More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
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Our breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
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“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
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Additionally, each book published by IntechOpen contains original content and research findings.
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We are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
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Simba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
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IntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
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Since the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
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More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\n
Our breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n
“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\n
Additionally, each book published by IntechOpen contains original content and research findings.
\n\n
We are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
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\n'}],latestNews:[{slug:"intechopen-supports-asapbio-s-new-initiative-publish-your-reviews-20220729",title:"IntechOpen Supports ASAPbio’s New Initiative Publish Your Reviews"},{slug:"webinar-introduction-to-open-science-wednesday-18-may-1-pm-cest-20220518",title:"Webinar: Introduction to Open Science | Wednesday 18 May, 1 PM CEST"},{slug:"step-in-the-right-direction-intechopen-launches-a-portfolio-of-open-science-journals-20220414",title:"Step in the Right Direction: IntechOpen Launches a Portfolio of Open Science Journals"},{slug:"let-s-meet-at-london-book-fair-5-7-april-2022-olympia-london-20220321",title:"Let’s meet at London Book Fair, 5-7 April 2022, Olympia London"},{slug:"50-books-published-as-part-of-intechopen-and-knowledge-unlatched-ku-collaboration-20220316",title:"50 Books published as part of IntechOpen and Knowledge Unlatched (KU) Collaboration"},{slug:"intechopen-joins-the-united-nations-sustainable-development-goals-publishers-compact-20221702",title:"IntechOpen joins the United Nations Sustainable Development Goals Publishers Compact"},{slug:"intechopen-signs-exclusive-representation-agreement-with-lsr-libros-servicios-y-representaciones-s-a-de-c-v-20211123",title:"IntechOpen Signs Exclusive Representation Agreement with LSR Libros Servicios y Representaciones S.A. de C.V"},{slug:"intechopen-expands-partnership-with-research4life-20211110",title:"IntechOpen Expands Partnership with Research4Life"}]},book:{item:{type:"book",id:"8970",leadTitle:null,fullTitle:"Tourism",title:"Tourism",subtitle:null,reviewType:"peer-reviewed",abstract:"Tourism was booming until 2019 when the COVID-19 pandemic hit. Since then, tourism and related industries have suffered from negative economic impacts. This book examines current challenges and opportunities in the tourism industry using case studies from different parts of the world. It also examines the challenges and obstacles faced by the tourism sector due to lack of environmental policies, high crime rates, and poverty.",isbn:"978-1-83962-173-4",printIsbn:"978-1-83962-172-7",pdfIsbn:"978-1-83962-174-1",doi:"10.5772/intechopen.82932",price:139,priceEur:155,priceUsd:179,slug:"tourism",numberOfPages:348,isOpenForSubmission:!1,isInWos:null,isInBkci:!1,hash:"4b086129cadc323ba152b00c6386c2c8",bookSignature:"Syed Abdul Rehman Khan",publishedDate:"October 6th 2021",coverURL:"https://cdn.intechopen.com/books/images_new/8970.jpg",numberOfDownloads:8639,numberOfWosCitations:0,numberOfCrossrefCitations:1,numberOfCrossrefCitationsByBook:0,numberOfDimensionsCitations:1,numberOfDimensionsCitationsByBook:0,hasAltmetrics:0,numberOfTotalCitations:2,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"June 2nd 2020",dateEndSecondStepPublish:"June 23rd 2020",dateEndThirdStepPublish:"August 22nd 2020",dateEndFourthStepPublish:"November 10th 2020",dateEndFifthStepPublish:"January 9th 2021",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6",editedByType:"Edited by",kuFlag:!1,featuredMarkup:null,editors:[{id:"254664",title:"Prof.",name:"Syed Abdul Rehman",middleName:null,surname:"Khan",slug:"syed-abdul-rehman-khan",fullName:"Syed Abdul Rehman Khan",profilePictureURL:"https://mts.intechopen.com/storage/users/254664/images/system/254664.jpg",biography:null,institutionString:"Xuzhou University of Technology",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"8",totalChapterViews:"0",totalEditedBooks:"6",institution:{name:"Nanjing University",institutionURL:null,country:{name:"China"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"71",title:"Hospitality Management",slug:"hospitality-management"}],chapters:[{id:"73393",title:"Challenges and Advances in the Planning of Tourism with Amazon River Dolphins in the Brazilian Amazon",doi:"10.5772/intechopen.93894",slug:"challenges-and-advances-in-the-planning-of-tourism-with-amazon-river-dolphins-in-the-brazilian-amazo",totalDownloads:488,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Being considered charismatic cetaceans are among the animals most sought after in tourist interactions that may involve observation, touch, swimming, and provisioning food. This tourism model has the potential to generate socioeconomic and conservationist benefits. However, when carried out in a disorderly manner, this can have a negative impact on cetaceans and tourists alike. In this chapter, we discuss the challenges and advances within the process of participatory planning of tourism with Amazon River dolphins (Inia geoffrensis). Our goal is to present strategies that can support the development of projects and public policies aimed at management of wildlife tourism in other areas. Since its implementation at the Anavilhanas National Park - Brazil, the activity had never had its impact monitored by any competent bodies, and this has led to problems and quick spreading to other sites. The rules and guidelines implemented in have significantly reduced risks for tourists and dolphins alike, improving tourist experience and promoting the awareness of animal life. However, many issues remain and need to be solved, especially in the protected areas. These include reduced staff levels, which limits the ability to implement and monitor planned actions. Such shortcomings lead to setbacks in the development of tourist activities with cetaceans.",signatures:"Marcelo Derzi Vidal, Priscila Maria da Costa Santos, Maria do Perpétuo Socorro Rodrigues Chaves and Robert Swett",downloadPdfUrl:"/chapter/pdf-download/73393",previewPdfUrl:"/chapter/pdf-preview/73393",authors:[{id:"322901",title:"Dr.",name:"Marcelo Derzi",surname:"Vidal",slug:"marcelo-derzi-vidal",fullName:"Marcelo Derzi Vidal"},{id:"329272",title:"MSc.",name:"Priscila Maria da Costa",surname:"Santos",slug:"priscila-maria-da-costa-santos",fullName:"Priscila Maria da Costa Santos"},{id:"329274",title:"Dr.",name:"Maria do Perpétuo Socorro Rodrigues",surname:"Chaves",slug:"maria-do-perpetuo-socorro-rodrigues-chaves",fullName:"Maria do Perpétuo Socorro Rodrigues Chaves"},{id:"329275",title:"Dr.",name:"Robert",surname:"Swett",slug:"robert-swett",fullName:"Robert Swett"}],corrections:null},{id:"76910",title:"From Eco to Sustainable Tourism, the Contradictions and Challenges of Nature-Based Tourism: The Case of Polar Cruises",doi:"10.5772/intechopen.96914",slug:"from-eco-to-sustainable-tourism-the-contradictions-and-challenges-of-nature-based-tourism-the-case-o",totalDownloads:318,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Polar tourism includes all leisure travel products set in the Antarctic and Arctic regions. As such, it is conditioned by an interest for nature in extreme settings (polar desert, cold climate, harsh travel conditions – when by sea. The Arctic adds an additional interest for indigenous cultures. Trying to met those tourism interests, a specialized cruise tourism branch developed in the late 1980s (thu sporadic cruises were held back from the XIXth century onward) providing exclusive access the most difficult and far distant latitudes of the High Arctic and opposite Antarctic coastline. In any form of tourism, operators must protect the resources their economic activities rest upon as any deterioration they suffer will sooner or later impact the experiente and its viability. Hence a paradox: how to protect the ecological (and cultural) integrity of these features for sustained competitiveness? Since its emergence, as an industry some 40 years ago, the polar cruising has followed trends in environmental and social management, referring in their marketing and travel policies to both eco- and sustainable tourism. Serving the wealthy customers, initially the well traveled elderly, the ship-based polar industry kept a simple programme of lecture and soft-oriented activities, namely inflatable cruising in icy bays and close-to-shore trekking. Yet, with an increasing clientele of younger middle-age tourists, operators have also diversified their excursion products to offer more sportive-oriented activities off-ship. As long as these activities were non-fuel based, the operators enforced their ecological management claims. But with more fuel-based activities (helicopter, Zodiac sightseeing), and therefore a more invasive approach to the sensitive ecosystems visited, can this industry continue to claim to be sustainable? Based on the sustainable claims made by two important polar cruise operators, this study ams to underlines that while the polar cruise industry, as a whole, might seek to improve its ecological footprint, there remains many contradiction between their will to be environmental and the desire to conquer the environment.",signatures:"Alain A. Grenier",downloadPdfUrl:"/chapter/pdf-download/76910",previewPdfUrl:"/chapter/pdf-preview/76910",authors:[{id:"324497",title:"Prof.",name:"Alain A.",surname:"Grenier",slug:"alain-a.-grenier",fullName:"Alain A. Grenier"}],corrections:null},{id:"73547",title:"The Light-Up of Dark Bali Tourism: A Qualitative Study",doi:"10.5772/intechopen.93389",slug:"the-light-up-of-dark-bali-tourism-a-qualitative-study",totalDownloads:367,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Tourism world currently stumbles due to Corona virus case that limits all human activities including those related to traveling. Various efforts in every country have been conducted to rebuild tourism to the normal condition; however, each country has its own obstacles. This study aims to create a strategic model in developing tourism based on cultural values or local wisdom to rebuild tourism passion to support economy. This study is a qualitative study using cultural approaches with ethnomethodology tools. Data are collected through in-depth interview with tourism actors: government and communities represented by traditional village leaders. The qualitative results indicate that cultural values summarized in a harmonization concept—harmony with God, harmony with fellow human being, and harmony with environment—become a model core that influences human behavior in developing tourism, namely: natural tourism, cultural tourism, spiritual tourism, culinary tourism, conference tourism, and so on. Traditional villages become the second pillar in developing tourism and it supports by local government. Another finding is that Bali will conduct a shift in tourism from cultural-based tourism to those that give more emphasis on natural tourism based on cultural and religious values as a promoter. The strategy will support health protocol related to physical distancing between tourists.",signatures:"I. Putu Astawa, Tjokorda Gde Raka Sukawati and I. Nyoman Gede Sugiartha",downloadPdfUrl:"/chapter/pdf-download/73547",previewPdfUrl:"/chapter/pdf-preview/73547",authors:[{id:"322956",title:"Prof.",name:"I. Putu",surname:"Astawa",slug:"i.-putu-astawa",fullName:"I. Putu Astawa"},{id:"322967",title:"Dr.",name:"Tjokorda Gde Raka",surname:"Sukawati",slug:"tjokorda-gde-raka-sukawati",fullName:"Tjokorda Gde Raka Sukawati"},{id:"323920",title:"Dr.",name:"I. Nyoman Gede",surname:"Sugiartha",slug:"i.-nyoman-gede-sugiartha",fullName:"I. Nyoman Gede Sugiartha"}],corrections:null},{id:"73254",title:"Opportunities and Obstacles in the Global Tourism Industry: A Story of Post-Covid-19",doi:"10.5772/intechopen.93683",slug:"opportunities-and-obstacles-in-the-global-tourism-industry-a-story-of-post-covid-19",totalDownloads:823,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"The rapid spread of Covid-19 has had far-reaching consequences for people’s daily lives in almost all parts of the world. Furthermore, it creates a negative impact on trade and economic activities, which further has spillover social problems, including unemployment and poverty. Moreover, in the tourism sector, millions of people lost their jobs, and hundreds of airlines are nearly bankrupt. This chapter is intended to investigate the link between the outbreak of Covid-19 and its effect on the tourism sector. The discussion reveals that due to the Covid-19, tourism sector declined sharply, but it provides an opportunity to transform our polluted world into a green one, which will have a significant and positive impact on global tourism in upcoming years. Finally, the chapter provides practical implications and recommendations, which will help policymakers to formulate an eco-friendly mechanism in the tourism sector.",signatures:"Syed Abdul Rehman Khan, Laeeq Razzak Janjua and Zhang Yu",downloadPdfUrl:"/chapter/pdf-download/73254",previewPdfUrl:"/chapter/pdf-preview/73254",authors:[{id:"254664",title:"Prof.",name:"Syed Abdul Rehman",surname:"Khan",slug:"syed-abdul-rehman-khan",fullName:"Syed Abdul Rehman Khan"},{id:"300373",title:"Dr.",name:"Zhang",surname:"Yu",slug:"zhang-yu",fullName:"Zhang Yu"},{id:"328959",title:"Dr.",name:"Laeeq Razzak",surname:"Janjua",slug:"laeeq-razzak-janjua",fullName:"Laeeq Razzak Janjua"}],corrections:null},{id:"74482",title:"Applying and Promoting the Seaport Quality System (SQS) and Spatial Interaction Model (SIM) for the Sustainable Development of the Recreational Seaport Industry in Malaysia",doi:"10.5772/intechopen.93765",slug:"applying-and-promoting-the-seaport-quality-system-sqs-and-spatial-interaction-model-sim-for-the-sust",totalDownloads:345,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"The recreational seaport industry carries out many critical functions, including transport circulation, logistics, commercial, and spatial ones. They influence local economic growth and determine the quality of recreational seaport. However, the definition of recreational seaport quality has remained elusive among the community, at present. Hence, this chapter explores the current literature by using the Systematic Literature Review (SLR) to derive at the definition of seaport quality based on three categories: seaport effectiveness, seaport reliability, and seaport governance, which will be main pillars for the development of marinas. This chapter proposes the Seaport Quality System (SQS) and Spatial Interaction Model (SIM) as a way to develop approaches and strategies that support sustainable planning and management of recreational seaports and marinas in countries with extensive coastlines. It is proposed that in order to offer sustainable and quality services, marinas must adopt the SQS model based on identifying and managing quality and risks. In addition, SIM can be utilised to improve the marinas operations by adopting key components in cruise activities, economic corridors and seaport regionalisation. The combination of both models are essential to enhance the growth momentum of marinas in this country.",signatures:"Mohamad Rosni Othman, Jagan Jeevan, Nurul Haqimin Salleh and Noor Azwa Noralam",downloadPdfUrl:"/chapter/pdf-download/74482",previewPdfUrl:"/chapter/pdf-preview/74482",authors:[{id:"322938",title:"Dr.",name:"Jagan",surname:"Jeevan",slug:"jagan-jeevan",fullName:"Jagan Jeevan"},{id:"329107",title:"Prof.",name:"Mohamad Rosni",surname:"Othman",slug:"mohamad-rosni-othman",fullName:"Mohamad Rosni Othman"},{id:"329109",title:"Dr.",name:"Nurul Haqimin",surname:"Salleh",slug:"nurul-haqimin-salleh",fullName:"Nurul Haqimin Salleh"},{id:"329110",title:"Ms.",name:"Noor Azwa",surname:"Noralam",slug:"noor-azwa-noralam",fullName:"Noor Azwa Noralam"}],corrections:null},{id:"74446",title:"Tourism Impact on Environmental Sustainability: A Focus on the Cruise Industry",doi:"10.5772/intechopen.93922",slug:"tourism-impact-on-environmental-sustainability-a-focus-on-the-cruise-industry",totalDownloads:186,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"The growth of the Global Economy and in particular the Caribbean Islands has been for the last two decades fueled by the cruise shipping industry. However, the growth in this industry gives rise to the expansion in ship size and the number of destinations. Unfortunately, the cruise line industry is responsible for the largest volume of waste, pollutants and destruction to marine lives when compared to other maritime industry sector. This chapter seeks to highlight the correlation between the industry and the growing global need for vibrant economies, a high quality of life, while protecting the environment and sustaining the world’s natural resources. A review of several literature has shown that within the last twenty years, the cruise lines have invested a lot of time and money correcting the negative environmental impacts created. Several proactive and green shipping initiatives designed to improve environmental management were successfully implemented by the industry. These diverse initiatives are group as follows: Research and Innovation, Corporate Social Responsibility (CSR) and Marketing, Awareness raising/environmental education initiative, and Green technologies. Emanating from these initiatives are: reduce or obviate of harmful environmental emissions and environmental management improvements and ultimately an environment that is experiencing an increased level of sustainability and economic activities.",signatures:"Kirkland Robert Anderson",downloadPdfUrl:"/chapter/pdf-download/74446",previewPdfUrl:"/chapter/pdf-preview/74446",authors:[{id:"267131",title:"Dr.",name:"Kirkland Robert",surname:"Anderson",slug:"kirkland-robert-anderson",fullName:"Kirkland Robert Anderson"}],corrections:null},{id:"73876",title:"Development of a Destination Image Recovery Model for Enhancing the Performance of the Tourism Sector in the Developing World",doi:"10.5772/intechopen.93854",slug:"development-of-a-destination-image-recovery-model-for-enhancing-the-performance-of-the-tourism-secto",totalDownloads:542,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"This chapter is based on a doctoral thesis on the development of a destination image (DI) recovery model for enhancing the performance of the tourism sector in Zimbabwe. The study was prompted by the failure of African destinations to develop DI image recovery models. A pragmatist paradigm, a convergent parallel mixed methodology research approach and a cross sectional survey were adopted. A sample of three hundred and nineteen comprising international tourists, service providers and key informants was used. A structured, semi-structured questionnaire and semi-structured interview guide were used respectively. Quantitative data was analyzed using the Statistical Package for Social Sciences (SPSS) and AMOS version 25 while qualitative data was analyzed using NVivo version 12. Tests were conducted using descriptive statistics, exploratory factor analysis, and confirmatory factor analysis. Structural Equation Modeling (SEM) was used to analyze the multiple independent variables. The major findings were that price, ancillary services and amenities significantly influenced affective image while ancillary services significantly influenced destination performance. The study recommended that the Ministry of Environment, Climate, Tourism and Hospitality Industry trains tourism stakeholders including the host community in order to achieve sustainable destination image recovery.",signatures:"Phillip Farayi Kanokanga, Marian Tukuta and Oliver Chikuta",downloadPdfUrl:"/chapter/pdf-download/73876",previewPdfUrl:"/chapter/pdf-preview/73876",authors:[{id:"324894",title:"Dr.",name:"Phillip Farayi",surname:"Kanokanga",slug:"phillip-farayi-kanokanga",fullName:"Phillip Farayi Kanokanga"},{id:"329365",title:"Prof.",name:"Marian",surname:"Tukuta",slug:"marian-tukuta",fullName:"Marian Tukuta"},{id:"329366",title:"Dr.",name:"Oliver",surname:"Chikuta",slug:"oliver-chikuta",fullName:"Oliver Chikuta"}],corrections:null},{id:"74265",title:"Contribution of Domestic Tourism to Sustainable Tourism Development",doi:"10.5772/intechopen.93646",slug:"contribution-of-domestic-tourism-to-sustainable-tourism-development",totalDownloads:664,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Tourism literature is awash with evidence of the value of domestic tourism to the tourism industry in general. However; there is limited knowledge of how domestic tourism is contributing towards sustainable tourism development especially in developing countries. This study explored the contribution of domestic tourism to sustainable tourism development in Zimbabwe, one developing country in Southern Africa. Using qualitative methodologies, data were collected and thematically analysed. The study revealed that domestic tourism has both positive and negative contributions to sustainable tourism development in unique ways. In conclusion, it was noted that without domestic