analyse how the introduction of RPAS affects one specific schedule.
This phase evaluates the way the integration of RPAS affects the airspace in a design or strategic phase. Thus, this analysis covers different input variables as the morphology or geometry and the main characteristics of the air traffic flow that operates at the airspace. The main results of this phase are:
thorough knowledge of the current airspace state, where it is intended to integrate RPAS jointly with conventional aircraft; and
identification of the airways and FLs that allows their segregated use for RPAS. The segregated use implies that the RPAS can fly without any affection to the conventional aircraft.
Design-phase indicators provide information about the state of the airways and the crossing points. They are the most elementary components to analyse the current operational situation of the airspace. These indicators separately evaluate the morphological and geometrical features of the airspace (static indicators) and their operation (dynamic indicators).
Static indicators provide information to analyse the current state of the airspace based on its morphology and geometry. The goal is to perform a prior analysis setting out the airspace design. Static indicators focus on the basic airspace components: airways and crossing points.
The complexity of an airway is characterised by the sections that are exposed to risk. The risk in an airway is modelled by the locations of the airway that are exposed to conflict with aircraft of other airways. These sections are denoted as critical sections (
where
The complexity of a crossing point depends on the number of intersections between the airway pairs that coincides at it and the angle between the airway pairs. In this way, combining both factors, it can be calculated the static indicator of crossing-point complexity:
where
Dynamic indicators focus on the operational features of the airspace. This allows analysing the operational characteristics of the air traffic flows to select the airway that favour or inhibit the RPAS integration.
This indicator provides information about the number of aircraft that operates an airway. It relates the real airway density
where
Taking into account the operational characteristics of the airspace, the dynamic indicator of crossing-point density provide an indicator of the number of aircraft that pass through it.
This indicator evolves from the previous dynamic indicators with a different goal.
Moreover, this indicator also works as a reference value to analyse the air traffic segregation by airways and FLs. Therefore, it is needed to calculate the total value for the whole airspace based on the sum of every airway conflict indicator:
The operational phase focuses on a different temporal horizon than the design phase. The operational phase is characterised by the disappearance of generic air traffic flows (modelled by airway density and average ground speed), and it entails a one-hour schedule. This schedule of air traffic fulfils the operational characteristics of the scenario, but each aircraft has its own characteristics (speed and entry time). Besides, this concept will relay on further work based on 4D trajectories. The operational phase allows the introduction of RPAS in specific schedules. Apart from analysing how this introduction affects the risk indicators, this phase provides the following results:
in-depth knowledge of the path evolution from the conventional aircraft schedule;
safety assessment for the RPAS integration for different schedules based on the risk indicators; and
identification of airways and FLs that favour or inhibit the introduction of RPAS based on the airway availability.
Operational-phase indicators provide information about the whole airspace. In this way, they permit to appraise the airspace situation by the RPAS integration. These indicators conclude if the integration of RPAS is feasible and the temporary restrictions.
where
Conflict severity (
This indicator aims to calculate the risk exposition of an aircraft flying an airway. This indicator is called airway availability because it links the time span the aircraft can safely fly an airway with the time span the aircraft can suffer a conflict. Knowing the airways that present higher availability (the time span the aircraft can safely fly without suffering a conflict), it can be extracted the airways that favour or inhibit the integration of RPAS.
the time duration of the TBWs depends on the crossing angle of the airways and the ground speed of the aircraft involved; and
the time location of the TBWs depends on the entry time of the conventional aircraft and RPAS, length of the airways, the ground speed and the distance between the airway entry-point and the crossing point.
Herein, the exposition time relates to a one-hour schedule. A minor TBW implies a bigger airway availability, which reduces the risk exposition. Moreover, airway availability is a novel indicator defined in this work. There is no previous knowledge about the threshold that this indicator should acquire. Then, the authors propose a division into four stretches (0–25%, 25–50%, 50–75% and 75–100%). Airways with airway availability greater than 50% are airways where RPAS could be included.
The risk-based framework was applied to the air traffic volume LECMPAU (Pamplona) in Spain. This airspace is constituted by 24 airways and 55 crossing points. The period of study was July and August 2016, and the operational data was obtained from NEST [31].
This section introduces the results of the design phase in the strategical horizon. This is the most valuable innovation of this work, and a further motivation is related to the fact that this methodology could also be applied to a pre-tactical phase. The design phase focused on a fix air traffic distribution for the whole day while in the pre-tactical phase, a temporary variation of the air traffic flow for a specific day could be considered. However, the application for a pre-tactical phase was out of the scope of this work. The process was as follows:
airways and crossing point were characterised based on the geometric information (length, angle and critical section) and operational information (air traffic flow and average speed); and
static and dynamic indicators were calculated for each airway and crossing point. With this information, we ordered and analysed which of them had a greater impact on safety.
Firstly, design-phase indicators are calculated for LECMPAU both for airways and for crossing points. However, for the sake of clarity, we only present the results for the airway due to the high number of crossing points. Table 1 shows the results for the design-phase indicators of the LECMPAU airways.