tourism, Zimbabwe as a tourism destination would be struggling to grow its tourism product offering and expand its market share on the global tourism market.",signatures:"Forbes Kabote",downloadPdfUrl:"/chapter/pdf-download/74265",previewPdfUrl:"/chapter/pdf-preview/74265",authors:[{id:"324488",title:"Dr.",name:"Forbes",surname:"Kabote",slug:"forbes-kabote",fullName:"Forbes Kabote"}],corrections:null},{id:"71919",title:"Vein Thrombosis Risk in Women and Travel",doi:"10.5772/intechopen.92229",slug:"vein-thrombosis-risk-in-women-and-travel",totalDownloads:700,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:1,abstract:"Deep vein thrombosis (DVT) of the lower limbs is a serious condition that can lead to pulmonary embolism (PE) in about 15–24% of cases. If it is not diagnosed/treated timely, nearly 15% of these PE are lethal. The relationship between travel and staying in the same position for a long time is well-known since World War II. Generally, it is more frequent in air flights. It is also associated with the economic downturn in airplanes because passengers have limited space and have greater difficulty of moving. It is estimated that approximately 1–6% of long-haul passengers arrive at their destination with a clot in their veins, but most DVT are asymptomatic.",signatures:"Panagiotis Tsikouras, Xanthoula Anthoulaki, Theodora Deftereou, Anna Chalkidou, Anastasia Bothou, Fotini Gaitatzi, Eleftherios Chatzimichael, Selma Gyroglou, Arsou Chalil Bourazan, George Stanulov, Spyridon Michalopoulos, John Tsirkas, Irene Babageogaka, Werner Rath, Georg-Friedrich Von Tempelhoff, Stefanos Zervoudis, Georgios Iatrakis, Georgios Galazios and Nikolaos Nikolettos",downloadPdfUrl:"/chapter/pdf-download/71919",previewPdfUrl:"/chapter/pdf-preview/71919",authors:[{id:"48837",title:"Prof.",name:"Panagiotis",surname:"Tsikouras",slug:"panagiotis-tsikouras",fullName:"Panagiotis Tsikouras"},{id:"229224",title:"Ms.",name:"Theodora",surname:"Deftereou",slug:"theodora-deftereou",fullName:"Theodora Deftereou"},{id:"229225",title:"Ms.",name:"Anna",surname:"Chalkidou",slug:"anna-chalkidou",fullName:"Anna Chalkidou"},{id:"229226",title:"Ms.",name:"Xanthoula",surname:"Anthoulaki",slug:"xanthoula-anthoulaki",fullName:"Xanthoula Anthoulaki"},{id:"229227",title:"Ms.",name:"Anastasia",surname:"Bothou",slug:"anastasia-bothou",fullName:"Anastasia Bothou"},{id:"229230",title:"Prof.",name:"Stefanos",surname:"Zervoudis",slug:"stefanos-zervoudis",fullName:"Stefanos Zervoudis"},{id:"229232",title:"Dr.",name:"Georgios",surname:"Iatrakis",slug:"georgios-iatrakis",fullName:"Georgios Iatrakis"},{id:"229233",title:"Dr.",name:"Georgios",surname:"Galazios",slug:"georgios-galazios",fullName:"Georgios Galazios"},{id:"290371",title:"Mrs.",name:"Fotini",surname:"Gaitatzi",slug:"fotini-gaitatzi",fullName:"Fotini Gaitatzi"},{id:"290372",title:"Mr.",name:"Ioannis",surname:"Tsirkas",slug:"ioannis-tsirkas",fullName:"Ioannis Tsirkas"},{id:"290373",title:"Mrs.",name:"Arsou",surname:"Chalil Bourazan",slug:"arsou-chalil-bourazan",fullName:"Arsou Chalil Bourazan"},{id:"290374",title:"Dr.",name:"Werner",surname:"Rath",slug:"werner-rath",fullName:"Werner Rath"},{id:"298197",title:"Mrs.",name:"Eirini",surname:"Bampageorgaka",slug:"eirini-bampageorgaka",fullName:"Eirini Bampageorgaka"},{id:"299669",title:"Prof.",name:"Georg-Friedrich",surname:"Von Tempelhoff",slug:"georg-friedrich-von-tempelhoff",fullName:"Georg-Friedrich Von Tempelhoff"},{id:"300195",title:"Mr.",name:"George",surname:"Stanulov",slug:"george-stanulov",fullName:"George Stanulov"},{id:"303163",title:"Dr.",name:"Spyridon",surname:"Michalopoulos",slug:"spyridon-michalopoulos",fullName:"Spyridon Michalopoulos"},{id:"317103",title:"Prof.",name:"Nikolaos",surname:"Nikolettos",slug:"nikolaos-nikolettos",fullName:"Nikolaos Nikolettos"},{id:"317104",title:"Dr.",name:"Selma",surname:"Gyroglou",slug:"selma-gyroglou",fullName:"Selma Gyroglou"},{id:"317105",title:"Mr.",name:"Eleftherios",surname:"Chatzinmichael",slug:"eleftherios-chatzinmichael",fullName:"Eleftherios Chatzinmichael"}],corrections:null},{id:"74326",title:"Manta Ray Tourism",doi:"10.5772/intechopen.93924",slug:"manta-ray-tourism",totalDownloads:453,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Manta rays are flagship species for marine conservation because of a number of threats including anthropogenic, overfishing, plastics (microplastics), over tourism, commercial trade (gills for medicine), and chaotic shipping lines where they often injured or killed. Because of these reasons, manta ray face risk of extinction and listed on the Red List of IUCN. A number of studies present the value of this fish estimated millions of dollars per year from tourism which show much greater valuable alive than dead. Responsible manta ray tourism encourages stakeholders to protect the species by generating incentives from tourism while develop conservations initiatives to protect the species. Desk study on current literatures were reviewed to identify the role of stakeholders in supporting the sustainable management of manta ray tourism. This chapter explored the operations of manta ray tourism in Indonesia as the study areas. In summary, to reach the positive contributions from manta ray tourism, there is an important role of co-management between stakeholders to ensure the sustainable operations and conservation of the ecology, economy, and socio-culture.",signatures:"Maulita Sari Hani",downloadPdfUrl:"/chapter/pdf-download/74326",previewPdfUrl:"/chapter/pdf-preview/74326",authors:[{id:"323655",title:"Dr.",name:"Maulita Sari",surname:"Hani",slug:"maulita-sari-hani",fullName:"Maulita Sari Hani"}],corrections:null},{id:"73258",title:"Effective Leadership in the 21st Century: Lessons for the Tourism Sector in the African Continent",doi:"10.5772/intechopen.93844",slug:"effective-leadership-in-the-21st-century-lessons-for-the-tourism-sector-in-the-african-continent",totalDownloads:371,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Although Africa has been one of the world’s fastest growing tourism regions, when comparing it to the rest of the world, tourism still lags behind. Tourism is a dynamic and a competitive industry that continues to develop whilst the tourists’ preferences are changing. Consequently, leading and managing in the tourism sector is of great importance, particularly in the 21st century. The purpose of this paper is to explore leadership concepts to draw lessons for the tourism sector in the African continent. Leadership in the African continent remains questionable and controversial; the nature of effective leadership has been the subject of great debate. The findings reveal the prominent African leadership concepts from Ubuntu, Culturally embedded values, Communalism, Common good and Paternalism as some of the existing leadership concepts that could be applicable to an effective leader in the 21st century in the tourism sector in Africa.",signatures:"Portia Pearl Siyanda Sifolo",downloadPdfUrl:"/chapter/pdf-download/73258",previewPdfUrl:"/chapter/pdf-preview/73258",authors:[{id:"324889",title:"Dr.",name:"Portia Pearl Siyanda",surname:"Sifolo",slug:"portia-pearl-siyanda-sifolo",fullName:"Portia Pearl Siyanda Sifolo"}],corrections:null},{id:"76067",title:"Model of Virtual Tourism as an Alternative of the Concept of Architecture Tourism Post Covid-19 in Bandung City, Indonesia",doi:"10.5772/intechopen.94015",slug:"model-of-virtual-tourism-as-an-alternative-of-the-concept-of-architecture-tourism-post-covid-19-in-b",totalDownloads:270,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"The tourism sector as one of the sectors that has been hit by the Covid-19 pandemic needs a new breakthrough to enter a new normal era. Amid the ongoing paradigm shift and a number of new protocols will be implemented to welcome the new normal conditions in the tourism industry. The tourism sector in Indonesia, which has been absorbing many jobs, has been hit hard by the Covid-19 pandemic. Coupled with the difficulty of predicting when the pandemic will end, it is necessary to take smart steps in maintaining the sustainability of the tourism industry in entering an era of new norms. The new normal era brings new roles, new roads and expectations in the tourism sector. Digitalization that is growing rapidly and rapidly requires adaptation to new conditions and rearranging business strategies and models so that they can survive in the new normal era by adjusting technological developments. The development method used in this research is the Multimedia Development Life Cycle (MDLC). MDCL as a method for designing multimedia tools by emphasizing the 6 stages of multimedia development. The tools used in this application are PT GUI, Eclipse, and Google Maps. The objects of historical and heritage buildings that become the Sate Building, Villa Isola, the Geological Building, the Asian Africa Museum, and the Merdeka Building are made using immersive photography techniques. By representing information in the form of panoramic images, the 3600 makes it easy for users to visually display information from historical and heritage buildings in Bandung. The tourism model is virtually a possible form of tourism in the future.",signatures:"Asep Yudi Permana, Aathira Farah Salsabilla Permana and Karto Wijaya",downloadPdfUrl:"/chapter/pdf-download/76067",previewPdfUrl:"/chapter/pdf-preview/76067",authors:[{id:"322928",title:"Dr.",name:"Asep Yudi",surname:"Permana",slug:"asep-yudi-permana",fullName:"Asep Yudi Permana"}],corrections:null},{id:"73374",title:"Eco-Cultural Tourism: Sustainable Development and Promotion of Natural and Cultural Heritage",doi:"10.5772/intechopen.93897",slug:"eco-cultural-tourism-sustainable-development-and-promotion-of-natural-and-cultural-heritage",totalDownloads:640,totalCrossrefCites:1,totalDimensionsCites:1,hasAltmetrics:0,abstract:"Ecotourism has the eradication of poverty and environmental protection at its core. Both of these goals were established by the United Nations in 2012 though their development began in the 1980s. The purpose of this chapter is to analyse, using a comparative methodology, global and local eco-cultural tourism (natural, rural and urban areas) in tourist destinations of countries with emerging economies (Asia: China, Malaysia, Thailand), developed countries (Europe: Spain), and developing nations (South America: Peru, Argentina, Bolivia). The working hypothesis states that local, sustainable planning, endorsed by all the tourist agents is required, and should be led by the load capacity and the economic and environmental balance (green economies and ideologies), in order to answer to poverty and climate change problems by means of Tourist Projects directed by governmental policies and administrations. The outcomes suggest a need for a logistical change of policies, to prevent economies from generating pollution and carrying out abrasive activities associated with tourism. This change will create sustainable tourist destinations, the inclusion of populations, and the protection and conservation of natural and cultural heritage.",signatures:"Violante Martínez Quintana",downloadPdfUrl:"/chapter/pdf-download/73374",previewPdfUrl:"/chapter/pdf-preview/73374",authors:[{id:"322905",title:"Mrs.",name:"Violante",surname:"Martínez Quintana",slug:"violante-martinez-quintana",fullName:"Violante Martínez Quintana"}],corrections:null},{id:"73647",title:"Gen Y: Emotions and Functions of Smartphone Use for Tourist Purposes",doi:"10.5772/intechopen.94245",slug:"gen-y-emotions-and-functions-of-smartphone-use-for-tourist-purposes",totalDownloads:539,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:1,abstract:"Smartphones have revolutionized the tourism industry due to their ability to create and improve the tourist experience, mostly among young users, especially those belonging to the Generation Y (Gen Y). Millennials, as the Generation Y is often referred to, stand out for their ability to travel more frequently and for longer periods, as well as for their often-addictive use of smartphones. Despite nomophobia is not a recent phenomenon, there are few research works on information and communication technologies and tourism that address the effects of smartphone use on the tourist experience. The objective of this exploratory study is to describe the feelings Gen Y experiences as a result of use smartphones during their travels, their tourism functionality, and the relationship between the two. The study is based on the application of an online survey to a representative sample. The results confirm the problems associated with smartphone use, especially among young people (16–19) and the existence of a correlation between smartphone use for tourist purposes and a positive travel experience. It has confirmed that they experience negative feelings and emotions. The study presents crucial information that destination marketing organizations can use to successfully integrate smartphones into their digital marketing and communication strategies.",signatures:"Alba-María Martínez-Sala, Concepción Campillo-Alhama and Irene Ramos-Soler",downloadPdfUrl:"/chapter/pdf-download/73647",previewPdfUrl:"/chapter/pdf-preview/73647",authors:[{id:"316359",title:"Dr.",name:"Alba-María",surname:"Martínez-Sala",slug:"alba-maria-martinez-sala",fullName:"Alba-María Martínez-Sala"},{id:"329491",title:"Dr.",name:"Concepción",surname:"Campillo-Alhama",slug:"concepcion-campillo-alhama",fullName:"Concepción Campillo-Alhama"},{id:"329492",title:"Dr.",name:"Irene",surname:"Ramos-Soler",slug:"irene-ramos-soler",fullName:"Irene Ramos-Soler"}],corrections:null},{id:"74136",title:"Tourism Routes for the Diversification of Rural Livelihoods: A Methodological Approach",doi:"10.5772/intechopen.94871",slug:"tourism-routes-for-the-diversification-of-rural-livelihoods-a-methodological-approach",totalDownloads:323,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Tourism routes are the configuration of resources and services into an experience. They are structured based on the characteristics of the local setting but also considering the tourists’ expectations motivations and interests. The objective of this manuscript is to present a methodological approach for the configuration and evaluation of tourism routes, using the municipality of Tenancingo, Mexico as a case study. The methodology followed consisted of three phases: 1) the identification and classification of resources; 2) the qualitative evaluation of the resources; and 3) the use of a route evaluation index to determine the suitability. The study concluded that the methodology allowed for the evaluation of different configurations, and the identification of the tourism route with the most potential, according to its characteristics.",signatures:"Emmanuel Mérida Velazquez, Tirzo Castañeda Martínez and Gandhi González-Guerrero",downloadPdfUrl:"/chapter/pdf-download/74136",previewPdfUrl:"/chapter/pdf-preview/74136",authors:[{id:"325521",title:"Prof.",name:"Gandhi",surname:"González-Guerrero",slug:"gandhi-gonzalez-guerrero",fullName:"Gandhi González-Guerrero"},{id:"337481",title:"M.Sc.",name:"Emmanuel",surname:"Mérida Velázquez",slug:"emmanuel-merida-velazquez",fullName:"Emmanuel Mérida Velázquez"},{id:"337482",title:"Dr.",name:"Tirzo",surname:"Castañeda Martínez",slug:"tirzo-castaneda-martinez",fullName:"Tirzo Castañeda Martínez"}],corrections:null},{id:"73267",title:"Developing a Rural Tourism Destination Brand Framework from the Perspective of a Relationship-Based Approach",doi:"10.5772/intechopen.93839",slug:"developing-a-rural-tourism-destination-brand-framework-from-the-perspective-of-a-relationship-based-",totalDownloads:502,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"The aim of this paper is to develop a destination brand framework for rural tourism destination. Bario a rural community in Sarawak (Borneo) in Malaysia was chosen as a study context. The choice of Bario over other pre-selected rural destinations is because of its unique remote destination. The primary data collection method for this paper was the in-depth interview with 48 participants; this was supplemented by participant observation and documentary evidence. From the perspective of relationship-based approach adopted in this paper, the findings outline three components for a theoretical construction of rural tourism destination brand framework that comprise tourism destination appeals, branding strategies, and stakeholders’ roles. Findings also indicate that the development of rural tourism destination brand should be from the bottom-up, where community-driven strategies can be most effectively delegated to the local leadership system and community’s association. Implications for practice and host community well-being are discussed in detail.",signatures:"Samuel Adeyinka-Ojo",downloadPdfUrl:"/chapter/pdf-download/73267",previewPdfUrl:"/chapter/pdf-preview/73267",authors:[{id:"323647",title:"Dr.",name:"Samuel",surname:"Adeyinka-Ojo",slug:"samuel-adeyinka-ojo",fullName:"Samuel Adeyinka-Ojo"}],corrections:null},{id:"71912",title:"Traveler’s Infections: Overview of Hepatitis B Virus Infection",doi:"10.5772/intechopen.92174",slug:"traveler-s-infections-overview-of-hepatitis-b-virus-infection",totalDownloads:550,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Hepatitis B virus (HBV) is a double-stranded circular DNA virus that infects the hepatocytes. HBV infection is considered as an important public health concern globally especially with one-third of the world’s population been infected. Local and international migrants are one of those population at high risk of the infection. Many factors interplay in the acquisition of HBV such as purpose of travel, destination endemicity rate of the virus, time of stay of the traveler, inadequate prevention and control measures, among others, understanding the genotypes of HBV is critical in correlating the evolution of the virus and migration of humans and also treatment responses of infected population. The symptom of the virus ranges from fever to jaundice and to a liver cirrhosis and hepatocellular carcinoma (HCC). Transmission of HBV is commonly via horizontal route in developing regions and in the developed regions; transmission occur more often among adults that use injectable drugs and high-risk sexual behaviors. Therefore, the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) have recommended HBV screening and vaccination to all travelers without an HBV immunization history before traveling to endemic regions. This chapter gives an overview on HBV as a potential traveler’s infection.",signatures:"Victor B. Oti",downloadPdfUrl:"/chapter/pdf-download/71912",previewPdfUrl:"/chapter/pdf-preview/71912",authors:[{id:"245062",title:"Mr.",name:"Victor B.",surname:"Oti",slug:"victor-b.-oti",fullName:"Victor B. Oti"}],corrections:null},{id:"73322",title:"Marketing Cultural Resources as a Tourism Product",doi:"10.5772/intechopen.93869",slug:"marketing-cultural-resources-as-a-tourism-product",totalDownloads:563,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"This chapter presents the marketing aspect of cultural tourism resources by taking evidence from Sidama, Southern Ethiopia. It identifies the major cultural tourism resources of Sidama, and assesses their market readiness state through the lenses of tourists. It also presents the profile of cultural tourists visiting endowments in Sidama using descriptive research approach. Brief introduction of marketing approaches to cultural tourism and a review of literature on cultural tourism products and cultural tourists is also provided. As to its significance, the chapter offers analysis of cultural tourism assets and their marketability as a tourism product in a developing destination context. Practical implications for sound cultural tourism marketing are also discussed in the chapter.",signatures:"Amare Yaekob Chiriko",downloadPdfUrl:"/chapter/pdf-download/73322",previewPdfUrl:"/chapter/pdf-preview/73322",authors:[{id:"324979",title:"Assistant Prof.",name:"Amare Yaekob",surname:"Chiriko",slug:"amare-yaekob-chiriko",fullName:"Amare Yaekob Chiriko"}],corrections:null}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},subseries:null,tags:null},relatedBooks:[{type:"book",id:"6939",title:"Terrorism and Developing Countries",subtitle:null,isOpenForSubmission:!1,hash:"ad19b1ce8023e63b593a1835e0ec744e",slug:"terrorism-and-developing-countries",bookSignature:"Syed Abdul Rehman Khan and Zhang Yu",coverURL:"https://cdn.intechopen.com/books/images_new/6939.jpg",editedByType:"Edited by",editors:[{id:"254664",title:"Prof.",name:"Syed Abdul Rehman",surname:"Khan",slug:"syed-abdul-rehman-khan",fullName:"Syed Abdul Rehman 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1. Introduction
Successful resuscitation in cardiac arrest (CA) requires discrete decision-making regarding circulation, airway, and breathing. It is crucial to identify and treat reversible causes of cardiac arrest during resuscitation in order to make decisions that reverse them and more efficiently achieve return of spontaneous circulation (ROSC) [1]. Bedside ultrasound (US) has emerged as an invaluable tool in the diagnosis and management of critically ill patients, including CA [2, 3]. US may aid to diagnose reversible causes of CA, such as pericardial tamponade, tension pneumothorax, or hypovolemia; guide procedures and other management strategies for quality resuscitation; and reveal signs that can serve in clinical context as prognosticators for the ability to achieve ROSC and longer term recovery. In critical care medicine, bedside US has been found to be faster and have greater sensitivity and specificity than conventional imaging, which is unavailable during resuscitation efforts [4].
Bedside US remains underutilized in resuscitation medicine as there is controversy as to how efficiently and reliably it can be implemented by clinicians, especially in a high-stakes and time-sensitive setting such as CA. This review will present literature that evaluates implementation of US in CA resuscitation and demonstrates the potential of US to improve patient outcomes.
2. Intra-arrest: US to identify reversible causes of cardiac arrest
The standard of care for advanced cardiac life support (ACLS) during pulseless electrical activity (PEA) or asystolic CA dictates that providers actively work to diagnose and treat the reversible causes of CA. The following is a summary of the role of US in detecting such reversible causes:
2.1. Tension pneumothorax
Tension pneumothorax (PTX) is a well-known etiology of CA, especially in chest trauma, and can be rapidly reversed with emergent evacuation of air by needle or tube thoracotomy. Approximately 1% of non-traumatic in-hospital CA events are caused by PTX [5]. A meta-analysis showed US to be more sensitive and specific than chest roentgenography (CXR) for detecting PTX, with a sensitivity and specificity of 91 and 98% as compared to 50 and 99% for CXR [6]. Another meta-analysis showed that consultant performed and clinician performed US examinations had similar sensitivity and specificity for PTX [7].
US can also investigate how an identified PTX is altering patient physiology, as clinicians can obtain sub-xiphoid cardiac windows to see inferior vena cava (IVC) engorgement with impaired right heart filling as obstructive physiology. Further research is looking into how the location of a specific lung US finding, called lung point, can be used to quantify PTX size, with initial data showing that lung points found in the mid axillary line of supine patients predicting a greater than 15% lung collapse size as measured by CT with a sensitivity of 83% and specificity of 82% [8]. The convenience and repeatability of bedside US for PTX makes it clinically useful in CA [7, 9].
2.2. Pericardial effusion with cardiac tamponade
Cardiac tamponade is a significant contributor to in-hospital CA, with reported incidence as high as 6% of in-hospital CA [5]. Performing cardiac ultrasound, or echocardiography, during chest compression holds, allows for rapid detection of pericardial effusion. Several studies have validated the diagnostic power of US in this setting, including in the hands of non-cardiologist physicians, with reported sensitivities ranging from 96 to 100% and specificities ranging from 87 to 98% [10, 11]. In the medical literature, outside of the case of CA, use of bedside US to detect tamponade physiology is widely supported. Internal medicine (IM) physicians with handheld US devices identified moderate to large pericardial effusions with moderate agreement (kappa of 0.51) compared to cardiologist-read formal echocardiography [12]. Detecting increased central venous congestion in this setting using the presence of IVC plethora on US has shown a sensitivity of 97% for predicting tamponade, with an understandably small specificity of 40% given the many causes of IVC plethora [13, 14]. Other US predictors of tamponade physiology relate to the enhanced ventricular interdependence seen in tamponade include right atrial collapse (sensitivity of 50–100%, specificity of 33–100%) [15, 16], left atrial collapse (sensitivity of 13%, specificity of 98%), and right ventricular collapse (sensitivity of 48–100%, specificity of 72–100%) [13, 17].
2.3. Pulmonary embolus with acute cor pulmonale
US diagnosis of acute cor pulmonale due to pulmonary embolism (PE) relies on identification of right ventricle (RV) enlargement as an important finding during targeted echocardiography. Outside of the case of CA, other findings of acute cor pulmonale in include septal flattening or leftward bowing and RV systolic dysfunction [18, 19] . The RV:LV end diastolic diameter ratio, D-sign, and McConnel’s sign are validated echocardiography patterns of acute cor pulmonale in PE [20]. Of course, these signs are a product of a large flow-obstructing PE altering hemodynamic physiology in the presence of spontaneous circulation, a factor that is not present at CA. However, several case reports and observational studies have reported that, even during CA, PE can still be identified using the same signs of disproportionate RV size and direct embolism visualization in the pulmonary artery, right atrium, or IVC as a homogenously echogenic structure independent of underlying anatomy (suggestive of thrombus presence [21–25]. Such findings may lead to change in management including use of thrombolysis, an intervention that could largely benefit mortality in these patients [26, 27]. Administration of thrombolytic agents during cardiopulmonary resuscitation (CPR) is a controversial and high-risk procedure that can produce serious complications, including fatal hemorrhage, leading to controversy in recommendations and guidelines [27]. Interestingly, studies have added contrast to early case reports of hemorrhage including a recent single-center retrospective analysis of 42 patients that found thrombolysis during CA yielded no significant difference in major and minor bleeding events [28]. In regards to resuscitation, one study on 42 CA patients with PE found higher rates of ROSC in patients who received emergent thrombolysis than those who did not (81% vs. 43%, p = 0.03) [29]. While prospective data in this setting is sparse, the largest randomized trial was performed by Böttiger et al. in 2008 with 1050 out-of-hospital CA patients. The trial was terminated early due to futility when no significant differences were detected between tenecteplase and placebo groups in 30-day survival, hospital admission rates, ROSC, 24-hour survival, survival to discharge, or neurologic outcomes [30].
Ultrasound can be used to view the lower extremity vessels for a rapid deep venous thrombosis (DVT) study without interfering with compressions or other resuscitation measures. Studies have shown that bedside US DVT exams performed by clinician-sonographers have similar speed and diagnostic accuracy as compared to a formal US study with a radiologist [31, 32]. One meta-analysis of 15 studies and nearly 7000 patients by Rodrigues et al. in 2016 showed that an abbreviated proximal-focused DVT study, had a pooled sensitivity of 41%, and specificity of 96% for DVT detection compared to 79 and 84% of a relatively time-intensive whole-extremity exam [33]. In this study, the positive likelihood ratio of the limited DVT studies was pooled at 11.9, suggesting a utility of this abbreviated study in settings such as cardiac arrest.
While there is a clear need for further research in this area, many sources are advocating for more widespread use of thrombolysis during CPR in CA patients, especially in those where intra-arrest US helps to diagnose PE early and identify those at the highest risk of mortality [34].
2.4. Hypovolemia
Perhaps one of the most commonly used applications of bedside US is the evaluation of intravascular volume status and prediction of fluid tolerance or responsiveness [11, 35]. During management of CA, imaging of the IVC can help a code-leader rapidly diagnose hypovolemic shock, a tool whose sensitivity and specificity can be enhanced by adding US images of the lung fields and basic cardiac windows in conjunction with US of the IVC [36].
Early studies involved viewing the IVC in dialysis patients and blood donors, showing differences in IVC diameters pre and post infusions with associated changes in vessel caliber with respiratory cycle thoracic pressure changes [37, 38]. The largest meta-analysis in support of IVC US showed a pooled sensitivity of 76% and specificity of 86% for the detection of fluid responsiveness, defined as improved cardiac output (CO) on cardiac catheterization [39]. In both spontaneously breathing and mechanically ventilated patients, IVC US has high sensitivity and specificity for assessing fluid volume and responsiveness, suggesting applicability in the setting of CA, where hypovolemia may be a reversible etiology of arrest [39, 40]. However, US interrogation of fluid responsiveness during CA requires the clinician to be aware of the altered hemodynamic physiology of CA, where there is significant venous congestion and an elevated central venous pressure associated with decreased cardiac output (CO) [41]. In addition, sonographers need to be aware of the comorbidities that decrease IVC imaging sensitivity for hypovolemia, such as an obstructive physiology such as cor pulmonale, cardiac tamponade, or a myocardial infarction with markedly decreased CO [41].
With this hemodynamic physiology of CA in mind, US evaluation of hypovolemia as a cause of CA can still be useful as IVC imaging can be coupled with rapid and sensitive interrogation of the thoracic, abdominal, and pelvic cavities. In these spaces, such a large volume of fluid can accumulate to where this could cause significant hypovolemia if blood loss into these spaces has decreased effective circulating volume [41, 42]. US evaluation for significant intra-abdominal and pelvic fluid accumulation is a widely accepted modality, with sensitivities ranging from 60 to 100% [43–46]. In the setting of CA, this technique can take place without interruption of compressions and has the potential to alter CA management [47].
3. Peri-arrest and post-arrest care: US to guide ACLS
Outside of its use in diagnosing reversible etiologies of CA, US has also been supported by the literature for guidance of interventions in the intra and peri-arrest period.
3.1. US to interrogate cardiac rhythm
During CA resuscitation, one can directly visualize the heart both during compressions and at pulse checks. This has allowed clinicians more insight into the physiology of each patient in addition to data provided by pulse palpation and electrical monitors. US has bolstered the clinical utility of categorizing electromechanical dissociation (EMD) into “true-EMD” vs. “pseudo-EMD.” Pseudo-EMD is defined as the sonographic evidence of intrinsic and coordinated myocardial and valvular movement in the absence of a palpable pulse [11, 48, 49]. Several authors have noted that this observation of pseudo-EMD is associated with a better prognosis for ROSC as compared to true-EMD, which shows no contractile movement of the heart. One such prospective observational study involving 49 intensive care unit (ICU) CA events showed pseudo-EMD to occur on US in 55% of PEA patients [48]. This study showed the rates of ROSC were 70% for those in pseudo-EMD compared to 20% for those in true EMD [48, 50]. This US distinction could aid clinicians in their prognostication and decisions to continue or halt resuscitative efforts, with implications to resource utilization. Alternatively, the finding of pseudo-EMD may support a clinical strategy of using vasopressors/inotropes to support this coordinated cardiac activity and better optimize cerebral and coronary perfusion pressures for achieving ROSC. While there is currently no data to support this practice in CA resuscitation, this approach has been utilized with success in shock patients [51, 52].
Similarly, authors have described resuscitative events where “pseudo-asystole” is identified as asystole on electrical cardiac monitor with asynchronous fibrillatory activity of the ventricles on echo, suggesting ventricular fibrillation (VF). In the existing case reports describing this finding, this immediately changed ACLS algorithm as unsynchronized defibrillation was indicated [53–55].
3.2. US to guide chest compressions
US has been suggested as a means to optimize the effectiveness of chest compressions and to increase accuracy and efficiency of pulse check intervals [56]. While there remains a paucity of data to support these uses, the potential demonstrated by early case reports warrants discussion. Effective chest compressions allow for adequate coronary and cerebral perfusion pressure during CA [57]. While ACLS guidelines state the optimal site of compressions is on the lower half of the sternum along the nipple line, some studies suggest significant anatomical variation among structures at this site [58]. One study of 30 out-of-hospital CA patients tested this site compared to three caudal alternatives and found that maximal end-tidal carbon dioxide was achieved at the AHA recommended site in only 1/3rd of their sample [59]. Another study using transesophageal echocardiography (TEE) observed 34 non-traumatic CA patients and identified the anatomic area of maximal compression (AMC) to be over the aorta or left ventricular outflow tract in all cases with a statistically significant linear association between LV stroke volume and AMC distance from the aortic valve [60]. In a swine model of cardiac arrest, animals randomized to have compressions centered over their LV, as identified by transesophageal echocardiography (TEE), had a greater proportion of ROSC and survival to 60 minutes compared to those that had compressions centered over their aortic root [61]. While more research in this area is needed, it is reasonable to predict a role for bedside ultrasound and echocardiography to be identifying appropriate positioning for chest compression efforts, either by trans-thoracic echocardiography (TTE) and/or TEE by viewing the anatomic landmarks directly.
3.3. US to guide pulse checks
Current ACLS guidelines state that pulse-checks during CA resuscitation should last no longer than 10 seconds. Some authors have called the accuracy of pulse palpation into question [62]. One study involving pulse palpation during cardiac bypass surgery (spontaneous vs. non-pulsatile blood flow) showed that, while health care providers with advanced levels of training had decreased decision delay, only 16.5% of the participants (34 of 206) were able to reach a confident decision about their patients’ pulse status within 10 seconds [63]. A similar earlier study in basic life support-trained personnel found that although sensitivity of all participants for central pulselessness approached 90%, specificity was only 55% [64]. While these studies have their limitations, they call attention to a potential role for ancillary devices to augment the accuracy of pulse palpation. Case reports have shown handheld Doppler US devices can allow for faster pulse checks in patients during in-hospital CA [62]. Other authors have already reported the utility of US performed concomitantly with pulse palpation to be effective in identifying perfusing heart rhythms [21]. While US in this exact context is not yet well studied, it seems of little risk but some benefit to use US to eliminate some of this intrinsic inaccuracy in pulse palpation during CA resuscitation.