Airway | ||
---|---|---|
UN858 | 1.00 | 0.0037 |
UM190 | 0.75 | 0 |
UP181 | 3.18 | 0.0090 |
UL176 | 4.40 | 0.0267 |
UQ262 | 5.42 | 0 |
UQ148 | 2.07 | 0 |
UN10 | 3.25 | 0.0267 |
UN857 | 3.92 | 0.0046 |
UL866 | 7.17 | 0.0005 |
UN995 | 7.03 | 0.0043 |
UN976 | 3.14 | 0.0275 |
UM601 | 2.84 | 0.0478 |
UM176 | 4.00 | 0 |
UQ57 | 3.04 | 0 |
UQ73 | 3.94 | 0 |
UT430 | 2.09 | 0 |
UP152 | 4.03 | 0.0034 |
UN725 | 1.21 | 0.0385 |
UQ400 | 1.18 | 0 |
UQ88 | 1.46 | 0 |
UL184 | 1.93 | 0 |
UQ424 | 1.45 | 0 |
UQ300 | 1.52 | 0 |
UQ268 | 2.10 | 0 |
Design-phase indicators for LECMPAU airways.
most of the values of
the lowest values referred to the airway UM190 (
the highest values were referred to as airways UQ262 (
Therefore, the highest values of the airway complexity static indicator were referred to the airways that concurred at crossing point PPN. Figure 1 shows a representation of the static indicators of the airway and crossing-point complexity.
Results of the design-phase analysis.
Regarding the dynamic indicator of airway density (
The airway segregation aimed to identify the airways (or geographical restrictions) that allowed the safe integration of RPAS because they did not generate conflicts with conventional aircraft. First, the total value for the whole airspace of the dynamic indicator of airway conflict (
Airway | |
---|---|
UM190 | 0.0108 |
UQ262 | 0.1449 |
UQ148 | 0.1567 |
UM176 | 0.0422 |
UQ57 | 0.0689 |
UQ73 | 0.0422 |
UT430 | 0.1210 |
UQ400 | 0.0304 |
UQ88 | 0.0304 |
UL184 | 0.0350 |
UQ424 | 0.0350 |
UQ300 | 0.0304 |
UQ268 | 0.1837 |
Base-scenario | 0.0037 |
Results of
As can be seen in Table 2, no airway was identified for its segregation.
The primary conclusion of the previous section was that no airway could be segregated at LECMPAU. In spite of this limitation, this work evaluated the existence of specific FLs that allowed the safe integration of RPAS. The process was similar to airway segregation but focusing on the FLs of interest: from FL250 to FL300.
There are five airways that could be segregated at different FLs for the integration of RPAS, see Figure 1. UQ400, UQ88 and UQ300 presented three FLs (260, 280 and 300) where RPAS could be integrated without any interaction with conventional aircraft. UM176 and UQ74 could be segregated at FL270 (see Table 3).
Airway | ||||||
---|---|---|---|---|---|---|
UN858 | 0 | 0 | 0 | 0 | 0 | 0 |
UM190 | 0.4903 | 0.5911 | 1.0445 | 1.7939 | 2.3628 | 1.1805 |
UP181 | 0.5528 | 0 | 0 | 0 | 0 | 0 |
UL176 | 0.2512 | 0 | 0 | 0 | 0 | 0 |
UQ262 | 0.4382 | 1.0439 | 1.5495 | 3.0551 | 2.7039 | 2.5510 |
UQ148 | 0.5058 | 0.9344 | 1.4552 | 2.7312 | 2.8154 | 2.9160 |
UN10 | 0 | 0.7542 | 0 | 1.1512 | 0 | 1.6451 |
UN857 | 0 | 0 | 0 | 1.0887 | 0 | 0.9156 |
UL866 | 0.9234 | 0.4331 | 1.1965 | 1.0887 | 0 | 0.9156 |
UN995 | 0 | 0 | 0 | 0 | 0 | 0 |
UN976 | 0 | 0 | 0 | 0 | 0 | 0 |
UM601 | 0 | 0 | 0 | 0 | 0 | 0 |
UM176 | 0.2564 | 0.2888 | 0.4519 | 0.9624 | 2.2068 | 1.4878 |
UQ57 | 0.3028 | 0.2888 | 0.5137 | 0.9624 | 2.4138 | 1.4878 |
UQ73 | 0.2564 | 0.2888 | 0.4519 | 0.9624 | 2.2068 | 1.4878 |
UT430 | 0.5058 | 0.4726 | 1.0935 | 1.9611 | 2.4653 | 2.5284 |
UP152 | 0.4903 | 1.0529 | 0 | 0 | 2.7128 | 1.5682 |
UN725 | 0 | 0 | 0.5137 | 0 | 0 | 0 |
UQ400 | 0.2351 | 0.1690 | 0.5137 | 0.8999 | 2.2080 | 0.7584 |
UQ88 | 0.2351 | 0.1690 | 0.5137 | 0.8999 | 2.2080 | 0.7584 |
UL184 | 0.6088 | 0.3702 | 0.9449 | 0.8999 | 2.6966 | 0.7584 |
UQ424 | 0.6088 | 0.3702 | 0.9449 | 0.8999 | 2.6966 | 0.7584 |
UQ300 | 0.2351 | 0.1690 | 0.5137 | 0.8999 | 2.2080 | 0.7584 |
UQ268 | 0.8537 | 1.3565 | 1.0451 | 2.5607 | 3.3841 | 3.3381 |
Values of
To study the operational phase, a real one-hour schedule was selected from the rush hour of LECMPAU at FL290. Table 4 shows the operational information of the schedule composed of four conventional aircraft and one RPAS. In this schedule, one RPAS is introduced by UM176 with a typical speed of 250 kts.
Aircraft | Airway | Entry time | ||
---|---|---|---|---|
1 | 12:13:56 | 290 | 310.13 | |
2 | 12:20:31 | 290 | 416.67 | |
3 | 12:25:00 | 290 | 420.11 | |
4 | 12:57:28 | 290 | 351.75 | |
12:30:00 | 290 | 250 |
Schedule of LECMPAU with one RPAS.
The first step was to calculate the TBWs that will underline the airway indicator and conflict detection. Table 5 provides de length or time span of the TBWs for the different aircraft that could interact between them.
Aircraft | 1 | 2 | 3 | 4 | |
---|---|---|---|---|---|
1 | — | 206 | — | — | — |
2 | 206 | — | 177 | 192 | 310 |
3 | — | 177 | — | — | 641 |
4 | — | 192 | — | — | — |
— | 310 | 641 | — | — |
Temporary-blocking windows (sec) for the base schedule.
The length of the TBWs increased with the RPAS due to its lower speed. The TBWs (i.e. the time exposed to conflict) almost doubled the value for conventional aircraft. Table 6 provides the temporary limits (initial and final) for the TBWs between aircraft pairs.
Aircraft | 1 | 2 | 3 | 4 | |
---|---|---|---|---|---|
1 | — | [12:15:18, 12:18:44] | — | — | — |
2 | [12:15:43, 12:19:08] | — | [12:20:19, 12:23:16] | [12:16:25, 12:19:37] | [12:14:12, 12:19:22] |
3 | — | [12:22:15, 12:25:12] | — | — | [12:14:40, 12:25:21] |
4 | — | [12:58:22, 13:01:34] | — | — | — |
— | [12:51:09, 12:56:19] | [12:49:39, 13:00:20] | — | — | |
Entry time | 12:13:56 | 12:20:31 | 12:25:00 | 12:57:28 | 12:30:00 |
Initial and final time of the TBWs.
According to the TBWs, aircraft with an entry time located inside the TBWs entailed a conflict between those aircraft pairs. In this example, there was no conflict between any aircraft. In the same way, there was no conflict; the indicator of conflict severity was zero.
However, airway availability was calculated for all airways taking into account base schedule, see Table 7.
Airway | |
---|---|
UN858 | 0.8932 |
UM190 | 1 |
UP181 | 0.1488 |
UL176 | 0.8917 |
UQ262 | 0 |
UQ148 | 0.2907 |
UN10 | 0.8901 |
UN857 | 0 |
UL866 | 0.6741 |
UN995 | 0.7024 |
UN976 | 0.7093 |
UM601 | 0.7007 |
UM176 | 0.5572 |
UQ57 | 0.3298 |
UQ73 | 0.6176 |
UT430 | 0.8931 |
UP152 | 0.4501 |
UN725 | 0.8911 |
UQ400 | 0.8890 |
UQ88 | 0.8736 |
UL184 | 1 |
UQ424 | 1 |
UQ300 | 1 |
UQ268 | 0.6802 |
Airway availability indicator for FL290.
The airway availability indicator decreased with the introduction of RPAS. In the case
This research developed a new risk-based framework to evaluate the safe introduction of RPAS in non-segregated airspace. The risk-based framework tackled two temporal horizons for the introduction of RPAS based on a design phase (strategical horizon) and an operational phase (tactical horizon). This innovative approach allowed considering the different variables that affected the aircraft operation at both temporal horizons, which ensured a hierarchical assessment. The design phase covered different input variables as the morphology or geometry, and the main characteristics of the air traffic flow. Meanwhile, the operational phase was characterised by the disappearance of generic air traffic flows (modelled by airway density and average ground speed) and focused on a one-hour schedule (constituted by conventional aircraft and RPAS). Different indicators were modelled depending on the temporal horizon. The design phase considered static and dynamic indicators (based on the airspace structure and generic air traffic flows). The operational phase considered three indicators: number of conflicts, conflict severity and airway availability. The application of the methodology was to detect geographical restrictions (airways that favour or inhibit the integration of RPAS) and temporary restrictions (when the RPAS can pierce into the airspace without generating any conflict).
This methodology was applied to Spanish airspace LECMPAU at different FLs from FL250 to FL300, which were the most favourable for RPAS integration due to their low density. The different static indicators ordered airways considering their complexity. LECMPAU was a complex scenario because of the high number of airways and crossing points. The airway segregation analysis concluded that no full airway could be segregated for RPAS; however, different FLs could be used considering their segregation for RPAS. The segregation of FLs for RPAS implied that they could operate these FLs without being exposed to conflict with conventional aircraft. A one-hour schedule of conventional aircraft was analysed for the introduction of one RPAS. Operational-phase indicators were assessed and based on the temporary-blocking windows, no conflict arose. The temporary-blocking windows provided the temporary restrictions for the integration of RPAS. Moreover, the airway availability indicator ordered the airway providing information about the airways that favoured (or inhibited) the introduction of RPAS with the operational-phase specific schedule. Regarding future research lines, the calculation of an independent and fixed value for conflict probability is crucial for the assessment of different airspaces and FLs. A further goal will be the analysis of the whole process to introduce flight plans of RPAS in non-segregated airspace ensuring safe scenarios.
This Project has been developed under the OIDATM (Observatory for the Advancement of Air Traffic Management) promoted by ISDEFE. Particularly, the authors would like to acknowledge Gonzalo Águeda and Cristina Altemir from UPM and Miguel A. Martín Blanco, Jaime Torrecilla, Maria Anta Garcia and Susana Duran Vizuete from ISDEFE.
The authors declare no conflict of interest.