3.4. US for endotracheal tube (ETT) placement confirmation
Verification of endotracheal intubation during ACLS can be accomplished with US of the neck. The usual methods of ETT placement verification have limitations when applied during cardiac arrest. Direct visualization is often not reliable especially if the intubation takes place during chest compressions due to the movements of the patient. Colorimetry methods can be misleading in the setting of a previously insufflated stomach, which is the case with the bag valve mask technique ongoing prior to intubation attempts or prior esophageal intubation with insufflation. Continuous waveform capnography remains as a reliable confirmatory method if this equipment is readily available. It can require time to set up and to evaluate the waveform over several breaths, which can be considered a limitation. US can distinguish an intubated trachea from and an intubated esophagus as each has distinct sonographic findings that can be rapidly attained.
Cadaver studies have shown that neck US findings of “double lumen sign” and “tube sliding” artifact can predict endotracheal or esophageal intubation with 100% sensitivity and 100% specificity [65]. The largest meta-analysis of studies with both adult patient and cadaveric models determined that bedside physicians and house staff had a pooled sensitivity and specificity of 93 and 97% [66]. US for ETT placement is especially useful when waveform capnography is not readily available [67] or if a conventional method is misleading, such as colorimetry-verified placement with continued hypoxia. Several authors have shown that US is quicker than conventional methodologies of ETT placement confirmation, demonstrating an average time to confirmation of 5.8 seconds, significantly faster than capnography at 11.8 seconds [68]. We advocate for enhancing testing characteristics by combining visualization of neck airway structures with lung field pleural sliding and respiratory diaphragmatic motion, which can be performed during pulse check.
3.5. US to guide post-ROSC hemodynamic management
Post-ROSC management includes the immediate initiation of hemodynamic support measures such as fluids, vasopressors, and inotropes. The ability to quickly utilize bedside US to evaluate fluid responsiveness and overall cardiac function can be clinically useful to guide this hemodynamic support.
Goal-directed echocardiography (GDE) is a concept that uses high-fidelity qualitative analysis, without Doppler technology or valvular measurements, to assess targeted cardiac windows in real-time with high sensitivity of identifying marked abnormalities and gross pathophysiology. GDE emphasizes grading LV function as normal, decreased, or very decreased, allowing bedside clinicians to make real-time evaluations upon which to guide management of CA [69]. Current literature supports agreement of GDE interpretations between formal consultant cardiologists and clinician-sonographers at the bedside. One such study demonstrated that, after a brief training course, novice sonographers with hand-held US at the bedside demonstrated 75% agreement with cardiologist in their formal-US study interpretations of LV dysfunction, compared to 83% intra-cardiologist agreement [12]. Thereby, in a CA resuscitation event, when a cardiologist is not always available, a relatively novice-level sonographer is sufficient for diagnostic capability.
Using this concept of GDE, clinician-sonographers can use US to better inform their post-ROSC hemodynamic management including the use of inotropes, pressors, and/or fluid support with the treating clinician acquiring selected TTE views to characterize pre-load and cardiac contractility in the immediately post-arrest period.
4. US for prognostication in CA
An important emerging area of current study in CA US involves using US data in prognostication for survival and neurological outcomes in CA. Despite best efforts during resuscitation, there is continued poor survivorship. The ability to prognosticate the patient’s likelihood of achieving ROSC can improve the practitioner’s ability to allocate resources and manage expectations of the treating team and patient’s caretakers.
The strongest literature supporting prognostic value of US in CA relates to the presence or absence of coordinated cardiac activity as noted by US [11, 48, 70]. Pooled data from over 500 patients showed that the presence of any cardiac kinetics by intra-arrest US had a positive likelihood ratio of 4.26 and negative likelihood ratio of 0.18 to predict ROSC [71]. Another observational study observed the survivorship of nearly 800 non-traumatic CA patients who received US examination as part of their resuscitative efforts upon presentation to the emergency room and showed presence of any cardiac activity on US was associated with ROSC, survival to admission, and survival to discharge [72].
Further areas of research into US in CA prognostication are looking outside the heart, including measuring optic nerve sheath diameter (ONSD) to predict a positive neurological outcome. ONSD was measured in CA patients 12–72 hours after ROSC and at 28 days after ROSC or discharge from the hospital before 28 days [73]. ONSD of less than or equal to 5.4 mm predicted a favorable neuro-functional prognosis as measures by Glasgow Outcomes Scale with a sensitivity of 83%, specificity of 73%, positive likelihood ratio of 3.1 and negative likelihood ratio of 0.23 [73].
5. Bedside CA US is feasible to be implemented today
The viability of using US during a cardiac arrest depends on the premise that non-radiologist and non- cardiologist physicians can obtain and rapidly interpret imaging data about patient anatomy and physiology with high diagnostic accuracy. Among the significant barriers to its implementation and widespread use are lack of confidence in usage of new technologies and inertia against supplementing traditional methods with new tools for guiding CA management.
In response to the issue of implementation, there is much known about the learning curve for non-radiologist and non-cardiologist practitioners to operate, interpret, and apply this bedside imaging technology. Authors from many different fields including emergency medicine, IM, and anesthesiology have conducted research to address this question of feasibility [74]. Multiple studies have shown that, after short-term (hours-days) educational sessions, novice and expert sonographers can perform without significant differences in sensitivity or specificity in challenging US applications such as ventricular function, volume status or cardiac tamponade [75].
Even at the trainee level, it has been shown that US is a technology which physicians can consistently learn. The Accreditation Council of Graduate Medical Education (ACGME) now requires that critical care ultrasonography be a mandatory component of critical care medicine fellowship training, surgical critical care fellowship training, and emergency medicine residencies [75, 76]. It is well established that this can be done successfully with a mixture of didactics, simulation, and hands on training [75]. In a 3 day critical care US course, 300 novice physicians were shown to proficiently acquire and interpret content from thoracic, vascular, and abdominal ultrasonography [77].
Integrating US techniques into CA management is simply a matter of targeted educational sessions focused on image acquisition, interpretation, and immediate application. After a 1-day training course in CA echocardiography given to novice clinicians of all training levels, the rate of US usage in CA management increased from 4.3 to 19.8% and that echocardiography during the CA event altered management in 70% of cases [78]. Another study found that novice sonographers as a part of an ACLS response team were able to integrate US into their management of cardiac arrest with images obtained and interpreted within an average of 8 min from CA alert activation and demonstrated strong image interpretation agreement with expert sonographers upon retrospective repeat interpretation [42]. In an analysis of CA events in the ICU where US was used in the setting of PEA or asystole, images of adequate quality were obtained during compressions in 100% of there were changes in management and diagnosis due to US findings in 51% of cases [48]. These data together suggest that US has significant potential to aid in CA resuscitation management and potentially improve patient mortality, morbidity, and outcomes [42, 72, 79, 80].
6. Overview of selected current CA US protocols
Implementation of US for CA will be most organized if a standardized approach can be systematically integrated into CA management. There are several example US for CA protocols noted to date, including our institutional protocol (Table 1). These protocols focus on rapidly identifying causes of PEA/asystole such as cardiac tamponade, PE, PTX, and hypovolemia. Each protocol is designed to minimally interfere with ACLS. Our institutional protocol includes the use of neck tracheal US for confirming placement of the ETT. Most protocols were estimated to take less than 1 min to complete and none endorse interruption of chest compressions beyond the standard time limit of a pulse check. One protocol advocates for saving a video loop of a subcostal view of the heart during a 10 s pulse check, allowing for repeat image analysis [42]. Most protocols emphasize knowledge of diagnostic limitations and careful image interpretation. The complexity of the protocols ranges from 1 view (echocardiography only) to 6 views (Table 1).
Title or author
Views obtained
# views
During CPR, pulse-checks, or both?
Estimated time of protocol
Notes/uniqueness
Ahmad
Lung, cardiac, Abd/IVC, deep venous, tracheal/ETT
5
Both
Cardiac: 10s Noncardiac: Variable Total: Variable, includes US at pulse checks, compressions, and post-ROSC
Only protocol to include verification of ETT placement.
Our institution is a tertiary care center in New York State, where all inpatient medicine CA resuscitative events are led by senior IM residents, variably with attending. Since early 2014, our institution has incorporated US training into CA management training for these team leaders. In addition, the pulmonary and critical care medicine (PCCM) fellowship program incorporates extensive educational experience in using bedside US in many aspects of critical care, including that for CA. Since the advent of use of this US protocol (Table 1 and Figure 1) for CA, our institution has conducted over 280 CA events and we have implemented use of handheld US devices when a trained clinician can participate. So far, we have received positive feedback from residents regarding the incorporation of ultrasound in CA. Finding a reversible cause has so far been rare. The largest impact has been that the use of US at CA allows code leaders to feel more comfortable stopping resuscitation by ruling out reversible etiologies of CA or findings that represent poor prognosis.
Figure 1.
CA US protocol at Stony Brook University medical center critical care ultrasound (SBUS) program.
7. Summary and conclusions
Bedside US has significant implications in the setting of guiding cardiac arrest management. In CA resuscitation, clinicians must make rapid, yet informed decisions about patient care in a fast-paced and high pressure environment. In the case of CA characterized by PEA/asystole, US can quickly assess for reversible causes. US can help physicians better interrogate cardiac rhythm or intrinsic cardiac activity, perform more effective chest compressions, reduce error in pulse checks, more rapidly rule out esophageal intubation, provide more tailored post-ROSC hemodynamic support, and provide assistance in prognostication. CA US allows clinicians to offer a higher level of care quality in concordance with, yet beyond, basic ACLS.
Incorporation of US at all CA may improve cost effectiveness and efficiency of hospital resource distribution. Rapid TTE improved the use of health care resources in patients with CA secondary to trauma, where patients who did not received US had a significantly higher mean cost of care, with an average of approximately $1100 less spent on the US examined group [81]. The prognostic value of US in CA carries an additional resource utilization benefit when considering effects such as ending futile resuscitative efforts earlier and redirecting valuable physician time, hospital personnel resources, as well as medication and equipment costs.
US has become a required part training and accreditation for several medical specialties and it has been consistently shown that physicians can learn US through targeted cumulative educational exposures, even starting at the residency and fellowship levels. It follows that emerging clinicians can be expected to gradually learn to apply these skills to the challenging clinical setting of CA. Most authors advocate for the adoption of a protocolized approach to US in CA as such an approach allows physicians to implement high-yield bedside US in conjunction with ACLS and with minimal interference. Protocolized approaches should include views of the heart to assess cardiac function and for pericardial fluid, IVC for volume status, lung fields to rule out PTX and fluid dependent spaces in the abdomen and pelvis for hemorrhage. Additionally, DVT study and airway confirmation by US may be employed.
Several authors agree that there is a paucity of research to evaluate differences in patient outcomes from US use in CA, therefore true benefits are difficult to assess [82]. Recent survey data identified that there is an existing perception that training in hemodynamic relevant US imaging takes too long, and that only specialized individuals can perform these examinations [83]. However, the literature reviewed here advocate against this criticism. Several authors have shown that time constraints do not prohibit a limited US study and that bedside clinicians can demonstrate success in learning US applications after simple educational interventions. Another barrier is the perception that US devices and sonographers will not be able to join an already crowded space. At our institution, we have found that an US provider, with either a portable or handheld US unit, can easily navigate a resuscitation event without interrupting ACLS. A dedicated sonographer is easily able to adopt to the needs of the resuscitation, change positions and deliver diagnostics to code leaders without interfering with team communications, medication administration, or procedural interventions.
We strongly support the role of US in guiding CA resuscitation management. In light of our and others’ experiences reporting US changes management in a majority of CA cases and we suggest that there needs to be support of ongoing research to investigate correlations of US to patient outcomes. US should be part of the standard of care in cardiac resuscitation events as it is currently one of the only means of real time diagnosis of several reversible causes of CA [84].
\n',keywords:"cardiac arrest, resuscitation, ultrasonography, echocardiography",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/57370.pdf",chapterXML:"https://mts.intechopen.com/source/xml/57370.xml",downloadPdfUrl:"/chapter/pdf-download/57370",previewPdfUrl:"/chapter/pdf-preview/57370",totalDownloads:1271,totalViews:294,totalCrossrefCites:0,totalDimensionsCites:0,totalAltmetricsMentions:0,introChapter:null,impactScore:0,impactScorePercentile:10,impactScoreQuartile:1,hasAltmetrics:0,dateSubmitted:"July 25th 2017",dateReviewed:"August 14th 2017",datePrePublished:null,datePublished:"December 6th 2017",dateFinished:"October 27th 2017",readingETA:"0",abstract:"Cardiac arrest (CA) is a high mortality event where the ability for clinicians to diagnose etiology and assess for intervention has a direct impact on patient outcomes. Bedside ultrasound (US) has emerged in current literature as a clinical tool to aid clinicians in CA resuscitation, though it remains underutilized. Reversible etiologies that can be efficiently diagnosed with US include tension pneumothorax, hypovolemia, pulmonary embolus with acute cor pulmonale, and cardiac tamponade. Other US findings may provide evidence in regard to prognosis. In this review, we present major applications of US in CA, compare existing protocols, and propose future research needs.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/57370",risUrl:"/chapter/ris/57370",book:{id:"5949",slug:"resuscitation-aspects"},signatures:"Marc Delaney, Bjorn Flora and Sahar Ahmad",authors:[{id:"218326",title:"Dr.",name:"Marc",middleName:null,surname:"Delaney",fullName:"Marc Delaney",slug:"marc-delaney",email:"madelaney@cnmc.org",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null},{id:"218461",title:"Dr.",name:"Bjorn",middleName:null,surname:"Flora",fullName:"Bjorn Flora",slug:"bjorn-flora",email:"Bjorn.Flora@stonybrook.edu",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:{name:"Stony Brook University",institutionURL:null,country:{name:"United States of America"}}},{id:"218462",title:"Dr.",name:"Sahar",middleName:null,surname:"Ahmad",fullName:"Sahar Ahmad",slug:"sahar-ahmad",email:"Sahar.Ahmad@stonybrookmedicine.edu",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:{name:"Stony Brook University Hospital",institutionURL:null,country:{name:"United States of America"}}}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Intra-arrest: US to identify reversible causes of cardiac arrest",level:"1"},{id:"sec_2_2",title:"2.1. Tension pneumothorax",level:"2"},{id:"sec_3_2",title:"2.2. Pericardial effusion with cardiac tamponade",level:"2"},{id:"sec_4_2",title:"2.3. Pulmonary embolus with acute cor pulmonale",level:"2"},{id:"sec_5_2",title:"2.4. Hypovolemia",level:"2"},{id:"sec_7",title:"3. Peri-arrest and post-arrest care: US to guide ACLS",level:"1"},{id:"sec_7_2",title:"3.1. US to interrogate cardiac rhythm",level:"2"},{id:"sec_8_2",title:"3.2. US to guide chest compressions",level:"2"},{id:"sec_9_2",title:"3.3. US to guide pulse checks",level:"2"},{id:"sec_10_2",title:"3.4. US for endotracheal tube (ETT) placement confirmation",level:"2"},{id:"sec_11_2",title:"3.5. US to guide post-ROSC hemodynamic management",level:"2"},{id:"sec_13",title:"4. US for prognostication in CA",level:"1"},{id:"sec_14",title:"5. Bedside CA US is feasible to be implemented today",level:"1"},{id:"sec_15",title:"6. Overview of selected current CA US protocols",level:"1"},{id:"sec_15_2",title:"6.1. Reflections on institutional experience",level:"2"},{id:"sec_17",title:"7. Summary and conclusions",level:"1"}],chapterReferences:[{id:"B1",body:'AHA MFH, Nolan JP. 2010 international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations. Circulation. 2010;122:S249'},{id:"B2",body:'Kendall JL, Hoffenberg SR, Smith RS. History of emergency and critical care ultrasound: The evolution of a new imaging paradigm. Critical Care Medicine. 2007;35:S126-S130'},{id:"B3",body:'Price S, Uddin S, Quinn T. Echocardiography in cardiac arrest. Current Opinion in Critical Care. 2010;16:211-215'},{id:"B4",body:'Lumb P. Critical Care Ultrasound: Expert Consult. Elsevier Health Sciences. 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Focused echocardiographic evaluation in life support and peri-resuscitation of emergency patients: A prospective trial. Resuscitation. 2010;81:1527-1533'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Marc Delaney",address:null,affiliation:'
Division of Pulmonary and Critical Care Medicine, Stony Brook University Medical Center, Stony Brook, New York, USA
Division of Pulmonary and Critical Care Medicine, Stony Brook University Medical Center, Stony Brook, New York, USA
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1. Introduction
Cellular senescence is best described as an inevitable irreversible proliferation arrest the phase of primary human fibroblasts in culture [1, 2]. The senescent phenotype is characterized by distinctive changes in morphology to become enlarged, flattened, and granular [2]. Multiple factors that activate senescence include various types of stress-related stimuli such as aberrant oncogenic signaling, oxidative stress, and DNA damage [2]. Moreover, the onset of senescence can be regulated by events such as epigenetic regulation, chromosome dynamics, protein degradation, mitochondrial mechanisms, and metabolic pathways. The molecular pathways of senescence differ considerably among cell types as well as different species [3].
Alternative splicing of pre-mRNAs is a process in which varied mRNA transcripts are generated to provide a major source of protein diversity in higher eukaryotes. Pre-mRNA splicing is a nuclear process that can be constitutive or alternative [4, 5]. Constitutive splicing involves the removal of introns and the joining of adjacent exons in the order of their arrangement. One of the core proteins involved in splicing is hnRNPA1 [5, 6]. Consequently, a single protein may be produced from a single pre-mRNA in constitutive splicing [7]. In contrast, in alternative splicing, the variable use of splice sites permits two or more mature mRNAs to be generated from the same pre-mRNA. Among the nuclear complexes primarily responsible for alternative splicing are heterogeneous nuclear ribonucleoproteins, small nuclear ribonucleoproteins snRNPs, and SR proteins [8, 9]. Splicing factors that play a crucial role through concentration changes or alterations of their expression patterns have significant impacts on mRNA alternative splicing [9, 10].
We have previously found that hnRNP A1 is significantly downregulated in cellular senescence [10] and can regulate the levels of the alternatively spliced INK4a locus that generates the mRNA isoforms, p16INK4a and p14ARF both of which are growth suppressors that are important in senescence [10, 11]. Increased expression levels of hnRNP A1 via over-expression can shift the expression pattern toward the p14ARF mRNA isoform [10].
We initiated this study to identify novel targets of hnRNP A1 and to further explore the role of hnRNP A1 in the modulation of gene expression during cellular senescence. The experimental approach used in this study was to identify the in vivo RNA targets bound in hnRNP A1 RNP complexes isolated from human fibroblasts. RNP complexes were isolated by a brief co-immunoprecipitation step with the 4B10 hnRNP A1-specific monoclonal antibody [12]. RNA species in these complexes were then subjected to reverse transcription followed by amplification. The products were then cloned and sequenced. Our findings suggest that hnRNP A1 is involved in the regulation of HDM2 gene expression. However, the involvement of hnRNP A1 in the regulation of HDM2 gene expression remains to be elucidated.
We sought to identify putative mRNA substrates for hnRNP A1 by identifying mRNA sequences that directly bind to the hnRNP A1 protein. We employed a modification of a procedure that had been used for the characterization of RNP complexes by Mili et al. [12]. We isolated hnRNP A1 protein complexes bound to their RNA targets from total young and senescent fibroblast cell lysates. To demonstrate that the complexes represented the majority of hnRNP A1-mRNP complexes found in cellular pools, we measured the mRNA levels of hnRNP A1 and actin in the isolated complexes by RT-PCR. Actin has been previously reported to be in hnRNP A1 RNP complexes; thus, we used actin as a positive control. The results in Figure 1A, show that actin and hnRNP A1 protein were present in lysates isolated from young and senescent IMR-90 fibroblasts. We assessed the protein level of hnRNP A1 in 4B10 RNP complexes isolated from young and senescent protein lysates. 4B10 RNP complexes reflect the results in panel A (Figure 1B) as hnRNP A1 was lower in senescent IMR-90 protein lysates following immunoprecipitation when compared with young cell lysates. These observations indicate that hnRNP A1 was present in the isolated RNP complexes. One known mRNA target of hnRNP A1 is its own RNA; therefore, we measured the level of hnRNP A1 mRNA by RT-PCR (Figure 1C). We found that the 4B10 monoclonal antibody immune-precipitated hnRNP A1 mRNA in RNP complexes from both young and senescent lysates. While the level of hnRNP A1 mRNA was lower in senescent RNP complexes, it was sufficient enough to determine putative mRNA targets.
Figure 1.
A. Expression of hnRNPA1 in young and old IMR90 fibroblasts. The endogenous level of hnRNP A1 protein is significantly lower in old IMR90 fibroblasts (major band at ~34 kDa is hnRNPA1). Equivalent amounts of protein (20 μg) were loaded. B. Post-immunoprecipitation levels of hnRNPA1 were also low in old cells, see the band at ~34 kDa. C. RT-PCR on cDNA transcribed from RNA isolated complexes, using hnRNPA1 and β-actin primer sets. In both young and old lysates, there appear to be comparable amounts of hnRNPA1 mRNA species in the isolated hnRNPA1-containing complexes.
2.2 Analysis of the hnRNPA1-mRNA complex
RNA that was isolated from the hnRNP A1-complexes was reverse transcribed and then amplified by PCR using random decamers to amplify all cDNA sequences. We used two different concentrations of cDNA template for PCR as it was not always possible to visualize PCR products in the senescent samples because the cDNA abundance is typically lower in these cells. There was a correlative increase in PCR products as shown in Figure 2 with an increase in cDNA template. PCR products were then immediately ligated into a pCR2.1cloning vector.
Figure 2.
Analysis of hnRNP A1 RNP complexes. A. RNA isolated from hnRNPA1 complexes was reverse transcribed to cDNA and amplified using random decamers as primers for the reaction. No amplification was observed in the absence of cDNA as indicated in the (−) lane.
There may also be a differential availability of hnRNP A1 protein in old cells as hnRNPA1 is modified by post-translational events, such as phosphorylation and methylation [13, 14]. There is an additional possibility that rearrangements of individual components in hnRNPA1-mRNA ribonucleoparticles may change during senescence, which could alter specific and non-specific mRNA sequences bound in the complex.
2.3 Identification of RNA species in hnRNP A1-mRNP complexes
We then determined the identity of the cloned inserts by sequencing. We found that there were partial mRNA sequences for four human genes bound in young and senescent RNP complexes. The identity of genes was identical for both young and old cells. Scores were considered to be positive if the similarity score was more than 200 [15]. The genes identified were Homo sapiens HDM2 gene (AF144029), intron 9 and exon 10, partial sequence; H. sapiens asthmatic clone 1 mRNA (AF095853), 3 UTR; H. sapiens D15S1506 ca repeat region (AF018071), complete sequence, and H. sapiens partial HR gene for hairless protein (AJ277249). As these four human genes were the only gene candidates identified, we chose to score the sequences by the number of times a positive hit occurred in a (BLAST) similarity search for individually isolated clones. The sequence for HDM2 that occurred most frequently was further analyzed to determine potential splicing enhancer/silencer elements by Zhang, et al. [16, 17] using the PESX utility (http://cubweb.biology.columbia.edu/pesx/). Figure 3 shows a schematic illustration of the HDM2 mRNA sequence that binds to hnRNP A1. The binding site is between exon 9 and intron 10. The regions marked in red are putative silencer sequences. We found that there were several matches to these sequences within the isolated HDM2 mRNA from hnRNPA1 RNP complexes.
Figure 3.
PESX analysis. PESX (putative exon spliyancer/silencers) is a utility that can predict regions of an mRNA that may be involved in exon splicing to enhance the inclusion or exclusion of an exon. In addition to the identification of the putative splicing regulatory sequences, the sequences were also scanned for putative hnRNPA1-binding sites [UAG (G/a)] based on known binding sites.
The identification of the human double minute 2 gene (HDM2) was of particular interest to us. It is the human homolog of the mouse double minute 2 (MDM2) and is a known oncogene [18, 19, 20]. It is a protein of ~90 kDa size and is usually localized in the nucleus of cells. Overexpression of HDM2 causes cells to proliferate uncontrollably as it facilitates the proteasomal degradation of p53 by acting as a ubiquitin ligase [21, 22].
The human murine double minute 2 (HDM2) gene is a 33-KB nucleotide sequence located on chromosome 12 (q14.3-q15). The gene consists of 12 exons and 11 introns [18, 20]. Transcription of the HDM2 gene is controlled by two different promoters, referred to as P1 and P2 that are P53-independent and P53-dependent, respectively [19]. The P1 promoter controls the basal expression of HDM2 and is positioned upstream in the first exon of the HDM2 gene [19, 20]. Transcription from the P2 promoter is highly regulated, responsible for the inducible expression of HDM2, and is found in the first intron [21]. The HDM2 transcript is translated into a protein of 491 amino acids with multiple sizes ranging from about 50 to 90 kDa [22]. HDM2 can be regulated positively by AKT, a serine-threonine kinase, leading to the repression of p53 activity. AKT phosphorylates HDM2, which leads to its nuclear entry and subsequent attenuation of p53 activity and p53 degradation [23]. p14 (ARF), a protein product from the CDNK2A locus, is a negative regulator of HDM2 [24]. The p14ARF protein binds to the central domain of HDM2, including the acidic region, leading to inhibition of the ability of HDM2 to act on p53 [25].
To determine whether the full-length HDM2 mRNA was bound to hnRNP A1 in RNP complexes, we isolated RNA from the complexes and used semi-quantitative to amplify full-length sequences. We found full-length HDM2 in total RNA and complexes isolated from young cells (Figure 4A). To provide an estimation of the nature of the HDM2 exons normally present in young fibroblasts, we amplified sequences from exon 1 to 2, exon 6 to 7, and exon 9 to 10. Figure 4B shows that of the regions amplified, the one that covered exon 9 to 10 was in the least abundance by a factor of 4 when compared with the most abundant region that spanned from exon 6 to 7. These results indicate that there may be preferential exon inclusion/exclusion in young fibroblasts in which hnRNP A1 is highly expressed.
Figure 4.
HDM2 mRNA variant expression levels. RNA was isolated from co- immunoprecipitated hnRNPA1 protein complexes from young IMR90 fibroblasts and reverse transcribed. Three independent replicates of cDNA were used as templates for detecting HDM2 regions: Exons 6–7, HDM2A; exons 1–2, HDM2B and exons 9–10, HDM2C. Taqman primer-probe sets specific for these regions of the HDM2 gene were used in qRT-PCR assays and compared to actin mRNA levels.
2.4 Identification of p16INK4a mRNA in hnRNPA1 complexes
We have previously shown that changes in the expression of hnRNP A1 regulate the alternative splicing and mRNA levels of two mRNA isoforms of the INK4a locus known as p14Arf and p16(INK4a) [10]. Both protein isoforms are growth suppressors and knockout of the INK4a gene allows cells to escape cellular senescence [11]. Our previous studies have shown [9] that overexpression of hnRNA1 results in a preferential expression of the p14Arf mRNA isoform, and an increase in the mRNA levels of both isoforms, thus suggesting a role for hnRNP A1 in control of cell proliferation and senescence [10]. In this study, we assessed the ability of hnRNP A1 to directly bind to INK4a transcripts in hnRNPA1 complexes. For this, we used AR5 cells and PRNS-1 (SV40-transformed clones of HS74 primary human bone marrow fibroblasts) since these cells express high levels of INK4a transcripts as compared with normal IMR-90 fibroblasts [26].
Figure 5 shows that the p16 transcript was amplified from hnRNPA1RNP complexes indicating that hnRNP A1 directly binds to p16 mRNA. We also measured the ability of hnRNP A1 to bind to actin mRNA as a positive control for our co-immunoprecipitation studies. Actin mRNA has been previously identified in hnRNPA1-RNP complexes [12]. Figure 5 shows that in addition to p16, we were also able to detect actin mRNA in the RNP complexes.
Figure 5.
Immunoprecipitation of specific mRNA hnRNP A1 RNP complexes. RNA was extracted from co-immunoprecipitated hnRNPA1 protein complexes isolated from AR5 and HS74-PRNS-1 cells and reverse transcribed. Primers set specific for p16 and actin were used to identify their respective mRNAs in hnRNP A1 RNP complexes. AR5 and HS74-PRNS-1 cells are SV40-transformed immortal cell lines.
2.5 Expression of HDM2 is modulated by hnRNPA1 expression levels
We next sought to determine whether hnRNPA1 modulated the expression of HDM2 mRNA levels. hnRNP A1 was overexpressed in young IMR-90 human fibroblast cells followed by real-time RT-PCR analysis using primer sets that amplified different regions of HDM2 mRNA. We observed a significant decrease in the full-length HDMd2 mRNA levels in cells overexpressing hnRNP A1 as compared with cells expressing the empty GFP Vector (Figure 6B). Downregulation of hnRNP A1 by siRNA transfection showed increased levels of HDM2 mRNA (Figure 6A). These results indicate that hnRNP A1 protein levels modulate the mRNA levels of HDM2. Since we had previously shown that hnRNP A1 expression and its subcellular distribution were altered during cellular senescence [27], we compared the endogenous HDM2 mRNA levels in young and senescent cells. We found that there was a significant increase in HDM2 mRNA levels in senescent cells as compared with young cells (Figure 6C). These results show that endogenous levels of HDM2 were consistent with our overexpression and siRNA results discussed earlier.
Figure 6.
Expression of HDM2 mRNA levels following alteration of hnRNP A1 expression. Panel a: Scrambled siRNA (control) or siA1 oligonucleotides were transfected into IMR-90 fibroblast cells. Real-time PCR was performed using primers for constitutive HDM2 mRNA (exon 1–3) and p53-inducible HDM2 mRNA (exon 2–3). RPLPO mRNA levels were used as an internal control. Panel B: Expression plasmids pEFGP empty vector or pEFGP-A1 were transfected into IMR-90 fibroblast cells. After 2 days of incubation, total RNA was extracted from cells and real-time PCR was performed with different sets of primers for detection of constitutive and p53-inducible HDM2 mRNA. RPLPO and GAPDH RNA levels were used as internal standards. Panel C: The steady-state endogenous levels of constitutive and p53-inducible HDM2 were measured in young and senescent mRNA.
We also investigated whether the protein level of HDM2 was modulated by the level of hnRNP A1 protein expression. To determine whether endogenous hnRNP A1 has an effect on HDM2 protein expression, scrambled siRNA or siA1 was transfected into IMR-90 fibroblast cells. We found that upon siRNA knockdown of hnRNP A1, the protein level of HDM2 was not altered as shown in Figure 7A. hnRNP A2, which has overlapping biochemical activity with hnRNP A1, when inhibited by siRNA interference, did not affect HDM2 expression. On the other hand, overexpression of hnRNP A1 in young cells transfected with GFP-A1 resulted in a slight decrease of HDM2 protein levels and an increase in p53 levels when compared with cells transfected with the GFP-Empty vector (Figure 7B). The increase in p53 protein levels may be a result of the decreased HDM2 expression. A direct correlation between protein and mRNA levels for any given gene is complicated by varying processes. For instance, studies conducted by various groups such as Vogel et al. [28] pointed out that transcription, mRNA export, decay, translation, and protein degradation are key processes in determining steady-state protein concentration [28].
Figure 7.
Effect of varying hnRNP A1 expression on HDM2 protein levels a. scrambled siRNA (control), siA1, siA2, or both oligonucleotides were transfected into IMR-90 fibroblast cells. About 30 μg of protein lysates were subjected to 12% SDS-PAGE and immunoblotted for hnRNPA1 and actin. The membranes were stripped and reprobed for total actin levels. B. IMR-90 cells were transiently transfected with GFP-hnRNP A1 and empty vector for 48 hours. Whole-cell lysates were prepared using RIPA lysis buffer. 30 μg of protein lysates were simultaneously subjected to 12% SDS-PAGE and immunoblotted for hnRNP A1, HDM2, p53 and actin. The membranes were stripped and reprobed for total actin levels. C. the relative protein levels was quantified by image density analysis software (image J), and the data were represented as mean value ± SEM (n = 3, and * p < 0.05 value).
2.6 Identification of HDM2 RNA sequences that bind to hnRNP A1
Our PESX analysis revealed that hnRNP A1 has a putative binding site within the intronic region between intron 9 and exon 10 of HDM2 (Figure 4). We obtained HDM2 constructs from Dr. Meek (University of Dundee). We performed biotin pull-down assay using MP4 construct that is similar to the MDM2-B isoform lacking p53-binding region followed by Western immunoblotting. We performed the biotin pull-down assay by first incubating the hnRNP A1 antibody (4B10) with Dynabeads Myone streptavidin. We also incubated the Biotinlabeled HDM2 mRNA (MP4 probe) or Biotinlabeled B-actin RNA probe with IMR-90 cell lysate and the mixture was incubated overnight. We performed Western blot analysis as described in Material and Methods to investigate the interaction between Biotinlabeled HDM2 mRNA and hnRNP A1. As shown in Figure 8, hnRNP A1 is directly associated with Biotinlabeled HDM2 mRNA (MP4 probe). The direct association between hnRNP A1 and Biotinlabeled HDM2 mRNA appears to be specific since these interactions were not observed with similar RNA-binding protein hnRNP A0. Furthermore, associations between hnRNP A1 and negative-control Biotinlabeled B-actin were not observed. These results demonstrate that hnRNP A1 has specific binding to Biotinlabeled HDM2 mRNA (MP4 probe). These results also show that hnRNP A1 can bind to the 3′ end of HDM2 as MP4 is the 3′ of HDM2 which is also a region devoid of HDM2 sequences upstream of exon 11.
Figure 8.
HDM2 transcript (MP4) binds to hnRNP A1. A. Schematic representation of full-length HDM2 and mini-protein “MP” fragment of HDM2 MP4. B. the interaction between hnRNP A1 and biotinylated HDM2 mRNA (MP4) was examined by biotin pull-down assay. Biotinylated HDM2 and actin probes were incubated with IMR-90 cell lysates. Interactions were analyzed by Western blotting. The protein-RNA probe streptavidin complexes were subjected to electrophoresis on a 12% SDS-PAGE. The 4B10 monoclonal antibody and anti-actin antibody were used to detect hnRNP A1 and actin, respectively.
3. Discussion
The role of hnRNP A1 during cellular senescence is unclear. Significant alterations in its levels, localization, and activity in senescent cells suggest that hnRNP A1 may contribute to the senescent phenotype [27]. However, only a few gene targets are known for hnRNP A1 [12]. This prompted us to search for additional mRNA targets for hnRNP A1 in young and senescent IMR-90 cells. We used an RNA co-immunoprecipitation protocol [12] to identify mRNA new targets for the hnRNP A1 protein. We found that hnRNP A1 is bound to several mRNAs not previously identified. Of particular interest to us was the observation that hnRNP A1 bound to HMD2 mRNA. Other RNA-binding proteins have been reported to bind to specific regions in HDM2 mRNA. La antigen, an RNA-binding protein, was found to interact with HDM2 5′UTR in a BCR/ABL cell line resulting in increased HDM2 expression [29]. It was further demonstrated that translational regulation contributed to the increased HDM2 levels in BCR/ABL cells [29]. Nucleolin, a multifunctional nucleolar protein with defined roles in ribosomal RNA processing, has also been reported to bind to the NLS/NES and RING domain of HDM2 [30]. The expression of HDM2 mRNA is transcriptionally regulated by p53 in response to stress such as DNA damage [31, 32]. We have found that the modulation of hnRNP A1 expression can regulate HDM2 mRNA levels.