Genetically encoded reporters such as fluorescent and bioluminescent proteins have achieved widespread success as useful research tools in life sciences, including cell biology [1, 2, 3], oncology [4, 5], cardiology [6, 7], neurology [8, 9], as well as infection and inflammation studies [10, 11]. Because of their sensitivity in quantitative measurement, both fluorescent and bioluminescent proteins remain the top choices for monitoring live cell processes in mammals. The most commonly used reporters include green/red fluorescent proteins or their genetic derivatives (GFP, RFP, YFP or mCherry) [12, 13, 14]. Insect or marine bioluminescent proteins (Firefly luciferase or Renilla/Gaussia luciferase) are used as well [15, 16, 17]. Through codon optimization, these reporters have been genetically engineered to monitor many physiological and disease processes such as cell communications [18, 19], protein and exosome secretion [20, 21], viral infection, inflammation, and apoptosis [5, 11, 22]. In most situations, a single type of reporter may provide better sensitivity and specificity over traditional methods such as Western blot analysis or polymerase chain reactions (PCRs). For example, fluorescent proteins may provide handy real-time monitoring when a fluorescence microscope is available. However, the signal quantification is less convenient and often requires sophisticated software or expensive FACS equipment [9, 23, 24]. On the other hand, the bioluminescent reporter is easily quantifiable and can be made amenable to laboratory automation by using a less expensive luminometer [18, 25, 26]. Therefore, the combination of both types of reporters may be superior, allowing for both visual monitoring and laboratory automation.
Recently, we have developed a novel dual-reporter circuit from the marine GFP and the firefly luciferase (Fluc) and demonstrated their usage in studying gene regulation and cell signaling in mammalian cells [27]. Additionally, we and others have shown that the marine
In this chapter, we describe the design and validation of a novel AAV vector-based dual-reporter format by a combination of GFP and Gluc for high throughput monitoring of inflammation in human cells. This new circuit has high sensitivity and specificity with little background noise in reporting inflammatory response. We demonstrate that the GFP allows for real-time monitoring and produces high-content data sets at individual cell levels using fluorescence microscope or FACS. In parallel, the secretory Gluc allows for the monitoring of inflammatory response at population levels and enables HTP analysis and laboratory automation with a luminometer or microplate reader. Together, this dual-reporter provides a robust and high throughput means to study inflammation in human cell or animal models.
Inflammatory cytokine TNFα was obtained from R&D Systems (Minneapolis, MN). Phorbol-12-myristate 13-acetate (PMA) was obtained from MilliporeSigma (St. Lois, MO). Luciferase assay reagent and fetal bovine serum (FBS) were obtained from ThermoFisher (Waltham, MA). AAV-DJ capsid protein and the helper free viral packaging system were obtained from Cell Biolabs (San Diego, CA). The serum-free UltraCULTRE complete medium was obtained from Lonza (Anaheim, CA).
The AAV-based dual-reporter circuit was constructed by DNA synthesis and a fusion technology as previously reported [24, 29]. The reporter circuit was flanked by inverted terminal repeats (ITRs). These two ITRs were synthesized by direct DNA synthesis from Genscript (Piscataway, NJ). We then built the genetic cassette according to the configuration from the 5′ to 3′-end: the transcription factor response elements (TREs) of NF-κB, a minimal CMV promoter sequences (mCMV), a dual-reporter with a self-splicing peptide (Gluc-T2A-GFP), the poly adenylation signaling sequences (Poly-A) (NF-κB reporter, GenBank Accession Number: MG786368). To evaluate the background noise, basal transcription activity and the inducible signal range, a promoterless reporter (background noise, MG786370), a minimal promoter (basal transcription activity, GenBank Accession Number: MG786371), and a full promoter CMV (signal range, GenBank Accession Number: MG786372) were built in a similar format. To assess the specificity, a dual-reporter responsive to growth factors (AP-1 reporter, GenBank Accession Number: MG786369) but not inflammatory stimuli was similarly designed and constructed. Final constructs were sequence-verified from ITR to ITR, and the annotated sequences can be retrieved from GenBank (MG786368–72).
Human embryonic kidney cells (HEK293), human liver cancer line (HepG2) and human glioblastoma line (U87) were obtained from ATCC (Manassas, VA). Cells were cultured in DMEM supplemented with 10% FBS, 2 mM GlutaMax and penicillin-streptomycin 100 U/mL. All cells were culture at 370C with 95% humid air and 5% CO2.
Cell culture transfections were conducted in 6-well plates as reported [27]. Cells growing at 50–70% confluency were transfected by combining reporter DNA (1–2.5 μg/well) with Lipofectamine (Thermo-Fisher) or FuGene 6 transfection reagents (Promega) for 24–72 h. Cells were then switched to fresh medium for cytokine treatment.
Reporter AAV were produced by transfecting HEK293 cells as reported [27]. Cells on culture dishes were transfected with a mixture of reporter DNA and helper AAV-reporter plasmids expressing Rep and Cap proteins. Twenty-four hours after the transfection, culture medium was changed to allow production of viral particles for additional forty-eight hours. The recombinant AAV viruses were prepared from the conditioned medium using an AAV concentration reagent according to the manufacturer’s instruction (System Biosciences, Palo Alto, CA). All AAV reporter viruses were packaged with AAV-DJ capsids, which have broad tropism in transduction [30].
Viral titration and multiplicity of infection (MOI) were determined by performing green cell fluorescent assay and PCR as reported [27, 31, 32]. Briefly, HEK293 cells on 12-well plates were transduced with serial dilutions of fCMV-Gluc-T2A-GFP control viruses. After 72 h, GFP-positive cells were visually scored under a fluorescence microscope. MOI of control virus was determined by GFP positivity of the transduced cells, while the MOI of reporter viruses was estimated by comparing the relative copy numbers of the reporter viruses to those of the GFP-positive control viruses [27].
Gaussia luciferase activity was assayed by a luminometer (Promega, Fitchburg, WI) as previously reported [18, 24]. Briefly, conditioned medium from treated cells were collected and subsequently cleared by centrifugation at 12,000 rpm for 5 min. The cleared supernatants were used for Gluc assay. For Gluc activities quantification, 100 μL of substrate was added to 5 or 10 μL of the conditioned medium and relative light units were recorded instantaneously [25].
Images of living cells were typically taken using fluorescence microscopy as reported [33]. To show the intensity of GFP expression, both fluorescent and phase contrast images were recorded. To compare expression levels of GFP, identical parameters including the exposure time, contrast and gain were kept identical within each set of experiments. When the fluorescence intensity was low, images were equally adjusted to show the relative GFP intensity.
Cultured HEK293 cells were sorted and quantified by using the Accuri C6 Cytometry (BD Biosciences, San Jose, CA). More than 10,000 events were recorded via a GFP channel. Triplicate samples were analyzed to ensure consistency in results. Data were processed by CFlow Plus software.
Relative GFP intensity was quantified using a Microplate Reader (BMG Labtech) following the cytokine treatment. To reduce background noise level, the conditioned media were removed and cells were washed with phosphate buffer. The relative GFP intensity was recorded for both treatment and control groups. For each sample, nine areas were measured and averaged by the OMEG 3.00R2 software.
Human cells were monitored in real-time under fluorescence microscopy. Fluorescent and phase contrast images were recorded under the same experimental conditions. For Gaussia luciferase assay, GFP quantification, and FACS analysis, the data are reported as mean ± SD (n = 3), unless stated otherwise.
Temporal monitoring and quantifying of inflammatory response at individual cell levels or within tissues is highly desirable [34, 35]. To accomplish this goal, we developed a new format of genetic circuit composed of transcription response elements (TREs), a minimal promoter (mCMV) and a dual-reporter (Gluc and GFP) (Figure 1A). According to this design, the TREs will respond to the binding of activated transcription factors such as NFκB; thus, they can switch the expression of reporter genes from an off-status to an on-status (Figure 1A). Since different signaling molecules may elicit distinct transcription factors (TFs). A careful choice of TREs will enable the construction of different genetic circuits for signaling monitoring. For instance, by using the binding sequences of NFκB as TREs (Figure 1B), this unique circuit may be able to monitor inflammation processes. Preferably, this genetic circuit will respond specifically to inflammatory molecules such as TNFα [36, 37, 38]. Taking advantage of different features of reporter proteins, the cell response can be real-time monitored by a number of HTP methods. For example, GFP may be imaged by fluorescence microscopy or quantified by microplate reader or FACS. Alternatively, secreted Gluc activity can be easily quantified by assaying a portion of conditioned medium, blood, or urine. Because the wave length of Gluc emission is longer than 600 nm, Gluc becomes a preferred imager for in vivo bioluminescence imaging (BLI) (Figure 1B).
System design and workflow of AAV-based dual reporters for high throughput monitoring of inflammatory response in human cells. (A) Schematic illustration of genetic circuit of the AAV-based dual reporter. The genetic circuit (5′ → 3′) is composed of the transcriptional responding elements (TREs), the minimal CMV promoter (mCMV), a chimeric gene coding dual reporter proteins Gluc and GFP with a T2A (self-cleavage peptide). Depending on the availability of transcription factor (NF-κB), this inflammatory circuit may be either in an off-status with only minimal expression of reporters when no NF-κB binds to its TREs (upper panel), or in an on-status with a high level of expression when NF-κB binds to its TREs stimulated by inflammatory cytokine TNFα (lower panel). (B) Workflow for HTP monitoring of inflammatory response with GFP and Gluc. Cellular response to inflammatory stimuli can be monitored and quantified by GFP reporter (high content microscopy, FACS, microplate reader, or luminometer). Alternatively, inflammatory signaling can also be quantified by Gluc reporter from a portion of conditioned medium (Luminometer or bioluminescent imaging, BLI).
To test our new system, we built two reporter circuits to monitor either inflammatory processes (NFκB) or cell growth signaling (AP1). Typically, 4–6 tandem reporters of TREs can be joined together via 6-bp linkers [27]. These TREs were inserted the upstream of the mCMV-driven dual-reporter. This reporter circuit was flanked with ITR to allow AAV packaging and production.
To determine the background noise, basal transcription activity, inducible signal range, and the signal-to-noise ratio of the new circuit, we further designed and constructed three additional vectors: (1) a promoterless vector to assess the background noise; (2) a minimal promoter vector to assess the basal activity; and (3) a full-length CMV promoter to assess the signal range (Figure 2A). We transfected these reporters into HEK293 and monitored the appearance of GFP and red fluorescent protein (RFP), which was co-transfected and served as an invariable control (driven by a constitutive EF1α promoter) (Figure 2B). As predicted, cells transfected by the promoterless circuit remained GFP negative for 48 h, indicating little background noise of this new AAV reporter circuit (Figure 2B, a, g). For the minimal promoter circuit, few cells were weakly positive for GFP, indicating low levels of basal expression (Figure 2B, b, h). In contrast, ∼80% of cells exhibited strong GFP fluorescence in the fCMV group (24–48 h) (Figure 2B, c, i). However, under the same experimental condition, the steady expression levels of control RFP remained consistent among different groups for both 24 h (Figure 2B, d–f) and 48 h (Figure 2B, j–l), suggesting the differential expression of GFP was attributed to the promoter usage rather than the differences caused by transfection discrepancy. Together, these data validated the functionality of our new circuit and confirmed that GFP could be used for reporting signaling strength in living human cells.