Posttranscriptional regulation of gene expression is important for the control of cellular processes such as cell proliferation, differentiation, development, and apoptosis [33]. RNA-binding proteins are the main regulators of post-transcriptional regulation [33]. hnRNP A1 is a multifunctional RNA-binding protein implicated in the regulation of major steps in posttranscriptional regulation of gene expression [14]. Upon observation that hnRNP A1 binds to HDM2 mRNA, we sought to determine whether the modulation of the expression of hnRNP A1 had an effect on steady state HDM2 mRNA levels. Given that our previous results demonstrated that overexpression of hnRNP A1 significantly decreased HDM2 mRNA levels, we next asked whether the HDM2 protein levels were also lowered when hnRNP A1 was overexpressed. Our Western blot analysis data revealed that overexpression of GFP-A1 slightly decreased HDM2 protein levels (Figure 7B), whereas knockdown of hnRNP A1 did not have effect on HDM2 protein expression. We also observed that p53 levels increased upon GFP-A1 overexpression suggesting that the levels of p53 protein were increased by the decreased expression of HDM2 (Figure 7B). Previous studies have shown that HDM2 regulates p53 by targeting it for degradation [19]. Recent studies have demonstrated that overexpression of hnRNP A1 led to a reduction of HDM2-FL transcript levels in HaCat cells [34]. More importantly, it was also shown that UVB irradiation increased the binding of hnRNP A1 to HDM2 pre-mRNA [34]. Our studies are consistent with these findings whereby we have found that overexpression of hnRNP A1 decreased HDM2 transcript levels.
In this study, our findings suggest that hnRNP A1 binds to the Biotinlabeled HDM2 mRNA probe (MP4) that includes part of exons 11 and 12 of HDM2 mRNA as shown in Figure 8A. Previous approaches as those used by S.J. Park et al. [35] demonstrated that hnRNP A1 associates with Drp1 mRNA at the 3′ UTR [35]. We applied this approach and found that hnRNP A1 binds to the HDM2 MP4 mRNA and that this binding was specific as hnRNP A0 did not bind to the HDM2 MP4 probe (Figure 8B). Our sequencing data of the HDM2 MP4 reveal that it contains a putative G/AGAAG nucleotide sequence similar to the 5′AGAAG 3′ high-affinity binding site found in the purine-rich 3′ splice site of c-src mRNA exon N1 [36]. hnRNP A1 has been shown to bind to these sites in c-H-ras and HIV TAT [37, 38]. Overall, our RNA-protein interaction experiments data strongly suggest that hnRNP A1 interacts with a region of HDM2 transcript corresponding to its 3′ UTR. It has been previously reported that RNA-binding protein RNPC1 binds to the 3′UTR region in HDM2 transcripts and inhibits its expression [39]. Thus, the HDM2 3′ UTR is bound by different RNA-binding proteins that might either repress or induce its expression. For example, HuR, an RNA-binding protein, has been shown to bind and stabilize HDM2 via its 3′ UTR. From our studies, we found that hnRNP A1 modulated the mRNA expression of HDM2. Therefore, the MP4 sequence maybe partially contributing to this modulation. Our findings are significant when taken in the context of RNA-binding protein contributing to the aging phenotype. Both HuR and hnRNPA1 are involved in regulating the senescent phenotype [40, 41]. Inhibiting HuR expression induces the senescent phenotype [41]. hnRNP A1 has been recently shown to antagonize cellular senescence through the SIRT1 pathway [42]. The research findings of the research project are important because they can add to the knowledge of the regulation of HDM2 gene expression.
4. Materials and methods
4.1 Cell culture and generation of senescent fibroblasts
The human lung fibroblast cell strain IMR90 from Coriell, NJ, was subcultured from early passage to terminal passage as previously described by Hubbard and Ozer [43] in Dulbecco’s modified Eagle’s medium and Ham’s F10 medium in a 1:1 mixture supplemented with 10% fetal bovine serum. IMR90 fibroblasts at population doubling <35 were used in all experiments and are considered comparable to young fibroblasts as determined by gene expression profiles previously performed [43]. Senescent IMR90 in all experiments was at a population doubling of 62. For transfection experiments, once cells had reached 90% confluence, either the expression plasmid pEFGP (Control) or pEFGP-A1 was transfected into IMR-90 cells in DMEM/F10 media without FBS/penicillin using Lipofectamine 2000 (Invitrogen) and incubate at 37°C in a CO2 incubator for 6 h.
4.2 RNA isolation and RNA-PCR to check for genomic contamination
After RNA was isolated as detailed above, to ensure that there was no genomic contamination, an RNA–PCR procedure was performed. 2 μL of template RNA was added to a PCR mixture using β-actin primers, which would detect genomic sequences if present. The total PCR reaction was composed of 2 μl of template RNA, 5 μl of 10× RT-PCR Buffer (100 mM Tris–HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2), 2.5 μl of dNTP mix (2.5 mM each dNTP), 0.25 μM each PCR primer, 0.5 unit of Thermostable DNA Polymerase (Novagen).
The primers for actin were: actin forward (5′CGCCGCCCTAGGCACCA3′) and actin reverse (5′TTGGCCTTAGGGTTCAGGGGGG 3′). For hnRNPA1, primer set were: hnRNP A1 forward (5′CTAAAGAGCCCGAACAGCTGAG 3′) and hnRNP A1 reverse (5′TCAGTGTCTTCTTTAATGCCACCA 3′). SYBR™ green stain. SYBR™ green can be visualized by blue fluorescence (Molecular Dynamics, Amersham) and quantified with ImageQuant software (Amersham).
5. Immunoblotting and protein analysis
Standard Western blotting protocols (Harlow et al. 1999) were to analyze specific proteins [44]. Protein extracts isolated from young and senescent fibroblasts were generated by washing cells were washed 3 times with 1X cold PBS and then, cultures were placed on ice. Cold RIPA (radioimmunoassay buffer containing NP-40 at 1%, sodium deoxycholate at 1%, sodium dodecyl sulfate at 0.1%, NaCl at 150 mM and Tris-HCl at 10 mM with protease inhibitors leupeptin at 0.1 μg/ml, pepstatin at 0.1 μg/ml, and phenylmethylsulfonyl fluoride at 1 mM) was added to culture dishes followed by scraping cells into cold microfuge tubes. The lysate was passed through a 21-gauge syringe needle to ensure complete lysis. Lysates were centrifuged at 10,000×g for 10 minutes at 4°C. The cleared lysate was collected and aliquots were prepared to estimate the amount of protein by the Bradford protein assay (Bio-Rad). Lysates were run on 8–12% acrylamide gels and then transferred in an electroblotting apparatus. Membranes (PVDF, Osmonics) were blocked with 5% non-fat milk in PBS. Monoclonal antibody 4B10 (1:10,000) was used to detect hnRNPA1/A1. An anti-actin monoclonal antibody (1:5000) was obtained from Chemicon. Antibodies specific for HDM2 were graciously provided by Dr. Jill Bargonetti. Secondary antibody, goat IgG, or mouse IgG conjugated with HRP were used for visualization of bands using the ECL kit (Amersham).
6. Overexpression of hnRNP A1 by transient transfection
Young IMR-90 cells were cultured in 10-cm plates. After approximately 24 h of incubation when the cells reached 90% confluence, the expression plasmid pEFGP (control) or pEFGP-A1 was transfected into IMR-90 cells in DMEM/F10 media without FBS/penicillin using Lipofectamine 2000 (Invitrogen) and incubated at 37°C in CO2 incubator for 6 h. We changed the media to DMEM with FBS and without penicillin and incubated for 48 h at 37°C.
7. RNA co-immunoprecipitation protocol
The RNA co-immunoprecipitation protocol was a modified version published by Mili et al. [12] that included a short immunoprecipitation step that minimized degradation of protein-associated RNA.
8. Confirmation of gene expression using real-time PCR
Real-time PCR experiments on selected genes were performed using an Applied Biosystems 7500 real-time PCR system that utilizes TaqMan gene expression assays for the following genes: mdm2; Human GAPD (GAPDH) Endogenous Control FAM/MGB (4333764F). Reactions were performed according to standard methods using the universal 10X PCR TaqMan mix, at a final reaction volume of 25 μL (Applied Biosystems).
9. Cloning protocol and sequencing
PCR products were ligated into the pCR 2.1 (Invitrogen, TA cloning kit) cloning vector that utilizes the single dT overhangs that are a by-product of PCR reactions catalyzed by Taq polymerase. The ligation reaction was performed at 14°C overnight using T4 DNA ligase and 3 μL of fresh PCR product (Invitrogen protocols).
10. Sequence analysis using BLAST and PESX
Vector sequences were subtracted from the sequences obtained. The rest of the sequence was compared against known sequences using the BLAST tool (www.ncbi.nlm.nih.gov). Sequences were chosen based on being previously identified as human genes and either in the coding regions or in the flanking regions of the mRNA sequences of known genes.
11. Biotin pull-down assay
Biotinylated transcripts were obtained by reverse transcription with the Maxiscript Kit (Invitrogen) according to manufacturer instructions and as previously described above in Section 3.2. The biotin pull-down assay was performed by first incubating hnRNP A1 antibody (4B10) with Dynabeads Myone streptavidin (Invitrogen) for 1 hour at 4 C. Also, incubated Biotinlabeled HDM2 mRNA (MP4 probe) or B-actin RNA probe with 25 μg of IMR-90 cell lysate for 1 h at RT. Following this incubation, we added the biotinylated RNA probes and protein lysate mixtures to the Myone streptavidin beads coated with 4B10 (hnRNP A1 antibody) and performed a second overnight incubation, immediately subjected the protein-RNA complexes to Western blot analysis as described in Section 2.4 to detect specific proteins bound to biotinylated transcripts.
Acknowledgments
We are grateful to Alisa Chalmers for technical assistance. This research was supported by NIH/NCI U54CA137788/U54CA132378 to KH and to NIH/NIMHD 3G12MD007603-30S2 and NIH RISE R25GM056833 to HM. We also thank Dr. Serafin Pinol-Roma for hnRNP specific antibodies and consultation on the RNA co-immunoprecipitation assays. We also thank Dr. Bargonetti for the HDM2 antibody and Dr. Meek for providing HDM2 plasmid constructs. Heriberto Moran and Shanaz A. Ghandhi contributed equally to this work.
Conflict of interest and financial disclosures
There are no conflicts of interest nor financial interest or benefits.
Abbreviations
HDM2
Human Double Minute 2
UTR
Untranslated region
hnRNPA1
Heterogeneous nuclear ribonucleoprotein A1
RNP
Ribonucleoprotein
MP4
Biotin-labeled HDM2 RNA probe
mRNP
Messenger ribonucleoprotein
\n',keywords:"senescence, fibroblasts, hnRNP A1, hdm2, RNP complexes",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/79668.pdf",chapterXML:"https://mts.intechopen.com/source/xml/79668.xml",downloadPdfUrl:"/chapter/pdf-download/79668",previewPdfUrl:"/chapter/pdf-preview/79668",totalDownloads:81,totalViews:0,totalCrossrefCites:0,dateSubmitted:"September 4th 2021",dateReviewed:"November 5th 2021",datePrePublished:"December 20th 2021",datePublished:null,dateFinished:"December 14th 2021",readingETA:"0",abstract:"hnRNP A1 is a member of the hnRNPs (heterogeneous nuclear ribonucleoproteins) family of proteins that play a central role in regulating genes responsible for cell proliferation, DNA repair, apoptosis, and telomere biogenesis. Previous studies have shown that hnRNPA1 had reduced protein levels and increased cytoplasmic accumulation in senescent human diploid fibroblasts. The consequence of reduced protein expression and altered cellular localization may account for the alterations in gene expression observed during senescence. There is limited information for gene targets of hnRNP A1 as well as its in vivo function. In these studies, we performed RNA co-immunoprecipitation experiments using hnRNP A1 as the target protein to identify potential mRNA species in ribonucleoprotein (RNP) complexes. Using this approach, we identified the human double minute 2 (HDM2) mRNA as a binding target for hnRNP A1 in young and senescent human diploid fibroblasts cells. It was also observed that alterations of hnRNP A1 expression modulate HDM2 mRNA levels in young IMR-90 cells. We also demonstrated that the levels of HDM2 mRNA increased with the downregulation of hnRNP A1 and decrease with the overexpression of hnRNP A1. Although we did not observe a significant decrease in HDM2 protein level, a concomitant increase in p53 protein level was detected with the overexpression of hnRNP A1. Our studies also show that hnRNP A1 directly interacts with HDM2 mRNA at a region corresponding to its 3′ UTR (untranslated region of a gene). The results from this study demonstrate that hnRNP A1 has a novel role in participating in the regulation of HDM2 gene expression.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/79668",risUrl:"/chapter/ris/79668",signatures:"Heriberto Moran, Shanaz A. Ghandhi, Naoko Shimada and Karen Hubbard",book:{id:"10935",type:"book",title:"Mechanisms and Management of Senescence",subtitle:null,fullTitle:"Mechanisms and Management of Senescence",slug:null,publishedDate:null,bookSignature:"Dr. Hassan M. Heshmati",coverURL:"https://cdn.intechopen.com/books/images_new/10935.jpg",licenceType:"CC BY 3.0",editedByType:null,isbn:"978-1-83969-051-8",printIsbn:"978-1-83969-050-1",pdfIsbn:"978-1-83969-052-5",isAvailableForWebshopOrdering:!0,editors:[{id:"313921",title:"Dr.",name:"Hassan M.",middleName:null,surname:"Heshmati",slug:"hassan-m.-heshmati",fullName:"Hassan M. Heshmati"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Results",level:"1"},{id:"sec_2_2",title:"2.1 hnRNP A1-messenger ribonucleoprotein (mRNP) complexes",level:"2"},{id:"sec_3_2",title:"2.2 Analysis of the hnRNPA1-mRNA complex",level:"2"},{id:"sec_4_2",title:"2.3 Identification of RNA species in hnRNP A1-mRNP complexes",level:"2"},{id:"sec_5_2",title:"2.4 Identification of p16INK4a mRNA in hnRNPA1 complexes",level:"2"},{id:"sec_6_2",title:"2.5 Expression of HDM2 is modulated by hnRNPA1 expression levels",level:"2"},{id:"sec_7_2",title:"2.6 Identification of HDM2 RNA sequences that bind to hnRNP A1",level:"2"},{id:"sec_9",title:"3. Discussion",level:"1"},{id:"sec_10",title:"4. Materials and methods",level:"1"},{id:"sec_10_2",title:"4.1 Cell culture and generation of senescent fibroblasts",level:"2"},{id:"sec_11_2",title:"4.2 RNA isolation and RNA-PCR to check for genomic contamination",level:"2"},{id:"sec_13",title:"5. Immunoblotting and protein analysis",level:"1"},{id:"sec_14",title:"6. Overexpression of hnRNP A1 by transient transfection",level:"1"},{id:"sec_15",title:"7. RNA co-immunoprecipitation protocol",level:"1"},{id:"sec_16",title:"8. Confirmation of gene expression using real-time PCR",level:"1"},{id:"sec_17",title:"9. Cloning protocol and sequencing",level:"1"},{id:"sec_18",title:"10. Sequence analysis using BLAST and PESX",level:"1"},{id:"sec_19",title:"11. Biotin pull-down assay",level:"1"},{id:"sec_20",title:"Acknowledgments",level:"1"},{id:"sec_23",title:"Conflict of interest and financial disclosures",level:"1"},{id:"sec_22",title:"Abbreviations",level:"1"}],chapterReferences:[{id:"B1",body:'Cristofalo VJ, Volker C, Francis MK, Tresini M. Age-dependent modification of gene expression in human fibroblasts. Critical Reviewes in Eukaryotic Gene Expression. 1998;8:43-80'},{id:"B2",body:'Francis R, Campisi J. Four faces of cellular senescence. The Journal of Cell Biology. 2011;192(4):547-556'},{id:"B3",body:'Wei W, Hemmer MR, Sedivy MJ. 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FEBS Letters. 2008;582(14):1977-1986'},{id:"B34",body:'Feng J, Li J, Tong L, Tan L, Wu S. The involvement of splicing Factor hnRNP A1 in UVB-induced alternative splicing of hdm2. Photochemistry and Photobiology. 2016;92:318-324'},{id:"B35",body:'Park SJ, Lee H. Jo YK, Shin JH, Kim, HB, Seo HM, Rubinsztein D., Koh JY, Lee EK, Cho DH. Heterogenous nuclear ribonucleoprotein A1 post-transcriptionally regulates Drp1 expression in neuroblastoma cells. Biochimica et Biophysica Acta 2015;1829(12):1423-1431'},{id:"B36",body:'Rooke N, Markovtsov V, Cagavi E, Black DL. Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1. Molecular and Cellular Biology. 2003;23(6):1874-1884'},{id:"B37",body:'Damgaard CK, Tange TO, Kjems J. hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure. RNA. 2002;8(11):1401-1415'},{id:"B38",body:'Guil S, Gattoni R, Carrascal M, Abian J, Stevenin J, Bach-Elias M. Roles of hnRNPA1, SR proteins, and p68 helicase in c-H-ras alternative splicing regulation. Molecular and Cellular Biology. 2003;23(8):2927-2941'},{id:"B39",body:'Xu E, Zhang J, Chen X. MDM2 expression is repressed by the RNA binding protein RNPC1 via mRNA stability. Oncogene. 2013;32(17):2169-2178'},{id:"B40",body:'Lee BP, Pilling LC, Emond F, Flurkey K, Harrison DE, Yuan R, et al. Changes in the expression of splicing factor transcripts and variations in alternative splicing are associated with lifespan in mice and humans. Aging Cell. 2016;15(5):903-913'},{id:"B41",body:'Hashimoto M, Tsugawa T, Kawagishi H, Asai A, Sugimoto M. Loss of HuR leads to senescence-like cytokine induction in rodent fibroblasts by activating NF-κB. Biochimica et Biophysica Acta. 2014;1840(10):3079-3087'},{id:"B42",body:'Wang H, Han L, Zhao G, Shen H, Wang P, Sun Z, et al. hnRNP A1 antagonizes cellular senescence and senescence-associated secretory phenotype via regulation of SIRT1 mRNA stability. Aging Cell. 2016;15(6):1063-1073'},{id:"B43",body:'Hubbard K, Ozer HL. Chapter 12; Senescence and immortalization of human cells. In: Studzinski G, editor. Cell Growth and Apoptosis: A Practical Approach. England: IRL Press; 1995. pp. 229-249'},{id:"B44",body:'Harlow E, Lane D. Using Antibodys: A Laboratory Manual. New York, NY: Cold Spring Harbor Laboratory Press; 1999'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Heriberto Moran",address:"hmoran@ccny.cuny.edu",affiliation:'
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IntechOpen’s Academic Editors and Authors have received funding for their work through many well-known funders, including: the European Commission, Bill and Melinda Gates Foundation, Wellcome Trust, Chinese Academy of Sciences, Natural Science Foundation of China (NSFC), CGIAR Consortium of International Agricultural Research Centers, National Institute of Health (NIH), National Science Foundation (NSF), National Aeronautics and Space Administration (NASA), National Institute of Standards and Technology (NIST), German Research Foundation (DFG), Research Councils United Kingdom (RCUK), Oswaldo Cruz Foundation, Austrian Science Fund (FWF), Foundation for Science and Technology (FCT), Australian Research Council (ARC).
Open Access publication costs can often be designated directly in the grants or in specific budgets allocated for that purpose. Many of the most important funding organisations encourage, and even request, that the projects they fund are made available at no cost to the wider public. IntechOpen strives to maintain excellent relationships with these funders and ensures compliance with mandates.
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In order to help Authors identify appropriate funding agencies and institutions, we have created a list, based on extensive research on various OA resources (including ROARMAP and SHERPA/JULIET) of organizations that have funds available. Before consulting our list we encourage you to petition your own institution or organization for Open Access funds or check the specifications of your grant with your funder to ascertain if publication costs are included. Where you are in receipt of a grant you should clarify:
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Please note that this list is not a definitive one and is updated regularly. To suggest possible modifications or the inclusion of your institution/funder, please contact us at funders@intechopen.com
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Please be aware that you must be a member, or grantee, of the institutions/funders listed in order to apply for their Open Access publication funds.
Open Access publication costs can often be designated directly in the grants or in specific budgets allocated for that purpose. Many of the most important funding organisations encourage, and even request, that the projects they fund are made available at no cost to the wider public. IntechOpen strives to maintain excellent relationships with these funders and ensures compliance with mandates.
\n\n
In order to help Authors identify appropriate funding agencies and institutions, we have created a list, based on extensive research on various OA resources (including ROARMAP and SHERPA/JULIET) of organizations that have funds available. Before consulting our list we encourage you to petition your own institution or organization for Open Access funds or check the specifications of your grant with your funder to ascertain if publication costs are included. Where you are in receipt of a grant you should clarify:
\n\n
\n\t
Does your institution already have a budget for covering Open Access publication costs?
\n\t
Does your grant list Open Access publication fees as legitimate direct/indirect costs?
\n
\n\n
If you are associated with any of the institutions in our list below, you can apply to receive OA publication funds by following the instructions provided in the links. Please consult the Open Access policies or grant Terms and Conditions of any institution with which you are linked to explore ways to cover your publication costs (also accessible by clicking on the link in their title).
\n\n
Please note that this list is not a definitive one and is updated regularly. To suggest possible modifications or the inclusion of your institution/funder, please contact us at funders@intechopen.com
\n\n
Please be aware that you must be a member, or grantee, of the institutions/funders listed in order to apply for their Open Access publication funds.
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Catarina Guedes and F. Xavier Malcata",authors:[{id:"83136",title:"Prof.",name:"F. Xavier",middleName:null,surname:"Malcata",slug:"f.-xavier-malcata",fullName:"F. Xavier Malcata"}]},{id:"30642",doi:"10.5772/34423",title:"Meiofauna as a Tool for Marine Ecosystem Biomonitoring",slug:"meiofauna-as-a-tool-for-marine-ecosystem-monitoring",totalDownloads:3912,totalCrossrefCites:22,totalDimensionsCites:84,abstract:null,book:{id:"1689",slug:"marine-ecosystems",title:"Marine Ecosystems",fullTitle:"Marine Ecosystems"},signatures:"Maria Balsamo, Federica Semprucci, Fabrizio Frontalini and Rodolfo Coccioni",authors:[{id:"100075",title:"Prof.",name:"Maria",middleName:null,surname:"Balsamo",slug:"maria-balsamo",fullName:"Maria Balsamo"},{id:"104309",title:"Dr.",name:"Federica",middleName:null,surname:"Semprucci",slug:"federica-semprucci",fullName:"Federica Semprucci"},{id:"104311",title:"Dr.",name:"Fabrizio",middleName:null,surname:"Frontalini",slug:"fabrizio-frontalini",fullName:"Fabrizio Frontalini"},{id:"104313",title:"Prof.",name:"Rodolfo",middleName:null,surname:"Coccioni",slug:"rodolfo-coccioni",fullName:"Rodolfo Coccioni"}]},{id:"35136",doi:"10.5772/29571",title:"Transmission Biology of the Myxozoa",slug:"transmission-biology-of-the-myxozoa",totalDownloads:2726,totalCrossrefCites:35,totalDimensionsCites:64,abstract:null,book:{id:"2052",slug:"health-and-environment-in-aquaculture",title:"Health and Environment in Aquaculture",fullTitle:"Health and Environment in Aquaculture"},signatures:"Hiroshi Yokoyama, Daniel Grabner and Sho Shirakashi",authors:[{id:"78409",title:"Dr.",name:"Hiroshi",middleName:null,surname:"Yokoyama",slug:"hiroshi-yokoyama",fullName:"Hiroshi Yokoyama"},{id:"83562",title:"Dr.",name:"Daniel",middleName:"Stefan",surname:"Grabner",slug:"daniel-grabner",fullName:"Daniel Grabner"},{id:"122643",title:"Dr.",name:"Sho",middleName:null,surname:"Shirakashi",slug:"sho-shirakashi",fullName:"Sho Shirakashi"}]},{id:"24078",doi:"10.5772/26795",title:"Photobacterium damselae subsp. damselae, an Emerging Pathogen Affecting New Cultured Marine Fish Species in Southern Spain",slug:"photobacterium-damselae-subsp-damselae-an-emerging-pathogen-affecting-new-cultured-marine-fish-speci",totalDownloads:3795,totalCrossrefCites:19,totalDimensionsCites:45,abstract:null,book:{id:"612",slug:"recent-advances-in-fish-farms",title:"Recent Advances in Fish Farms",fullTitle:"Recent Advances in Fish Farms"},signatures:"A. Labella, C. Berbel, M. Manchado, D. Castro and J.J. Borrego",authors:[{id:"67855",title:"Prof.",name:"Juan J.",middleName:null,surname:"Borrego",slug:"juan-j.-borrego",fullName:"Juan J. Borrego"},{id:"71146",title:"Dr.",name:"Alejandro",middleName:null,surname:"Labella",slug:"alejandro-labella",fullName:"Alejandro Labella"},{id:"71148",title:"Dr.",name:"Concepcion",middleName:null,surname:"Berbel",slug:"concepcion-berbel",fullName:"Concepcion Berbel"},{id:"71149",title:"Dr.",name:"Manuel",middleName:null,surname:"Manchado",slug:"manuel-manchado",fullName:"Manuel Manchado"},{id:"71151",title:"Dr.",name:"Dolores",middleName:null,surname:"Castro",slug:"dolores-castro",fullName:"Dolores Castro"}]}],mostDownloadedChaptersLast30Days:[{id:"35141",title:"Antibiotics in Aquaculture – Use, Abuse and Alternatives",slug:"antibiotics-in-aquaculture-use-abuse-and-alternatives",totalDownloads:19357,totalCrossrefCites:138,totalDimensionsCites:293,abstract:null,book:{id:"2052",slug:"health-and-environment-in-aquaculture",title:"Health and Environment in Aquaculture",fullTitle:"Health and Environment in Aquaculture"},signatures:"Jaime Romero, Carmen Gloria Feijoo and Paola Navarrete",authors:[{id:"72898",title:"Dr.",name:"Jaime",middleName:null,surname:"Romero",slug:"jaime-romero",fullName:"Jaime Romero"},{id:"79684",title:"Dr.",name:"Paola",middleName:null,surname:"Navarrete",slug:"paola-navarrete",fullName:"Paola Navarrete"},{id:"83411",title:"Dr.",name:"Carmen",middleName:null,surname:"Feijoo",slug:"carmen-feijoo",fullName:"Carmen Feijoo"}]},{id:"69948",title:"Floating Cage: A New Innovation of Seaweed Culture",slug:"floating-cage-a-new-innovation-of-seaweed-culture",totalDownloads:974,totalCrossrefCites:2,totalDimensionsCites:2,abstract:"Eucheumatoid cultivation continues to expand with a variety of methods that can increase production. This chapter will discuss an innovation in seaweed cultivation of the genus Eucheuma, which is the prime marine commodity in the tropical regions of the world. Research conducted during 2015-2017 and 2019 in Southeast Sulawesi Province, Indonesia, provided an overview of the use of floating cage that showed very significant growth results. The research result showed that the growth rates of Eucheuma denticulatum and Kappaphycus alvarezii in floating cage seemed faster and resulted in better thallus morphology. Daily production of E. denticulatum and K. alvarezii that were cultivated in floating cage was higher than daily production of E. denticulatum and K. alvarezii cultivated on longline. Specific growth rate (SGR) of E. denticulatum and K. alvarezii cultivated by using floating cage method was also higher than E. denticulatum and K. alvarezii cultivated by using longline method. Moreover, the cultivation by using floating cages produces good growth rates with no effect of herbivore attacks.",book:{id:"8928",slug:"emerging-technologies-environment-and-research-for-sustainable-aquaculture",title:"Emerging Technologies, Environment and Research for Sustainable Aquaculture",fullTitle:"Emerging Technologies, Environment and Research for Sustainable Aquaculture"},signatures:"Ma’ruf Kasim, Abdul Muis Balubi, Ahmad Mustafa, Rahman Nurdin, Rahmad Sofyan Patadjai and Wardha Jalil",authors:[{id:"309893",title:"Prof.",name:"Maruf",middleName:null,surname:"Kasim",slug:"maruf-kasim",fullName:"Maruf Kasim"},{id:"313040",title:"MSc.",name:"Abdul Muis",middleName:null,surname:"Balubi",slug:"abdul-muis-balubi",fullName:"Abdul Muis Balubi"},{id:"313041",title:"MSc.",name:"Wardha",middleName:null,surname:"Jalil",slug:"wardha-jalil",fullName:"Wardha Jalil"},{id:"313042",title:"MSc.",name:"Ahmad",middleName:null,surname:"Mustafa",slug:"ahmad-mustafa",fullName:"Ahmad Mustafa"},{id:"313043",title:"MSc.",name:"Rahman",middleName:null,surname:"Nurdin",slug:"rahman-nurdin",fullName:"Rahman Nurdin"},{id:"313044",title:"MSc.",name:"Rahmat Sofyan",middleName:null,surname:"Patadjai",slug:"rahmat-sofyan-patadjai",fullName:"Rahmat Sofyan Patadjai"}]},{id:"62842",title:"Integrated Rice and Aquaculture Farming",slug:"integrated-rice-and-aquaculture-farming",totalDownloads:1919,totalCrossrefCites:2,totalDimensionsCites:3,abstract:"The burning problems like scarcity of food for ever-growing human population in the present world are addressed by adapting various methods for production of protein, carbohydrate, oils and other food materials. One of the methods to produce high amount of food is integrated farming including rice-aquaculture farming, which produces protein and carbohydrate as major components besides others. Rice-aquaculture farming produces grain (carbohydrate) and animal protein without affecting the quality and quantity of rice yield on the same piece of land and renders additional financial gain besides main crop (rice) like conventional monoculture. The aquatic species grown in the integrated culture are mainly distinct types of fishes, selected crustaceans and other selected species. Profitable rice-aquaculture integrated farming is popular in Asian countries than in Western countries. However, the integrated rice-aquaculture farming has its own limitations. The type of methods, culture species, influencing factors, and pros and cons of rice-aquaculture integrated farming are discussed in the present chapter.",book:{id:"7229",slug:"aquaculture-plants-and-invertebrates",title:"Aquaculture",fullTitle:"Aquaculture - Plants and Invertebrates"},signatures:"Pamuru Ramachandra Reddy and Battina Kishori",authors:[{id:"242524",title:"Dr.",name:"Ramachandra Reddy",middleName:null,surname:"Pamuru",slug:"ramachandra-reddy-pamuru",fullName:"Ramachandra Reddy Pamuru"},{id:"255022",title:"Dr.",name:"Kishori",middleName:null,surname:"Battina",slug:"kishori-battina",fullName:"Kishori Battina"}]},{id:"24074",title:"Embryonic and Larval Development of Freshwater Fish",slug:"embryonic-and-larval-development-of-freshwater-fish",totalDownloads:7466,totalCrossrefCites:1,totalDimensionsCites:2,abstract:null,book:{id:"612",slug:"recent-advances-in-fish-farms",title:"Recent Advances in Fish Farms",fullTitle:"Recent Advances in Fish Farms"},signatures:"Faruk Aral, Erdinç Şahınöz and Zafer Doğu",authors:[{id:"25600",title:"Prof.",name:"Faruk",middleName:null,surname:"Aral",slug:"faruk-aral",fullName:"Faruk Aral"},{id:"29132",title:"Dr.",name:"Zafer",middleName:null,surname:"Dogu",slug:"zafer-dogu",fullName:"Zafer Dogu"},{id:"39952",title:"Dr.",name:"Erdinc",middleName:null,surname:"Sahinoz",slug:"erdinc-sahinoz",fullName:"Erdinc Sahinoz"}]},{id:"68966",title:"Novel Biofloc Technology (BFT) for Ammonia Assimilation and Reuse in Aquaculture In Situ",slug:"novel-biofloc-technology-bft-for-ammonia-assimilation-and-reuse-in-aquaculture-in-situ",totalDownloads:1951,totalCrossrefCites:2,totalDimensionsCites:8,abstract:"Ammonia is one of the most harmful risks for success of fish and shrimp culture. There is no effective solution for harmlessness of ammonia in traditional aquaculture operations except exchanging water, which would bring negative effects on environment, or fixing expensive equipment. Biofloc technology (BFT) that appeared in recent years supplies a novel solution for this issue without exchanging huge water and fixing equipment. This technology could assimilate ammonia almost in real time with many other supplemental benefits. Because of the very high nutritional value for fish and shrimp, bioflocs, the by-product of BFT, could also be reused as a complemented food in situ or a gradient for feedstuff to replace expensive fishmeal or be processed to pellet diet to feed fish and shrimp directly. 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He was elected a Yangtze River Scholars Distinguished Professor in 2013, a member of the International Statistical Institute (ISI) in 2016, a member of the board of the International Chinese Statistical Association (ICSA) in 2018, and a fellow of the Institute of Mathematical Statistics (IMS) in 2021. He received the ICSA Outstanding Service Award in 2018 and the National Science Foundation for Distinguished Young Scholars of China in 2012. He serves as a member of the editorial board of Statistics and Its Interface and Journal of Systems Science and Complexity. He is also a field editor for Communications in Mathematics and Statistics. His research interests include biostatistics, empirical likelihood, missing data analysis, variable selection, high-dimensional data analysis, Bayesian statistics, and data science. He has published more than 190 research papers and authored five books.",institutionString:"Yunnan University",institution:{name:"Yunnan University",country:{name:"China"}}},{id:"1177",title:"Prof.",name:"António",middleName:"J. R.",surname:"José Ribeiro Neves",slug:"antonio-jose-ribeiro-neves",fullName:"António José Ribeiro Neves",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/1177/images/system/1177.jpg",biography:"Prof. António J. R. Neves received a Ph.D. in Electrical Engineering from the University of Aveiro, Portugal, in 2007. Since 2002, he has been a researcher at the Institute of Electronics and Informatics Engineering of Aveiro. Since 2007, he has been an assistant professor in the Department of Electronics, Telecommunications, and Informatics, University of Aveiro. He is the director of the undergraduate course on Electrical and Computers Engineering and the vice-director of the master’s degree in Electronics and Telecommunications Engineering. He is an IEEE Senior Member and a member of several other research organizations worldwide. His main research interests are computer vision, intelligent systems, robotics, and image and video processing. He has participated in or coordinated several research projects and received more than thirty-five awards. He has 161 publications to his credit, including books, book chapters, journal articles, and conference papers. He has vast experience as a reviewer of several journals and conferences. As a professor, Dr. Neves has supervised several Ph.D. and master’s students and was involved in more than twenty-five different courses.",institutionString:null,institution:{name:"University of Aveiro",country:{name:"Portugal"}}},{id:"11317",title:"Dr.",name:"Francisco",middleName:null,surname:"Javier Gallegos-Funes",slug:"francisco-javier-gallegos-funes",fullName:"Francisco Javier Gallegos-Funes",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/11317/images/system/11317.png",biography:"Francisco J. Gallegos-Funes received his Ph.D. in Communications and Electronics from the Instituto Politécnico Nacional de México (National Polytechnic Institute of Mexico) in 2003. He is currently an associate professor in the Escuela Superior de Ingeniería Mecánica y Eléctrica (Mechanical and Electrical Engineering Higher School) at the same institute. His areas of scientific interest are signal and image processing, filtering, steganography, segmentation, pattern recognition, biomedical signal processing, sensors, and real-time applications.",institutionString:"Instituto Politécnico Nacional",institution:{name:"Instituto Politécnico Nacional",country:{name:"Mexico"}}},{id:"428449",title:"Dr.",name:"Ronaldo",middleName:null,surname:"Ferreira",slug:"ronaldo-ferreira",fullName:"Ronaldo Ferreira",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/428449/images/21449_n.png",biography:null,institutionString:null,institution:{name:"University of Aveiro",country:{name:"Portugal"}}},{id:"165328",title:"Dr.",name:"Vahid",middleName:null,surname:"Asadpour",slug:"vahid-asadpour",fullName:"Vahid Asadpour",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/165328/images/system/165328.jpg",biography:"Vahid Asadpour, MS, Ph.D., is currently with the Department of Research and Evaluation, Kaiser Permanente Southern California. He has both an MS and Ph.D. in Biomedical Engineering. He was previously a research scientist at the University of California Los Angeles (UCLA) and visiting professor and researcher at the University of North Dakota. He is currently working in artificial intelligence and its applications