System setup and performance analysis. (A) Design and construction of three AAV-based dual reporters for system testing. The dual-reporter circuit is flanked by inverted terminal repeats (ITR), which allows for packaging into recombinant AAV. Promoterless, minimal promoter (mCMV) and full CMV (CMV)-driven dual reporter circuit are shown from top to bottom. (B) Expression of dual-reporters in live HEK293 cells. The GFP expression (green) in HEK293 cells were recorded with a fluorescence microscope following transfections of either a promoterless, an mCMV, or an fCMV-driven reporter at 24 h (a–c) and 48 h (g–i), which were co-transfected with a positive control plasmid DNA expressing RFP (d–f for 24 h; j–l for 48 h). Arrows indicate GFP- or RFP-positive cells. The bottom panels show the corresponding phase-contrast images for each group. (C) The Gluc activity from the conditioned medium was determined by a luciferase assay following the transfection of three reporters at the same time points. The luciferase activity was expressed as relative light units (RLU), normalized against protein input, and presented as fold increase over untreated control (mean ± SD, n = 3) with statistical significance of P<0.001, using student’s T-test. The bottoms panels are representative phase-contrast images for each treatment group.
In parallel, we also examined the Gluc activities from the conditioned media to determine if the Gluc activities were similarly regulated depending on the promoter types. As shown in Figure 2C, the promoterless circuit showed a very low background noise while the mCMV promoter circuit exhibited a significant increase in Gluc activities (∼30.9-fold at 24 h, ∼56.9-fold at 48 h). A marked ∼2255- or ∼3847-fold increase in Gluc activity was recorded for the fCMV promoter circuit. These data confirmed that the new reporter format has low levels of background noise and a broad signal range with a signal-to-noise ratio of ∼3847:1.
Next, we tested whether the dual-reporter system was specific in monitoring inflammation with a commonly used transfection protocol. We conducted a comparative study on two distinctive pathways, the inflammation (NFκB) and cell growth (AP-1). These two pathways have been shown to be specifically activated by proinflammatory cytokine (TNFα) and cancer promoting reagent (PMA), respectively [18, 39]. Accordingly, we co-transfected HEK293 cells using each of these reporters along with EF1α-driven RFP as invariable reference to determine specific effects of TNFα and PMA on reporter activations. After cells were transfected with NFκB-Gluc-2A-GFP reporter, treatment of cells with 10 ng/mL TNFα induced a marked increase in GFP levels (Figure 3A, b). In parallel, a 42-fold increase in Gluc activity was detected, indicating an activation of the inflammatory pathway (Figure 3B). As expected, very few GFP-positive cells were present in the wells treated with PMA, suggesting specific activation of NFκB by TNFα but not PMA (Figure 3A, c). Again, no significant change in Gluc activities was observed in mock control or cells treated with PMA (Figure 3B). Conversely, after cells were transfected with the AP1-Gluc-2A-GFP reporter, treatment of cells with 50 ng/mL PMA induced a significant increase in GFP levels (Figure 3C, c), in line with a 3.8-fold increase in Gluc activities (Figure 3D). In contrast, neither mock-control (Figure 3C, a) nor TNFα (Figure 3C, b) caused any significant changes in the expression levels of GFP or Gluc (Figure 3D). Additionally, the co-transfected invariable reference RFP showed little changes among different groups (Figure 3A, C, middle panels), excluding the possibility that such differences were caused by discrepancies in transfection efficiency. Taken together, these results confirm the specificity of our new reporter circuit in monitoring inflammation using a simple transfection protocol.
The specificity of the AAV-based dual-reporters by transfection. HEK293 cells were transfected with NF-κB reporter for 24 h. Cells were then switched to low serum medium in the presence or absence of either TNFα (10 ng/mL) or PMA (50 ng/mL) for 24 h. The images of GFP (A: a–c), RFP (A: d–e), and phase (A: lower panel) were recorded 24 h after the treatment. The corresponding luciferase activity was assayed using the conditioned medium collected from a control, TNFα or PMA treatment group (B). In a separate set of experiments, HEK293 cells were transfected with AP-1 reporters for 24 h, followed by the treatment of either TNFα (10 ng/mL) or PMA (50 ng/mL) for additional 24 h. The GFP (B: a-c), RFP (B: d-e), and phase (B: lower panel) images were recorded and the corresponding luciferase activity was also assayed and graphed (D). Arrows indicates the GFP- or RFP-positive cells. The luciferase activity was expressed as relative light units (RLU) and presented as fold increase over untreated control (mean ± SD, n = 3). *** denotes P<0.001, using student’s T-test.
Following successful monitoring of cell signaling with a transfection protocol, we further test whether the new AAV-based circuit could be successfully packaged into delivery viral particles to deliver the reporter circuit to various cells [40, 41, 42]. For virus packaging, we used our established protocol to generate recombinant reporter viruses [27]. For cell transduction, packaged viruses (∼ 1× MOI viruses) were used to transduce HEK293 cells for 24 h. Following the transduction, cells were washed and switched to UltraCULTURE for the treatment of TNFα or PMA. Similar to the transfection experiment, the NFκB reporter circuit showed a marked increase in Gluc activity (85-fold over control) in response to the TNFα treatment but not PMA (Figure 4A). Conversely, the AP-1 reporter circuit showed a smaller but significant increase in Gluc activity (9.6-fold over control) for the treatment of PMA but not TNFα (Figure 4B). These results validated the specificity of this dual-reporter using a highly effective transduction protocol. Consistent with low basal level of Gluc activities, we also observed a low GFP expression in control cells, which increased in their intensities in response to TNFα treatment but not to PMA. Likewise, the GFP intensity increased in response to PMA but not to TNFα treatment in the AP-1 system, indicating specific response to its corresponding singling stimuli. It is worth noting that both GFP intensity and Gluc activity appeared to be low for cells with transduction in comparison to transfection. However, the relative fold-increase of Gluc appeared to be more prominent (85-fold vs. 42-fold increase for NF-κB and 9.6-fold vs. 3.8-fold increase for AP-1), which suggests that the lower background noise may increase the detection sensitivity using viral transduction protocol. Nevertheless, our data support the notion the new format of reporter circuit could be used to monitor and quantify inflammation or cell growth signaling by either simple transfection or highly efficient transduction protocols.