in medical signal processing. In addition, he is using digital signal processing in medical imaging and speech processing. Dr. Asadpour has developed brain-computer interfacing algorithms and has published books, book chapters, and several journal and conference papers in this field and other areas of intelligent signal processing. He has also designed medical devices, including a laser Doppler monitoring system.",institutionString:"Kaiser Permanente Southern California",institution:null},{id:"169608",title:"Prof.",name:"Marian",middleName:null,surname:"Găiceanu",slug:"marian-gaiceanu",fullName:"Marian Găiceanu",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/169608/images/system/169608.png",biography:"Prof. Dr. Marian Gaiceanu graduated from the Naval and Electrical Engineering Faculty, Dunarea de Jos University of Galati, Romania, in 1997. He received a Ph.D. (Magna Cum Laude) in Electrical Engineering in 2002. Since 2017, Dr. Gaiceanu has been a Ph.D. supervisor for students in Electrical Engineering. He has been employed at Dunarea de Jos University of Galati since 1996, where he is currently a professor. Dr. Gaiceanu is a member of the National Council for Attesting Titles, Diplomas and Certificates, an expert of the Executive Agency for Higher Education, Research Funding, and a member of the Senate of the Dunarea de Jos University of Galati. He has been the head of the Integrated Energy Conversion Systems and Advanced Control of Complex Processes Research Center, Romania, since 2016. He has conducted several projects in power converter systems for electrical drives, power quality, PEM and SOFC fuel cell power converters for utilities, electric vehicles, and marine applications with the Department of Regulation and Control, SIEI S.pA. (2002–2004) and the Polytechnic University of Turin, Italy (2002–2004, 2006–2007). He is a member of the Institute of Electrical and Electronics Engineers (IEEE) and cofounder-member of the IEEE Power Electronics Romanian Chapter. He is a guest editor at Energies and an academic book editor for IntechOpen. He is also a member of the editorial boards of the Journal of Electrical Engineering, Electronics, Control and Computer Science and Sustainability. Dr. Gaiceanu has been General Chairman of the IEEE International Symposium on Electrical and Electronics Engineering in the last six editions.",institutionString:'"Dunarea de Jos" University of Galati',institution:{name:'"Dunarea de Jos" University of Galati',country:{name:"Romania"}}},{id:"4519",title:"Prof.",name:"Jaydip",middleName:null,surname:"Sen",slug:"jaydip-sen",fullName:"Jaydip Sen",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/4519/images/system/4519.jpeg",biography:"Jaydip Sen is associated with Praxis Business School, Kolkata, India, as a professor in the Department of Data Science. His research areas include security and privacy issues in computing and communication, intrusion detection systems, machine learning, deep learning, and artificial intelligence in the financial domain. He has more than 200 publications in reputed international journals, refereed conference proceedings, and 20 book chapters in books published by internationally renowned publishing houses, such as Springer, CRC press, IGI Global, etc. Currently, he is serving on the editorial board of the prestigious journal Frontiers in Communications and Networks and in the technical program committees of a number of high-ranked international conferences organized by the IEEE, USA, and the ACM, USA. He has been listed among the top 2% of scientists in the world for the last three consecutive years, 2019 to 2021 as per studies conducted by the Stanford University, USA.",institutionString:"Praxis Business School",institution:null},{id:"320071",title:"Dr.",name:"Sidra",middleName:null,surname:"Mehtab",slug:"sidra-mehtab",fullName:"Sidra Mehtab",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y00002v6KHoQAM/Profile_Picture_1584512086360",biography:"Sidra Mehtab has completed her BS with honors in Physics from Calcutta University, India in 2018. She has done MS in Data Science and Analytics from Maulana Abul Kalam Azad University of Technology (MAKAUT), Kolkata, India in 2020. Her research areas include Econometrics, Time Series Analysis, Machine Learning, Deep Learning, Artificial Intelligence, and Computer and Network Security with a particular focus on Cyber Security Analytics. Ms. Mehtab has published seven papers in international conferences and one of her papers has been accepted for publication in a reputable international journal. She has won the best paper awards in two prestigious international conferences – BAICONF 2019, and ICADCML 2021, organized in the Indian Institute of Management, Bangalore, India in December 2019, and SOA University, Bhubaneswar, India in January 2021. Besides, Ms. Mehtab has also published two book chapters in two books. Seven of her book chapters will be published in a volume shortly in 2021 by Cambridge Scholars’ Press, UK. Currently, she is working as the joint editor of two edited volumes on Time Series Analysis and Forecasting to be published in the first half of 2021 by an international house. Currently, she is working as a Data Scientist with an MNC in Delhi, India.",institutionString:"NSHM College of Management and Technology",institution:{name:"Association for Computing Machinery",country:{name:"United States of America"}}},{id:"226240",title:"Dr.",name:"Andri Irfan",middleName:null,surname:"Rifai",slug:"andri-irfan-rifai",fullName:"Andri Irfan Rifai",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/226240/images/7412_n.jpg",biography:"Andri IRFAN is a Senior Lecturer of Civil Engineering and Planning. He completed the PhD at the Universitas Indonesia & Universidade do Minho with Sandwich Program Scholarship from the Directorate General of Higher Education and LPDP scholarship. He has been teaching for more than 19 years and much active to applied his knowledge in the project construction in Indonesia. His research interest ranges from pavement management system to advanced data mining techniques for transportation engineering. He has published more than 50 papers in journals and 2 books.",institutionString:null,institution:{name:"Universitas Internasional Batam",country:{name:"Indonesia"}}},{id:"314576",title:"Dr.",name:"Ibai",middleName:null,surname:"Laña",slug:"ibai-lana",fullName:"Ibai Laña",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/314576/images/system/314576.jpg",biography:"Dr. Ibai Laña works at TECNALIA as a data analyst. He received his Ph.D. in Artificial Intelligence from the University of the Basque Country (UPV/EHU), Spain, in 2018. He is currently a senior researcher at TECNALIA. His research interests fall within the intersection of intelligent transportation systems, machine learning, traffic data analysis, and data science. He has dealt with urban traffic forecasting problems, applying machine learning models and evolutionary algorithms. He has experience in origin-destination matrix estimation or point of interest and trajectory detection. Working with large volumes of data has given him a good command of big data processing tools and NoSQL databases. He has also been a visiting scholar at the Knowledge Engineering and Discovery Research Institute, Auckland University of Technology.",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"314575",title:"Dr.",name:"Jesus",middleName:null,surname:"L. Lobo",slug:"jesus-l.-lobo",fullName:"Jesus L. Lobo",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/314575/images/system/314575.png",biography:"Dr. Jesús López is currently based in Bilbao (Spain) working at TECNALIA as Artificial Intelligence Research Scientist. In most cases, a project idea or a new research line needs to be investigated to see if it is good enough to take into production or to focus on it. That is exactly what he does, diving into Machine Learning algorithms and technologies to help TECNALIA to decide whether something is great in theory or will actually impact on the product or processes of its projects. So, he is expert at framing experiments, developing hypotheses, and proving whether they’re true or not, in order to investigate fundamental problems with a longer time horizon. He is also able to design and develop PoCs and system prototypes in simulation. He has participated in several national and internacional R&D projects.\n\nAs another relevant part of his everyday research work, he usually publishes his findings in reputed scientific refereed journals and international conferences, occasionally acting as reviewer and Programme Commitee member. Concretely, since 2018 he has published 9 JCR (8 Q1) journal papers, 9 conference papers (e.g. ECML PKDD 2021), and he has co-edited a book. He is also active in popular science writing data science stories for reputed blogs (KDNuggets, TowardsDataScience, Naukas). Besides, he has recently embarked on mentoring programmes as mentor, and has also worked as data science trainer.",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"103779",title:"Prof.",name:"Yalcin",middleName:null,surname:"Isler",slug:"yalcin-isler",fullName:"Yalcin Isler",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRyQ8QAK/Profile_Picture_1628834958734",biography:"Yalcin Isler (1971 - Burdur / Turkey) received the B.Sc. degree in the Department of Electrical and Electronics Engineering from Anadolu University, Eskisehir, Turkey, in 1993, the M.Sc. degree from the Department of Electronics and Communication Engineering, Suleyman Demirel University, Isparta, Turkey, in 1996, the Ph.D. degree from the Department of Electrical and Electronics Engineering, Dokuz Eylul University, Izmir, Turkey, in 2009, and the Competence of Associate Professorship from the Turkish Interuniversity Council in 2019.\n\nHe was Lecturer at Burdur Vocational School in Suleyman Demirel University (1993-2000, Burdur / Turkey), Software Engineer (2000-2002, Izmir / Turkey), Research Assistant in Bulent Ecevit University (2002-2003, Zonguldak / Turkey), Research Assistant in Dokuz Eylul University (2003-2010, Izmir / Turkey), Assistant Professor at the Department of Electrical and Electronics Engineering in Bulent Ecevit University (2010-2012, Zonguldak / Turkey), Assistant Professor at the Department of Biomedical Engineering in Izmir Katip Celebi University (2012-2019, Izmir / Turkey). He is an Associate Professor at the Department of Biomedical Engineering at Izmir Katip Celebi University, Izmir / Turkey, since 2019. In addition to academics, he has also founded Islerya Medical and Information Technologies Company, Izmir / Turkey, since 2017.\n\nHis main research interests cover biomedical signal processing, pattern recognition, medical device design, programming, and embedded systems. He has many scientific papers and participated in several projects in these study fields. He was an IEEE Student Member (2009-2011) and IEEE Member (2011-2014) and has been IEEE Senior Member since 2014.",institutionString:null,institution:{name:"Izmir Kâtip Çelebi University",country:{name:"Turkey"}}},{id:"339677",title:"Dr.",name:"Mrinmoy",middleName:null,surname:"Roy",slug:"mrinmoy-roy",fullName:"Mrinmoy Roy",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/339677/images/16768_n.jpg",biography:"An accomplished Sales & Marketing professional with 12 years of cross-functional experience in well-known organisations such as CIPLA, LUPIN, GLENMARK, ASTRAZENECA across different segment of Sales & Marketing, International Business, Institutional Business, Product Management, Strategic Marketing of HIV, Oncology, Derma, Respiratory, Anti-Diabetic, Nutraceutical & Stomatological Product Portfolio and Generic as well as Chronic Critical Care Portfolio. A First Class MBA in International Business & Strategic Marketing, B.Pharm, D.Pharm, Google Certified Digital Marketing Professional. Qualified PhD Candidate in Operations and Management with special focus on Artificial Intelligence and Machine Learning adoption, analysis and use in Healthcare, Hospital & Pharma Domain. Seasoned with diverse therapy area of Pharmaceutical Sales & Marketing ranging from generating revenue through generating prescriptions, launching new products, and making them big brands with continuous strategy execution at the Physician and Patients level. Moved from Sales to Marketing and Business Development for 3.5 years in South East Asian Market operating from Manila, Philippines. Came back to India and handled and developed Brands such as Gluconorm, Lupisulin, Supracal, Absolut Woman, Hemozink, Fabiflu (For COVID 19), and many more. In my previous assignment I used to develop and execute strategies on Sales & Marketing, Commercialization & Business Development for Institution and Corporate Hospital Business portfolio of Oncology Therapy Area for AstraZeneca Pharma India Ltd. Being a Research Scholar and Student of ‘Operations Research & Management: Artificial Intelligence’ I published several pioneer research papers and book chapters on the same in Internationally reputed journals and Books indexed in Scopus, Springer and Ei Compendex, Google Scholar etc. Currently, I am launching PGDM Pharmaceutical Management Program in IIHMR Bangalore and spearheading the course curriculum and structure of the same. I am interested in Collaboration for Healthcare Innovation, Pharma AI Innovation, Future trend in Marketing and Management with incubation on Healthcare, Healthcare IT startups, AI-ML Modelling and Healthcare Algorithm based training module development. I am also an affiliated member of the Institute of Management Consultant of India, looking forward to Healthcare, Healthcare IT and Innovation, Pharma and Hospital Management Consulting works.",institutionString:null,institution:{name:"Lovely Professional University",country:{name:"India"}}},{id:"1063",title:"Prof.",name:"Constantin",middleName:null,surname:"Volosencu",slug:"constantin-volosencu",fullName:"Constantin Volosencu",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/1063/images/system/1063.png",biography:"Prof. Dr. Constantin Voloşencu graduated as an engineer from\nPolitehnica University of Timișoara, Romania, where he also\nobtained a doctorate degree. He is currently a full professor in\nthe Department of Automation and Applied Informatics at the\nsame university. Dr. Voloşencu is the author of ten books, seven\nbook chapters, and more than 160 papers published in journals\nand conference proceedings. He has also edited twelve books and\nhas twenty-seven patents to his name. He is a manager of research grants, editor in\nchief and member of international journal editorial boards, a former plenary speaker, a member of scientific committees, and chair at international conferences. His\nresearch is in the fields of control systems, control of electric drives, fuzzy control\nsystems, neural network applications, fault detection and diagnosis, sensor network\napplications, monitoring of distributed parameter systems, and power ultrasound\napplications. He has developed automation equipment for machine tools, spooling\nmachines, high-power ultrasound processes, and more.",institutionString:'"Politechnica" University Timişoara',institution:null},{id:"221364",title:"Dr.",name:"Eneko",middleName:null,surname:"Osaba",slug:"eneko-osaba",fullName:"Eneko Osaba",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/221364/images/system/221364.jpg",biography:"Dr. Eneko Osaba works at TECNALIA as a senior researcher. He obtained his Ph.D. in Artificial Intelligence in 2015. He has participated in more than twenty-five local and European research projects, and in the publication of more than 130 papers. He has performed several stays at universities in the United Kingdom, Italy, and Malta. Dr. Osaba has served as a program committee member in more than forty international conferences and participated in organizing activities in more than ten international conferences. 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Her areas of interest and knowledge include natural language processing (NLP), detection of impersonation in social networks, semantic web, and machine learning. Dr. Esther Villar made several contributions at conferences and publishing in various journals in those fields. 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He is currently a principal researcher in data analytics and optimisation at TECNALIA (Spain), a visiting fellow at the Basque Center for Applied Mathematics (BCAM) and a part-time lecturer at the University of the Basque Country (UPV/EHU). His research interests gravitate on the use of descriptive, prescriptive and predictive algorithms for data mining and optimization in a diverse range of application fields such as Energy, Transport, Telecommunications, Health and Industry, among others. In these fields he has published more than 240 articles, co-supervised 8 Ph.D. theses, edited 6 books, coauthored 7 patents and participated/led more than 40 research projects. 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Currently working as an Assistant Professor in the Department of Mathematics, Institute of Applied Science, Mangalayatan University, Aligarh. She taught so many courses of Mathematics of UG and PG level. Her research Area of Expertise is Functional Analysis & Sequence Spaces. She has been working on Ideal Convergence of double sequence. She has published 17 research papers in National and International Journals including Cogent Mathematics, Filomat, Journal of Intelligent and Fuzzy Systems, Advances in Difference Equations, Journal of Mathematical Analysis, Journal of Mathematical & Computer Science etc. She has also reviewed few research papers for the and international journals. 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