The specificity of AAV-based reporters by transduction. HEK293 cells were transduced with either NFκB (A) or AP-1 (B) reporter AAV (MOI = 1) for 24 h. Cells were then switched to low serum medium in the presence or absence of TNFα (10 ng/mL) or PMA (50 ng/mL) for additional 24 h. In separate experiments, following 24 h transduction with NFκB reporter AAV, HEK293 cells were subject to either the treatment of increasing concentration of TNFα (0, 0.1, 1, 5 10, and 50 ng/mL) for 24 h (C), or in the presence of TNFα (10 ng/mL) for 0, 3, 7, 24, 48, and 72 h (D).The luciferase activity was determined using conditioned medium, and expressed as relative light units (RLU) or presented as fold increase over untreated control (mean ± SD, n = 3). ** denotes P<0.01, while *** denotes P<0.001, using student’s T-test.
We next performed the dose-response and time-course experiments to examine the sensitivity of our inflammatory reporter circuits in HEK293 cells. Following the transduction of cells with either AAV-based NFκB reporter or control plasmid for 24 h, cells were treated with incremental amount of TNFα (0.01, 0.1, 1, 5, and 10 ng/mL) for 24 h. As low as 0.01 ng/mL TNFα induces a significant rise of Gluc activity (3-fold over control, Figure 4C). As expected, higher TNFα concentrations (0.1–10 ng/mL) elicited more robust responses (15–105-fold over control) in a dose-dependent fashion (Figure 4C). In agreement with Gluc activities, GFP images exhibited a similar pattern of dose-dependent increase of GFP expression levels in responding to TNFα.
To temporally examine the activation of the NFκB reporter, we conducted a time-course study on the effects of TNFα using the established transduction protocol. We used a dosage of 10 ng/mL of TNFα because this dosage can induce a robust response in the above dose-response experiments. As early as 3 h following TNFα treatment, Gluc activity started to rise significantly (5-fold over control, Figure 4D). During the first 24 h, Gluc activity steadily increased (5–64-fold over control) (Figure 4D). Together, these results show that our new dual-reporter responds to inflammatory stimuli in both a dose- and time-dependent fashion, hence demonstrated the usefulness of the secreted Gluc for temporal monitoring of inflammation with our new reporter circuit in living human cells.
To further explore whether our new dual-reporter circuit is amenable to HTP applications, we conducted a series of high content experiments that consisted of multiple dose- and time-course studies. Following the transduction of AAV-based NFκB reporters, cells were treated with incremental concentrations of TNFα (0, 0.01, 0.1, 1, 5, 25, 100 ng/mL). At multiple time-points (0, 1, 3, 6, 9, 24, 32, and 48 h), a small portion of culture medium was collected to determine the Gluc activity. As shown in Figure 5A, both time- and dose-dependent responses to the treatment of TNFα were detected. However, the cellular response to TNFα appeared to be attenuated in the following 24–48 h, indicating the maximum possible stimulation achieved at 24 h time-point. It is important to note that cellular response demonstrated a typical early response (Figure 5B). At doses of 0.1 ng/mL and above, significant increases (Figure 5B) in Gluc activities were apparent. Even at the lowest dose of 0.01 ng/mL, a small increase (1.4-fold over control) in Gluc activities was detected at 6 h (Figure 5B), supporting a receptor-mediated quick activation model. Additionally, the secreted reporter Gluc enabled us to conduct these multi-dose (eight dosages) and multi-times (8 time-points) experiments in a triplicate format (three biological repeats), yielding a total of 192 data points, rendering a reliable and informative pattern of response.
(A) HEK293 cells respond to TNFα stimuli in a time- and dose-dependent manner. Following transduction with NF-κB reporter virus (MOI = 1) for 24 h, HEK293 cells were treated with increasing concentration of TNFα (0, 0.01, 0.1, 1, 5, 10, 25, 50 and 100 ng/mL). A small portion of conditioned medium was collected at the indicated time-points (0, 1, 3, 6 and 9 h). The luciferase activity was determined using the conditioned medium, and expressed as relative light units (RLU) or presented as fold increase over untreated control (mean ± SD, n = 3). (B) Inset of control and treatment concentrations of 0.1 and 0.01 ng/ml. ** denotes P<0.01, while *** denotes P<0.001, using student’s T-test.
We next examined the individual cell response to inflammatory cytokines using FACS as a HTP tool. HEK293 cells were transduced with AAV-based NFκB reporters, and the GFP emission was quantified by FACS. As expected, cells in the background group (mock transfection) were GFP-negative (Figure 6A, left panel), while reporter-transduced cells showed weak GFP expression in the absence of TNFα (Figure 6A, middle panel), but high GFP expression in the TNFα-treatment group (Figure 6A, right panel), demonstrating a robust cellular response. In parallel, our cytometry data revealed high intensity of GFP signal at individual cell levels: 0% in background group vs. 26% in control group vs. 83.6% in TNFα group (Figure 6B). A marked shift of GFP intensity following TNFα treatment was apparent when these graphs were merged (Figure 6C). Additionally, the individual response could also be summarized and averaged to evaluate cellular response as a heterogeneous population, which showed ∼40-fold increase in TNFα group over control (Figure 6D). Together, our results demonstrated that flow cytometry can be used to assess inflammatory response at both individual cell and population levels using our novel reporter circuit.
High-content quantification of inflammatory response by the flow cytometry. Following transduction with NFκB reporter virus (MOI = 1) or mock transduction (background control) for 24 h, HEK293 cells were switched to serum-free medium in the presence or absence (b) of TNFα (10 ng/mL) for additional 24 h. Response of individual cells was imaged by fluorescence microscopy (A) and further quantified by FACS analysis (B, C). The average response was calculated and graphed (D). The data was presented as mean ± SD, n = 3.
Next, we examined whether a more commonly available microplate reader can be an alternative readout tool compared to the expensive cytometer for GFP quantification. Following the transfection of HEK293 cells with our NFκB reporter, the GFP intensity was recorded by fluorescent microscopy and the GFP emission was quantified with a microplate reader equipped with a laser lamp and a GFP signal detector. As expected, TNFα treatment induced a drastic increase in the GFP intensity over control (Figure 7A) with a 16.7-fold increase in GFP signal measured by a microplate reader. To further examine whether our NFκB reporter can be used to monitor the inflammation response in other cell types, we transfected two additional human cell types (U87 and HepG2) with this reporter and quantified their response to TNFα treatment. Similar to HEK293 cells, we observed marked increases (9.2-fold and 18.3-fold over control) in GFP intensity in U87 (Figure 7B) and HepG2 (Figure 7C) cells, respectively. Together, our results validated another convenient approach for signal quantification by using less expensive Microplate reader, which is easily amenable to laboratory automation.
Quantification of inflammatory response by microplate reader. Three types of human cells were separately transfected with NF-κB reporter for 24 h and then switched to serum-free complete medium in the presence or absence of TNFα (10 ng/mL) for additional 24 h. The GFP (A–C: a, b) and phase (A–C: c, d) images were taken and the corresponding GFP intensity was further quantified by a microplate reader and graphed (e–g). The data was presented as mean ± SD, n = 3.
We report the development of a new AAV-based dual-reporter circuit for live monitoring of cell signaling that is fundamental to both physiology and pathology. Our system combines two functionally complementary reporters (Gluc and GFP), which enables HTP applications and laboratory automation. The distinctive feature of this dual-reporter from our previous ones [18, 27] is the introduction of Gluc, which is a secretary form of luciferase and can be easily retrieved from conditioned medium for quantification [17]. Retaining the GFP reporter preserves the capability of both real-time imaging and single cell analysis [34]. Through comprehensive examination and validation using a fluorescence microscope, flow cytometer, and microplate reader, we demonstrated that our new system is robust and provides attractive advantages over existing methods. These advantages include a wide detection range, low background, high sensitivity and better specificity, and multiple gene delivery options.
The successful development of a genetic circuit requires a sound strategy and an ability to monitor molecular singling in a highly sensitive and specific manner. To study inflammation, we chose the NFκB because it is an important transcription factor in regulating cellular responses with a rapid-activation property [43, 44, 45]. In most types of cells, NFκB exists as a dimer in the cytoplasm in an inactive status via interaction with IκB inhibitor proteins. NFκB can be activated by various inflammatory molecules, including TNFα, IL-1β, or bacterial lipopolysaccharides (LPS) [44, 45, 46]. Upon binding of these stimuli to their respective receptors, the IκB kinase becomes activated and phosphorylates IκB proteins, which in turn are ubiquitinated and degraded by proteasomes. Once the IκB is degraded, the NFκB complex is free to migrate into nucleus where it binds to its response element and turns on the expression of specific genes that mediate inflammatory responses [47, 48]. Traditionally, methods for the study of inflammation are invasive in nature. Two commonly used methods are Western blot analysis for evaluating IκB activation and RT-PCR for quantifying effector gene expression, both requiring lysis of cells. Additionally, these methods are cumbersome and low in throughput [43, 48]. To overcome these limitations, Lee et al. created a RFP- NFκB reporter cell line, which allows researchers to monitor the NFκB translocation from the cytoplasm to the nucleus [49]. Although this reporter may permit real-time monitoring, its readout is one of the early events of inflammatory signaling, namely the NFκB translocation, rather than the biological endpoint [49]. Due to the oscillation of NFkB, this process may not correlate well with the biological response. Differing from RFP- NFκB reporter, our new system monitors the transcriptional activation, the final step in producing the biological response. Thus, our new system will produce biologically relevant data. Moreover, our dual-reporter format may preserve the signaling history at both individual cell (GFP) and population (Gluc) levels, yielding complimentary data sets as demonstrated in this study (Figures 3–5). Most importantly, the endpoint quantification is a direct measurement of NFκB activation in terms of GFP intensity or Gluc activity. Those measurements can be obtained via high content FACS analysis or laboratory automotive microplate reader, in a sensitive and specific manner.
Transcriptional activation and control of gene expression is the common focal point that converges on a variety of signaling pathways. Here, we demonstrate that our dual-reporter system can be used to monitor two critical signaling pathways mediated by either by NFκB (inflammation) or AP-1 (cell proliferation and/or differentiation). By the same token, our genetic circuit can be easily modified to report other critical signaling processes such as cancer (P53 or Myc), dyslipidemia (SREBP1 or PPAR), brain development (OCT4 or PAX6) and endocrine function (ER or AR). Hence, this new genetic circuit will have wide applicability and represents a promising platform for studying cell signaling in live cell or animal models.
We developed and validated a new dual-reporter circuit for real-time monitoring and quantification of inflammatory signaling in various mammalian cells. The new system is readily amenable to noninvasive manipulations allowing high throughput applications and laboratory automation.
We thank Dr. Yan Jiang for critically reviewing the manuscript and helpful comments.
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